On the morning of the first day, I saw that my brother did a simple cell seed plate experiment, it is not difficult to tell the truth, but it needs a more delicate operation, let a person with no academic qualifications to train, and it will not take long to train it, but why do graduate students do the same thing, is that they can observe various phenomena from a higher dimensional perspective, not only know what it is, but also know why it is so, that is, why do this experiment instead of that, I do this experiment to illustrate what kind of point of view, how we organize these points of view to become a reasonable logic, and finally form a kind of story。 Of course, we still need to explore this slowly. We still need to prepare for various experiments in a down-to-earth manner, such as cell culture, such as reagent preparation, and so on. If you want to fly, you need to practice flapping your wings honestly. -

In the afternoon, review a previously read article IKK-interacts-with-rictor-and-regulates-mTORC2 in the graduate student lounge. Generally speaking, this kind of literature has a relatively good logical chain, although it is actually very painful for me not to accumulate, because there are many preconditions that are omitted, but I am entangled in the specific construction of this logical chain, and I still don't understand many self-evident things, and the background knowledge provided by those cited literature is completely confused. But this pain is something I have to bear, and who makes me like to think at a philosophical level? If I don't put it into practice, I can go crazy. Of course, in the process, I also started to go crazy. - IKK inhibition was found ---association-between-mTOR-and-rictor was also inhibited, possibly because IKK interacts with -rictor and thus competes with mTOR, thereby reducing the expression of mTORC2. Discovery of novel mTORC2-related signaling pathways, IKK regulates mTORC2 activity, such as affecting the cytoskeleton by phosphorylating AKT-at-the-serine-473- and affecting mTORC2 assembly. IKK binds tightly to rictor, and IKK-inhibition decreases --- mTORC2 expression level --- mTORC2 activity. - For now, activating/inhibiting the expression of a gene/protein to observe the expression of the gene protein associated with it is a relatively basic experimental treatment. Of course, different levels of observation are required, such as protein expression (western-blot), tissue sectioning, in vivo processing, and even animal experiments. It is really important to have observable changes in indicators to guide the experiment. - Of course, according to my usual urine, I wondered if the process of "IKK-inhibition --- decrease in mTORC2 expression level --- decrease in mTORC2 activity" can be represented by one sequence, and then I will collect other relationship sequences, such as "IKK activation - XX protein expression level decreases - YY activity decreases" , we can construct a certain tree structure, and then according to a certain operation, such as A inhibition - B activation equivalent to A activation - B inhibition, we can construct a relatively macroscopic long sequence, of course, this is a multi-possibility path, we can determine through experiments.

Day 3: Today and yesterday together are a good experience of going through a complete western-blot. After all, I always felt very clean and neat when I read the literature before, but I thought of the struggle of various experiments behind this. In order to make a word make-sense, a lot of work is waiting for you, but this is also the realm we pursue. - Many of the reagents in the laboratory are prepared by themselves, considering the various errors, it seems that the reason for the low reproducibility of biological experiments is seen, which is a bit like traditional Chinese medicine, and success and failure are also confused. Of course, I'm more inclined to think that a successful experiment is a collapsed wave function, a concrete path emergence. -

