godfather of surgery

Chapter 1304 You're pretty good at guessing.

After Zhang Lin left the lab excitedly, Yang Ping turned his attention back to the "identity map" on the big screen.

Even if this is indeed a common target for pancreatic cancer, designing a K factor that can accurately identify it is extremely difficult, let alone ensuring the safety, specificity, and delivery efficiency of the new K factor…

Yang Ping analyzed: "We are now facing several technical challenges—the three-dimensional structural analysis of the protein complex; the design of a K factor with high affinity and specificity based on this structure; and the stability and delivery system of the K factor in vivo. Designing the K factor is the most difficult, and although we have the current K factor as a reference, it is still an extremely difficult task."

If even the professors say it's difficult, then it really is difficult.

“Structural analysis can be left to me,” Lu Xiaolu volunteered. “Our institute just installed a cryo-electron microscope, a 300kV Titan Krios, equipped with the latest direct electron detector. If the sample is well prepared, it can theoretically be resolved to near-atomic resolution.”

"How many samples do we need?" Song Ziming asked.

"The purified protein complex needs to be at least 0.5 mg, and the purity must be above 95%," Lu Xiaolu stated the figures.

Tang Shun gasped: "Purify 0.5 milligrams of membrane protein complex from pancreatic cancer cells? How many cells would that require to culture?"

“At least… 500 liters of culture capacity.” Xu Zhiliang quickly calculated in his mind, “Moreover… the purification yield of membrane proteins is usually less than 1%, so in reality… more is needed.”

“At the normal speed, this would take at least two months, but using our amplification technology can speed up the culture process. You bring me the sample cells, and I will culture them. Cell culture is our specialty.” Tang Shun took over the cell culture work.

“Perhaps there’s another approach that can save time and get the observation started immediately,” Griffin, who had been silent all along, suddenly spoke up; his Chinese was fluent.

Everyone looked at him.

Griffin walked to the whiteboard, picked up a pen, and said, “When I was in the United States, I participated in a project on unconventional structural analysis. At that time, we were dealing with a membrane receptor that was extremely difficult to purify. In the end, we used the in-situ structural analysis method, which was not to purify the protein, but to observe it directly on the cell membrane in a near-native state.”

He drew a simple diagram: "Specifically, we label the target protein with gold nanoparticles and use cryogenic electron tomography to create three-dimensional images of local areas of intact cells. Although the resolution is not at the atomic level, it is sufficient to see the general conformation of the protein and key interaction interfaces. With this information, we can use computational simulations to complete the atomic details."

"Is there any precedent for this method?" Song Ziming asked.

“A paper published in Nature in 2019 resolved the in-situ structure of the NMDA receptor in the cellular synapse with a resolution of 4.5 angstroms,” Yang Ping accurately quoted. “The key lies in the labeling technology and image processing algorithms. Gold labeling may interfere with the native conformation of proteins, but we now have a better option—gene-encoded mini-tags, such as ALFA-tag or SpyTag, which can covalently bind specific nanolabels with less interference.”

Griffin was surprised that Professor Yang had even dabbled in such obscure papers; his knowledge was so broad.

Lu Xiaolu's eyes lit up: "If we can see the structure of this identity lock in situ on cancer cells, it can not only guide the design of K factor, but also verify its state in the real tumor microenvironment. This is more meaningful than proteins that may be inactivated or denatured after purification."

“But the technology is very challenging,” Griffin said cautiously. “Cryo-electron computed tomography (cryo-electron computed tomography) has extremely high requirements for sample preparation, low image signal-to-noise ratio, and complex data processing…”

“We can give it a try,” Yang Ping decided. “Lu Xiaolu, you’re in charge of this direction. Communicate more with Griffin and start by exploring the conditions with a small number of cells.”

"Yes, Professor." Lu Xiaolu nodded seriously.

Griffin was very excited that his opinion had been approved by Professor Yang, which was a highlight in his academic career that he could boast about.

After arranging the structural analysis, Yang Ping turned to another challenge: "Even with the structure, designing the K factor is a challenge. Traditional antibodies or small molecules are difficult to meet the requirements. Antibodies are too large and cannot penetrate the tumor matrix; small molecules lack specificity and are prone to off-target effects."