Appendix 1 day's reflection notes:- For the various conditions of the biological organism, such as normal physiological and pathological changes of diseases, according to the game theory and natural selection model we introduced, we try to imitate the collapse of the wave function to form a specific path, that is, with the sequence as the basic object of operation, and the competitive game between the sequences can form a certain matrix, thus representing different new states, and the different elements of these matrices have a certain correspondence with the various health conditions of the body. Just as the convergence property of energy minimization is also related to the principle of the existence of continuous functions, Brouwer's fixed point principle states that there is a stable fixed point, and the connection between the different objects of the matrix is the Nash equilibrium, that is, the stable state formed by the sequential competitive game. The sequence of multi-level equilibrium formation is the macroscopic life cycle of organisms. - Specifically, there are various anti-XX mechanisms, such as antioxidant and so on. From the perspective of the network, there are different levels of resistance, that is, there is a competitive game. And we acknowledge relativity, i.e., what is generally thought to be a specific anti-xx protein, but the gene protein can exhibit the opposite effect in other contexts. Based on the above ideas, we decided to use the structure of the sequence to explain these biological mechanisms. We imagine that there is a sequence, each of which corresponds to a specific gene protein, and the specific expression level is a specific sequence relationship, so the expression pattern of the corresponding gene protein is an object of operation. - First of all, we have to prove that this assumption is not too outrageous, and we think that some of the literature on cell biology that we have read can support it. By constructing a certain relationship between specific gene proteins and macroscopic biological processes, a certain intrinsic relationship is sought, that is, possible proteins with high expression probability. This is an emergent path, based on a series of experiments. For example, IKK inhibition---association-between-mTOR-and-rictor is also inhibited, possibly because IKK interacts with -rictor and thus competes with mTOR, thereby reducing mTORC2 expression. Discovery of novel mTORC2-related signaling pathways, IKK regulates mTORC2 activity, such as affecting the cytoskeleton by phosphorylating AKT-at-the-serine-473- and affecting mTORC2 assembly. IKK-inhibition --- decreased mTORC2 expression level --- mTORC2 activity was a sequence-related emergence. We can obtain this high-dimensional relationship from the changes in the expression levels of specific gene proteins. Not strict certification. - "IKK-inhibition --- decrease in mTORC2 expression level --- decrease in mTORC2 activity" This process can be represented by one sequence, and then I collect other relationship sequences, such as "IKK activation - XX protein expression level decreases - YY activity decreases" , we can construct a certain tree structure, and then according to a certain operation, such as A inhibition - B activation equivalent to A activation - B inhibition, we can construct a relatively macroscopic long sequence, of course, this is a multi-possibility path, we can determine through experiments. - Thus we can think of concrete biological processes as equilibrium states formed by the game of multi-level sequences. - We can use the medium to partially illustrate. Complete medium can only proliferate cells, while the addition of β glycerol, vitamin C, and dexamethasone can cause cells to differentiate, and these components can be understood as a change in the pattern of their final expression due to the effect on the sequence. Of course, the specific action pathways are multi-layered, and our ideal is to be able to integrate large-scale data, that is, the summary of the basic sequence relationships can lead to specific pathways, or even fixed-point intrinsic pathways. We are able to have an impact at the macro level through the impact of this pathway, that is, to build a relatively high correlation between basic research and clinical research. This is not nonsense, see the various receptor antagonists that can be produced as drugs to promote medical treatment. Of course, the most brilliant achievement of this immobility point is the antagonistic effect of antibiotics on microorganisms, and what we need today is multi-level coupling to approximate this effect. One of them, the cocktail therapy for AIDS, is a good one.

Day 4: In the afternoon, we will officially do qPCR, that is, all kinds of reagents, and then put them on the machine. Speechless. During the three hours of waiting, we added the tip of the gun with my brother, processed the cells, and then rested. We went back to the lounge and caught my sister and pestered her to ask about the idea behind the experiment. The answer is to look at the literature, look at the logical reasoning and experimental proof of other people's articles. Of course, I was still very greedy and didn't want to have a general grasp of the overall subject research during the short apprenticeship, so I took out the literature I had read before, and asked why such an elegant logic could be obtained through such a large number of ugly and chaotic experiments, and it turned out that it was based on a lot of pathways discovered by the work of predecessors, and then the specific topic was to choose a specific object to deal with, and then there were certain changes that could reveal a certain relationship, which is the subtitle of the aesthetic result in the article. Therefore, I think that the experiment without aesthetic sense is actually my lack of level, and I can see the forest for the trees. This reminds us once again of the importance of the basics of the background, and that we can only communicate meaningfully if we have a common language of communication. -As for the selection of topics, it takes the help of the tutor and your own efforts to determine the direction, and then you collect various materials and read, and constantly determine the final idea in the process. In a word, a large amount of literature research. The next thing is the very detailed details, and it is difficult to communicate without small peers. - Experimental technology is art, all kinds of theories are the way, there is no way to achieve nothing, there is no way to know. We need techniques to verify our Dao, after all, "the avenue is easy to obtain, but the small technique is difficult to find", the former is the macro structure of the network, and the latter is the collapse of the specific network path, which is the emergence of probability.

Day 5: Brother told us a little bit about the principles behind his experiments, the forest I talked about earlier, the big picture behind a series of complex experiments. In fact, it is to narrow the scope of various databases to determine the target that is relevant to one's own purpose, and in this case, to find proteins related to diseases. Protein chips were used to screen out related proteins with large changes in expression, and some of these proteins were selected for various experiments such as western-blot to verify their correlation with changes at various levels of disease. The purpose of the senior brother is to find an unreported protein that can build a certain correlation with macroscopic diseases, from in vitro cell experiments to xenotransplantation in nude mice in vivo to the detection of clinical samples and so on to prove its relevance, so as to finally provide a theoretical basis for drug targets. The process is the search for new knowledge, and no one knows what will happen. The trial and error of this process reminds me of a game in the last lesson of our team building, which is to find a safe path in a grid full of mines, and this process requires many failures to find the only definite pathway, which corresponds to the formation of a certain protein pathway, that is, the mechanism that our senior brother wants to get. It is foreseeable that the senior brother will face the torture of countless failures before he can find the final mechanism, publish articles, and successfully graduate. One of the big takeaways is to keep your feet on the ground, and I've been ambitious to do a big integration, and it's embarrassing to float in mid-air. Although it is good to say that teenagers have dreams, mature people need to break down their goals into small goals that can be executed in order to finally integrate and complete the final goal. I still need to keep learning. - Continue to do the qPCR that failed yesterday. The RNA is unstable, so it is reverse transcribed into DNA, and then PCR can be amplified in large quantities, and we can indirectly get RNA information. -In this process, the amount of each substance needs to be carefully designed. Today I was going to do two sets and use different mixes (one of the reagents of the kit), and it turned out that it seemed that there was not enough cDNA for transcription. Then the primers seem to be added incorrectly. Then we'll see what happens. Well, the result of the three-hour wait is still-like data. Senior brother will continue to work hard tomorrow, which is also a growth process that constantly eliminates mistakes.

Day 6: Brother gives us a chance to do a complete Western-blot. The glue that was prepared yesterday was found to be unusable today, so it can only be matched again. So we waited and waited, after all, the real operation time is not long, the waiting time is long, and the interval between different experiments still needs to be prepared for the next set of experiments. -During this period, the senior brother took us to see the results of yesterday's western-blot, and the results were terrible. Experiments are not just one after another, but also mistakes. At first, the glue was unevenly matched, and then we were dumbfounded in the darkroom watching the results come out. Senior brother can only do it again, and a few days of failure will be in vain. - And then we go ahead and do it. It was almost 3 o'clock when I ran electrophoresis after adding samples, and we could finally have lunch, and the takeaway gave me the experience of a full banquet, which was really tearful. What made me cry even more was when I ran to see how the electrophoresis was doing after eating, only to find that the bands were wavy. Hell, our glue was uneven, the initial experiment was wrong, and our next experiment was meaningless. One wrong step, one wrong step.

When we watched my brother do the experiment, it was called a comfort, and I thought it was very simple. I didn't expect that the kung fu is outside the poem! The addition of various reagents is very simple, but how to add, what order and what concentration ratio requires experience, and why there is also a lot of attention and knowledge. Anyway, when we did it ourselves, there were all kinds of rushes, all kinds of plausible, and there were many things that we should have paid attention to but didn't notice. When it came to the setting of various parameters of the machine, it was also full of laboratories running around to find senior brothers and sisters. It can be said that reality has given me a hard slap in the face for dreamers. Reality requires a down-to-earth approach. The result of our western-blot is out, and it can only be said that ten thousand arrows pierce the heart. The protein bands of 36 and 88KD did not come out, and the result was ugly. I was confident that I might be able to do better when I saw my brother doing the experiment, but now I think about it, where did I get my confidence? The success rate and results of the experiments I did in school were not bad, but this is completely different from the current situation where I have to prepare many experimental materials by myself. This may be similar to the situation when we come out of society in the future, the arrogance at the beginning is actually a kind of illusory self-confidence, and only after various tests of reality can we recognize how many pounds and taels the real self has.