“Perhaps we can draw inspiration from nature,” Song Ziming said thoughtfully. “Professor, do you remember the concept of molecular glue you mentioned before? Certain natural compounds can pull two proteins that are not normally bound together. If we could find or design a molecule that can specifically bind the identity lock of pancreatic cancer cells to the killer receptors of immune cells…”

Yang Ping pondered for a moment: "Factor K is a completely different theory from these things. Before K therapy was rolled out, weren't we always looking for a new Factor K? We found nothing! Now we have found the identity lock of pancreatic cancer. According to our research results on osteosarcoma, this identity lock must correspond to a key. This key must exist, we just haven't found it yet. I will do this part of the work."

Is the professor going to take action personally?

The research process of the vaccine enhancer X-2 is still fresh in our minds. The professor led several newly graduated doctoral students to overcome numerous difficulties and make X-2 make a stunning debut.

Now the professor is personally researching Factor K for pancreatic cancer? Doesn't that mean the project is already halfway to success? That's Song Ziming's intuition.

……

At 11 p.m., most of the lights in the institute's experimental building were off, except for the lights in the structural biology laboratory.

Lu Xiaolu stood in front of the cryo-electron microscope control console, his eyes fixed on the constantly refreshing data stream on the screen. He had been working continuously for fourteen hours, but his mind remained highly focused. Sample preparation was progressing smoothly than expected; he had employed a modified cryogenic fixation method that preserved the native state of cell membrane proteins to the greatest extent possible.

On the screen, the pre-processed tomographic image gradually becomes clear. Among the outlines of countless organelles, some distinct granular structures are distributed on the cell membrane; these are the PANC-ID1 complexes labeled with SpyTag.

“The initial resolution is estimated to be around 20 angstroms.” Griffin’s voice came from behind him as he walked over with two cups of coffee. “It’s enough to see the general shape and aggregation, but to see the interface clearly, you need at least 10 angstroms.”

Lu Xiaolu took the coffee and said thank you: "The first round of data collection is complete. Next is 3D reconstruction and classification. I wrote a new image alignment algorithm, which may improve the signal-to-noise ratio."

"You can even write algorithms?" Griffin asked, somewhat surprised.

Lu Xiaolu rarely showed a smile: "When I was in Germany, I minored in computational biology. Structural biology is now half experiment and half computation."

The two sat side by side in front of the workstation. Lu Xiaolu brought up the interface of the program he had written. The code was concise and efficient, and the comments were clear, showing a solid foundation in computer science.

"This clustering algorithm is very innovative," Griffin commented after carefully reading it. "You not only classify by shape, but also add local membrane curvature and the distribution of neighboring proteins as features, which can better distinguish between real signals and noise."

“The function of membrane proteins is often influenced by the microenvironment,” Lu Xiaolu explained. “The same protein may have different conformations in flat membrane regions and in regions with high curvature, and its accessibility can also be altered by interactions with neighboring proteins. The K factor we designed will ultimately work in a real tumor environment, so this information is very important.” Through his conversation with Lu Xiaolu, Griffin felt that this young man’s level should be world-class, but as an expert in oncology himself, Griffin had no impression of Lu Xiaolu before. In fact, he had never heard Lu Xiaolu’s report during his several visits to China to attend academic conferences. This shouldn’t be the case.

Griffin glanced at Lu Xiaolu: "You are also engaged in tumor research. Why haven't I read your papers before? I have also communicated with Chinese scholars. How come I have never heard of your name before? That shouldn't be the case."

Lu Xiaolu had a long story to tell: "I only achieved some results after coming to the Sanbo Research Institute. Before that..." He didn't finish his sentence, and the laboratory was quiet for a moment.

“I’m sorry,” Griffin said.

He realized that Lu Xiaolu had something he couldn't say, perhaps he was a late bloomer who hadn't achieved much before.

After a short break and ending their casual conversation, Lu Xiaolu turned her attention back to the screen and started the 3D reconstruction program. Thousands of two-dimensional projection images began to be calculated, aligned, and superimposed, gradually constructing a 3D model of a local area of ​​the cell.

Time passed by, and at 2 a.m., the initial 3D reconstruction was completed.

On the screen, a stunning microscopic world unfolds: the cell membrane of cancer cells is not a smooth plane, but rather covered with undulations, depressions, and protrusions. Within these dynamic membrane structures, the PANC-ID1 complex is embedded like tiny locks.

Lu Xiaolu magnified one of the complexes, and at the current resolution, it can be seen that it consists of three main subunits: a transmembrane core, a large extracellular domain, and a smaller, irregularly shaped accessory protein.

“This small one should be the unknown protein you discovered.” Griffin pointed to the irregular module. “It seems to be connected to the core via flexible connections, like it’s oscillating on the membrane surface.”