Day 9: The senior brother first summarized the various errors of qPCR. First of all, the software operation is not proficient, and some of the settings are wrong, which is the result of the senior brother asking the teacher. There may also be operational issues. And it may be of a different species than the primers that the senior brother asked for from the senior sister. This suggests that we should be careful about the sources when we need materials, and it is best to look up the literature. - Today, Senior Brother gave us two documents to read (⊙o⊙). I feel like my grade is different. Before, I always felt that there was a layer of separation when I was looking for articles by myself, and I didn't seem to be able to read it, so I read a lot of articles in one go, and it was regarded as accumulation. I found that it is still very necessary, it doesn't matter if you don't understand it now, you will understand it slowly after reading more. After a few days of internship in the laboratory, I found that I was able to understand an article at a higher level. For example, western-blot verifies and even discovers the connection between different objects by combining the changes in the amount of expression of different proteins and the relationship between protein pathways revealed in the original literature. If there is already a pathway ABCD, if it is processed to a certain extent, such as inhibiting the expression of B, it is theoretically possible to find a decrease in the expression of C, if not, it means that there are other regulatory mechanisms, which can be done for the next step. - And I have always emphasized the beauty of the data, such as the western-blot strips must be uniform and straight, but now I don't have such high requirements, probably because I have personally done these experiments, I know how difficult it is to produce data, and I am more tolerant.

Day 12: The results of qPCR are very strange, I found that the expression of a certain gene has decreased significantly, more than 100 times, I was excited at the time, no, this may be a more valuable discovery, maybe finally able to achieve the target of a certain drug! It seems that I still think too much as always, always thinking that others are wrong, but it is more likely that I am wrong. But I was still not reconciled, so I went to Google Scholar to search for literature, and found that the expression of this gene increased by 2 times. Although this is a measurement of human cells, we do mouse cells, but according to my brother, the expression is not much worse, so our RANKL expression has dropped by 100 times, which is inconsistent with the existing literature. On the premise that we can make credible assumptions about this document, there is a high probability that something is wrong with us. Of course, as a dead rationalist, we can still consider that there is a low probability that this is a new discovery that has not been made before/that the previous person was wrong. But for now, it's better not to be so ambitious, for me, the probability of 1 in 10,000 is equal to non-existent. - There may always be some problems with the results of the experiment, which requires the completion of the experiment, and the experimental materials are not said to be available, and the cultivation takes time. Therefore, while doing experiments, we should pay attention to the cultivation of echelons. In this way, every few days is a cycle, maintaining a constant supply. And the cryopreservation of cells is also necessary.

Day 13: When preparing for RT, i.e., reverse transcription, the senior brother should ensure that the relative concentration of RNA is the same, that is, different concentrations of RNA plus different doses. We use a computer to calculate to a few tenths of a microliter, of course, there is no use in it, we still have to round up to a few tenths of a microliter, because the accuracy of the dispensing gun is limited. And specific biological experiments don't need that much precision. You don't know how fortunate I am at this moment! The calculation of various processes of organisms is relatively vague, which provides a guarantee for the maximization of the adaptability of organisms to the environment, and can eventually form a certain distribution within a certain range, rather than various calculations like mathematics that can be accurate to n decimal places. Although the coupling of multi-level distributions may lead to a certain window period and require very precise conditions, generally speaking, there is still a certain probability that the results of our treatment of variables will occur, and we can confirm the final relationship between the variables by adjusting the relative proportions of different variables in the reaction system, such as setting a certain gradient. In other words, you can toss freely, because the final expression is the result of a Markov sequence expression, which can be understood as a certain distribution, and as long as it exceeds a certain threshold, there can be an observed effect. And this is the reason why we apply different treatments but have the same effect, which is the construction of equivalence relations for different paths.

Day 15: It looks like we're going to do a literature review on this little topic. In other words, I need to select various existing research objects, such as bone protectin, to observe the effects of disease on genes that form a coupling relationship under the premise of equilibrium in the competitive game of different objects. My ideal is that the effect of each object on gene expression is probabilistic, and then the interaction of multiple objects can iterate the probabilities like Bayesian formulas.

Day 19: A review of the literature to be done. - First of all, of course, the general background, the harm of osteoporosis to the population, etc., and then you can cite a few articles that I have not read seriously but many people want to cite, such as (NIH-Consensus, -2001).-(WHO, -Press-Release 1999) and so on. And then there's the question of how much it means to brag about the disease. As-a-result-it’s-of-great-importance-to-release-the-clinical-and-social-and-economic-pressure-of-the-modern-world-by-exploring-the-cellular-and-molecular-mechanism-of-osteoporosisOnly disease-related research is more of interest. - And then there's the more specifics. I intend to reconstruct the idea of disease from a game theory perspective, that osteoporosis can be broken down into a competitive game between osteoblasts and osteoclasts, and the equilibrium reached by them shifts in the direction of osteoclast formation. On this basis, we continue to delve into the molecular level, the RANKL/RANK system. -We-think-that-the-pathophysiology-changes-happen-when-the-basal-balances-between-some-key-genes/proteins-expression-were-to-be-damaged.-In-this-case， -we-would-like-to-concert-about-the-disease-called-osteoporosis，-which-can-be-caused-by-the-imbalance-between-the-expression-level-of-OPG，-RANKL-and-RANK.-After-we-explain-the-whole-picture-of-the-disease,-we-then-hope-to-pick-out-all-the-possible-targets,-like-the-osteoprotegerin,-receptor-activator-of-nuclear-factor-kappa-B-(RANK)-and-RANK-ligand, -which-are-crucial-to-osteoclastogenesis -and-osteoblastogenesis (osteoblastogenesis), -bone-regeneration -bone-resorption, -that-is-the-key-to-the-balance-of-osteoclast-and-osteoblast-including-absolute-amount-and-relative-ratio-of-any-kind. -How-illness-can-be-caused-is-that-the-equilibriums-of-such-competitions-between-coupling-pair-go-beyond-the-normal-range.-Such-equilibriums-can-be-further-explained-in-the-level-of-genes-and-proteins，-and-it’s-the-way-how-we-know-the-processes-of-bone-physiology-and-pathophysiology.- As-far-as-we-concert，-we-think-it’s-the-way-to-build-a-great-system-using-the-same-way-Newton-built-his-calculus.- Different-genes-and-proteins-react-lonely-can-be-regarded-as-the-partial-derivative，-which-is-the-way-how-we-do-our-research， -but-nowadays-we-are-trying-to-add-up-the-sum-effect-of-multi-gene/protein-to-get-a-better-understanding，-and-by-this-way-can-we-get-to-know-how-to-guide-the-medicine. -We-can-change-the-relative-rate-of-different-molecular-so-as-to-change-the-relative-rate-of-osteoblast-and-osteoclast. ,-then-the-balance-is-responsible-for-osteoporosis,-cancer-induced-bone-destruction(skeletal-metastasis)(etc.),-and-so-on. -The-equilibrium-between-bone-resorption-and-new-bone-formation-can-be-combined-with-different-effects-like-tumor-cells，-cytokins，-and-so-on. -What-matters-most-is-the-pattern-of-OPG-and-RANKL-expression-RANKL/RANK-signaling-is-related-to-osteoclast-formation，-activation-and-survival-in-many-situation. -OPG-protects-bone-from-excessive-resorption-by-binding-to-RANKL-and-preventing-it-from-binding-to-RANK.-So，-the-relative-expression-of-RANKL-and-OPG-determines-the-physiology-and-pathophysiology-in-bone.- Different gene protein expression patterns can be associated with disease, and selective changes to this expression pattern can be used for treatment. Disease is the selective expression of the normal state, and the disease also has the expression of the normal partial state, which is the fixed point of coupling. Specific selective expression is a kind of macro-level coordination, that is, selectively applying different treatments to different objects. - OPG, RANKL-and-RANK system, regulates bone resorption and bone formation, and the fixed point level is stem cells. Below the cellular level are the expression and binding of various protein levels, such as receptor-activator-of-NF-jB-ligand-(RANKL)-and-osteoprotegerin- (OPG) and other cytokines, and can construct certain relationships with certain signaling pathways. Of course, there is a multi-action pathway for multi-action objects, that is, a specific biological process is related to many protein processes, and these protein processes are not only related to these processes, but also related to other processes. - Normal bone formation—bone reformation—OPG, RANKL-and-RANK activation relationship matrix on the regulation of osteoclasts—respective effects—comprehensive effects—specific mechanisms, effects on transcription factors, effects on membrane receptors, effects on signaling pathways, overall homeostasis, equivalence construction of different state sequences: inhibition of bone resorption = bone formation (equilibrium movement), activation of bone formation = inhibition of bone resorption. The existing inhibition of bone resorption has been effective in clinical treatment, but we can continue to apply some treatment to its equivalent sequence in depth, after all, the reflex law can still be partially established (the inhibition of inhibition is the same as the activation part). The only way to avoid the long-term malignant effects that may arise from the disruption of this equilibrium (inhibition of RANKL) is to avoid the possible long-term malignant effects of this antagonist OPG and its monoclonal antibody, and at the same time we apply a certain treatment to its equivalent sequence, which is to avoid the possible long-term malignant effects of this balance break (inhibition of RANKL-The-RANKL/RANK/OPG-SYSTEM-IS-A-LOW-DIMENSIONAL-NETWORK-OF-THE-WHOLE-BODY, -which-can-explain-many-aspects-of-bone-biology— that-is-any-disease-comes-from-the-damage-of-homestasis，-which-can-be-regarded-as-result-of-different-subjects-completing-，-and-achieve-the-Nash’s-equilibrium.- In-a-way，-we-can-use-the-lineal-algebra-to-make-this-concept-more-clearer:-different-subjects，-like-RANKL/RANK/ OPG-,-are-the-bases,-and-its-lineal-combination-can-correspond-to-any-dots-in-the-space,-that-means-it-can-explain-all-the-situations-of-bone-biology.