“This might be a regulatory mechanism,” Lu Xiaolu said thoughtfully. “If the conformation of this accessory protein is variable, then the opening and closing state of the lock might be adjustable. For the K factor to function, it may need to be stable in the open state.”

Just as the two were focused on their analysis, the laboratory door was gently pushed open.

Zhang Lin peeked in, saw someone, and paused for a moment: "Dr. Lu, Dr. Griffin, you're still here?"

"What are you doing here again?" Lu Xiaolu asked.

"I... I couldn't sleep, so I got up and wandered around and saw the lights on in this lab. I wonder who will leave the lights on after get off work?" Zhang Lin scratched his head sheepishly. "Since the lights are on here, I re-analyzed the data from the breathing experiment during the day using the machine learning methods I just learned, and I found an interesting correlation. I wanted to show it on the big screen."

"You sleep in the lab?" Lu Xiaolu asked, quite puzzled.

Zhang Lin nodded: "Xiao Wu and I both sleep in the lab. How can we do experiments without sleeping in the lab? The early bird catches the worm."

Lu Xiaolu couldn't help but feel a little admiration; they were quite serious and not just joking around.

"What correlation?" Lu Xiaolu asked.

“When hand posture changes, not only does the breathing pattern change, but heart rate variability also changes simultaneously.” Zhang Lin walked in and opened his laptop. “Look, this is the frequency domain analysis when the thumb and index finger are pinched together. Low-frequency power increases and high-frequency power decreases, which is a manifestation of relatively enhanced sympathetic nerve activity. When the thumb and ring finger are pinched together, the situation is exactly the opposite—it is more like a pattern of parasympathetic nerve activation.”

Lu Xiaolu and Griffin exchanged a glance; this certainly went beyond a simple "attention diversion" explanation.

"You mean, certain gestures can directly regulate the balance of the autonomic nervous system?" Lu Xiaolu became serious.

“The data suggests this possibility, but I’m not sure about the mechanism,” Zhang Lin said. “Perhaps it’s through some kind of somatosensory-autonomic reflex that hasn’t been clearly identified yet, or… wait, what are you looking at?”

His attention was drawn to the 3D structural model on the main screen: "Is this the target of pancreatic cancer? Wow, it's so clear!"

"The preliminary model has a very low resolution," Lu Xiaolu said.

Zhang Lin leaned closer to the screen, examined it carefully for a while, and suddenly said, "This little waving protein... does it have some areas of positive charge accumulation on its surface?"

Lu Xiaolu was taken aback: "How did you figure that out? The current resolution can't show the charge distribution."

“I didn’t see it, I guessed it.” Zhang Lin pointed to a few slightly raised areas on the structural model. “Look at these places, they form a concave-convex-concave pattern in three-dimensional space. If I were a naturally evolved lock, to specifically bind with other molecules, I would probably use charge complementarity. The attraction between positive and negative charges is one of the simplest and most direct interactions.”

Griffin seemed to understand something: "Computational chemistry methods can be used to predict surface electrostatic potential, Professor Lu, why don't we give it a try?"

Lu Xiaolu nodded, brought up another program, and based on the existing low-resolution structure, he used AlphaFold2 to predict the all-atom model of the complex, and then calculated the surface electrostatic potential distribution.

The result was surprising. Just as Zhang Lin had guessed, the surface of the wobbly little protein did indeed have three obvious positive charge enrichment areas, arranged in a triangle.

“This is very likely the binding interface.” Lu Xiaolu’s voice was filled with excitement. “If the K-factor is to identify this target, its binding surface should have a corresponding region of complementary negative charges. You’re quite good at guessing.”

Zhang Lin chuckled: "Looks like my unorthodox observational skills are sometimes useful after all."

Lu Xiaolu patted his shoulder: "Scientific discoveries sometimes require a different perspective. By the way, your discovery that your gesture affects the autonomic nervous system might be explained by neuroanatomy. The palm and fingers have abundant mechanoreceptors and proprioceptors, and their afferent nerves have extensive projections in the spinal cord and brainstem, which could theoretically affect the autonomic nervous nuclei."

"Really?" Zhang Lin's eyes lit up. "Dr. Lu, could you guide me through some literature?"

"Let's do it tomorrow, it's too late now." Lu Xiaolu glanced at the time; it was already 3 a.m. "Everyone should go back and rest."

Griffin then said, "If you need, I can introduce you to a neurology expert in the United States."