-Then there is the specific effect of the object on the system, and the ability to observe its possible needs through certain indicatorsand then go down to the molecular level according to the previous research ideas. Effects-of-Phytoestrogen-a-ZAL-and-Mechanical-Stimulation-on-Proliferation,-Osteoblastic-Differentiation,-and-OPG/ Specific sequence of action, effects of phytoestrogens Phytoestrogen-a-ZAL on various aspects of MC3T3-E1-Pre-Osteoblasts osteoblasts: proliferation, osteoblast differentiation, OPG/RANKL expression pattern. Bone formation also has the effects of estrogen and mechanical stress. -a-ZAL inhibits proliferation and promotes differentiation (increased staining of ALP RNA), Runx2-mRNA- expression increases, and the -OPG/RANKL ratio is upregulated, i.e., the balance shifts to inhibit osteoclast. - Mechanical stress promotes proliferation and differentiation, Runx2-protein expression (also a marker of osteoblast differentiation), and the OPG/RANKL ratio is upregulated. - Its co-effect, i.e. the coupling operation of the sequence, inhibits proliferation. High concentration and intensity promoted ALP (osteoblast differentiation marker), a-ZAL and high intensity mechanical stress promoted RUNX2 expression and decreased the OPG/RANKL ratio, while a-ZAL and low intensity mechanical stress increased the OPG/RANKL ratio. - Theoretically, the OPG/RANKL ratio up-regulation is a principle of treatment, and osteoporosis can be treated with a-ZAL and mechanical stress of appropriate intensity. The underlying mechanism of this possible treatment is actually the interaction of various signaling pathways, which we can continue to deal with. Such as Changing-RANKL/OPG-mRNA-expression-in-differentiating-murine-primary-osteoblasts. - Effects that can be produced by changing the ratio of RANKL/OPG expression. The receptor acts as an immobile point, and the combined action of its ligand and other antagonists provides the receptor to lead the antagonism to produce an effect. Therefore, we can introduce different objects according to the signaling pathway, such as estrogen and vitamin D, and observe their effects on various expressed gene proteins in the cell, so as to construct a certain logical relationship. - Effectiveness of sequence calculations: OPG is continuously upregulated in osteoblast differentiation, and RANKL-mRNA can be promoted by vitamin d, while vitamin d has no effect on the expression of OPG in osteoblasts. RANKL/OPG-ratio, an indicator of osteoclast production, can be expressed by vitamin d, which is of course reduced by mineralization (inhibition of osteoblasts) - Conclusion: vitamin d - increases the expression level of RANKL - promotes osteoclast formation. The ratio of mature and immature cells is related to the physiology of macroscopic bones, which is a balanced mobile treatment of the microenvironment. Associated with bone resorption and formation. We should also consider the location of these cells. - After proposing the effects of the above different factors (phytoestrogens, vitamin D, mechanical stress, etc.) on the balance of osteoblasts/osteoclasts, we found that the existing literature is more biased towards the role of osteoclasts, and as the two ends of the balance, in fact, the roles of both sides should be explored at the same time, i.e., RANKL/RANK/ OPG is a protein system at the osteoclast level, and theoretically there should be a protein system at the osteogenic level, such as various cytokines and receptors, which can significantly antagonize various osteoclast effects and promote the differentiation of osteoblast levels rather than inhibit the differentiation of osteoclasts. After all, the interface of this osteocyte organism should not only have RANK receptors. So we need to consider the influence of other signaling pathways, because this is essentially a probabilistic network. - At the cellular level, the factors secreted by adipocytes promote the proliferation of osteoblasts and can influence osteoclast formation through some OPG/RANKL expression ratio. Its specific mechanism of action is the change of specific protein expression patterns, which causes changes in signaling pathways. The specific exploration is still old-fashioned, the protein that plays an inhibitory role and the previously known work, first look at the role played by specific objects, the factors secreted by adipocytes increase MC3T3-E1-2.8 times, and the cell proliferation of pre-osteoblasts is 1.5 times, and then through groping to observe which proteins can inhibit these processes, it is found that the inhibitors of FGFR1-and-PI3K tyrosine kinase can reduce its proliferation, indicating that this process is related to the signaling pathway related to FGFR1-and-PI3K, the above is the description of the phenomenon, and then the specific cell model, animal models and even clinical testing and experiments. The increasing scale of this dimension requires us to accumulate more information in order to play a meaningful guiding role. - Adipocytes were able to secrete bFGF, and bFGF could promote the proliferation of related osteoblasts, and the proportion of OPG/RANKL expression was significantly increased (9-fold), indicating that the osteoclast effect was reduced. This suggests that the relationship between osteoblasts and osteoclasts can be mediated by adipocytes, the protein system at the osteogenic level that we mentioned earlier. （Adipocyte-secreted-factors-increase-osteoblast-proliferation-and-the-OPG/ RANKL-ratio-to-influence-osteoclast-formation)--Then we need to collect more literature to discover the possible effects of protein pathways, and then we can make our own scientific hypotheses based on this. - And finally, the summary. -In-summary，-the-relative-concertration-of-osteoblast-and-osteoclast-play-an-important-part-in-osteogenesis-and-bone-reabsorption，-******- differences-in-bone-density-and-mass，-which-is-highly-related-to-bone-diseases-such-as-osteoporosis.-As-we-look-deeper， -we-can-find-out-the-expression-level-of-some-genes-and-proteins-have-high-correlation-with-the-relative-amount-of-osteoblast-and-osteoclast: -RANKL-and-RANK-regulate-the-osteoclastogensis-while-OPG-blocks-such-progress-by-inhibiting-RANK-expression-via-binding-RANKL. -And-other-cytokins-can-contribute-to-such-progress-for-their-impacts-on-the-signal-pathway，-which-is-a-higher-dimensional-structure-that-can-account-for-different-effects-of-a-varity-of-genes-and-proteins.-For-example，-the-secretion-of-OPG-is-affected-by-Wnt/β-catenin-pathway.-We-know-that-such-pathways-are-always-related-to-such-network.-As-a-result， -we-can-choose-our-therapy-to-treat-diseases-through-changing-the-balance-of-coupling-pairs-like-RANKL-and-OPG. -We-can-use-different-antibody/ antagonist-to-inhibit-the-expression-or-agonist-to-promote-the-expression-so-that-the-balance-of-osteoblast-and-osteoclast-can-be-changed,-bolcking-the-pathologic-pathway-.- theoretically this is the overall framework of my review, which is very brief, and not enough literature research has been doneMany of the ideas are based on many concepts of mathematics and hope to be used in the study of biology, so they are still very immature, so please understand.

The existing model of biological research (statement, limited to a part of my understanding) can be idealized as a certain sequence: primary factor - object of action (generally a system formed by a multi-level game) - effect of action (observation of various marker formulas). As mentioned above, estrogen, vitamin D, fat cell secretion factors, and so on. But this is the univariate level of calculus, I think of the joint action of multiple variables, of course, this process will inevitably produce a certain amount of interaction, and many of the results of multivariate of calculus cannot be directly used (of course, ideally, we can apply it if there is no interaction, but biology just has many interactions, no way). Combined with the orthogonal experiments and meta-analyses I have studied before, I wondered if it is possible to apply a certain computational model to simulate the effects of these factors through certain calculations, and finally provide an individualized treatment plan: ABCDE+FNEWD+ECWNJ+JHJCD=HELP (sequence operation, the specific form is to be determined), that is, different proportions of various drugs (like the substrate of linear algebra) are privately customized. This is my vision. After all, my computing power is limited, and knowing that estrogen induces osteocyte differentiation and cyclophosphamide inhibits osteoblast differentiation, I can speculate that cyclophosphamide may exert an effect on estrogen receptors to inhibit osteoblast differentiation. But in my opinion, this is far from enough, I need more data, make more inferences, select meaningful pathways for the various possible action sequences generated in it, and I need basic research that can play a guiding role in the clinic.

Day 20: Speaking of which, I have some doubts about the significance of expressing different genes and proteins, and the significance of up-regulation or down-regulation of expression is actually just a selective expression of Markov sequences, and we need to get its general distribution at the statistical level, which is a relatively high-dimensional picture. Moreover, the change in expression levels at different times may not necessarily be continuous, and we are currently only based on the assumption that the expression levels of different genes are constant at different times, which is likely to cause various false positives. Because the expression of a related gene is relatively constant due to the influence of various relationships, this can be used as a marker, and the expression pattern of more genes changes periodically, and we can only observe the expression of genes with large changes in cycles. In other words, the genes we selected must have the above characteristics in order to produce better results, which can be regarded as the level of intrinsic characteristics. But other gene expressions should be based on this fixed point distribution pattern. A simpler expression is to make logical inferences through this observation of the difference in the amount of expression, much like a blind man touching an elephant. But at present, it seems that this is a local optimal solution, with a certain probability of approximating the global optimum, that is, an accurate description of the whole. I can only hope that my further learning algorithms can help me break through this methodology, and the limitations of paradigms, such as the BLAST algorithm for biological information, may be a good breakthrough point.

Day 21: The senior brother and they had different influences on these rats before, the control group had a normal diet, the experimental group was eating sausages, and there were low-dose and high-dose groups. After a period of time and a month, it is now necessary to observe its impact through various indicators, that is, it is necessary to measure different indicators of different parts of the digestive system: esophagus, stomach, rectum, colon, liver, respectively, to do pathological sections and part of the tissue liquid nitrogen preservation, and then to do metabolome analysis. It is hoped that through the analysis of these indicators, we can draw conclusions about the possible impact of sausage on the human digestive system, so as to guide our eating habits, and to be able to discover possible changes in metabolic pathways, which can provide certain targets for the next treatment, such as which pathways are up-regulated and down-regulated, and we can antagonize this change to treat. - I've never been happy with this model, but there's no better way. Moreover, this measurement is a change in expression within the cell, relatively ignoring the higher level of cell-to-cell connections and even the relative relationship of tissues, after all, the latter is more difficult to measure and less stable. Don't think about it so much now.

Day 22: While waiting for electrophoresis, my brother took us to search for some proteins that may be related to the relationship between osteoporosis according to the mechanism, that is, A may be related to the BCDEF process, and G may be related to the F process, so we can construct a possible relationship between A and G. A more specific example is the expression of a specific gene in skeletal muscle, because bone and muscle are relatively integrated (muscle atrophy and osteoporosis have a certain correlation), so the gene protein may be associated with osteoporosis ref1 simple theoretical derivation, not sure whether it is right or wrong, so it needs to be verified experimentally). Theoretically, if you find these proteins that have not been reported, then it may be a treasure, of course, it may also be that others can't do it so they are not reported, this is the legendary pit. So we all got our weapons, pubmed, Google-scholar, springer, sciencedirect, and waited to return to a blank page. It's a bit of a pit that I can't find it in pubmed, but I find a lot of them in google-scholar, which is embarrassing. Theoretically, you can go through all the databases to determine the next direction.

1The-role-of-sarcopenia-with-and-without-fracture

Twenty-third day: A little personal thought: injured mice are in a state of stress, their normal physiological functions will change compared with the general function, which requires us to observe and understand these levels of data on a larger scale, our current scientific research paradigm can only understand the univariate like partial derivation, and the accumulation of knowledge of the former is also needed for the simultaneous consideration of multiple variables. In other words, we just don't have the ability and desire to understand these data (the task is not to explore stress changes), and we can't just want to run before we learn to walk. After all, injured mice may be related to changes in their brains, social formation of mice, etc., which can be related to many studies of neurobiology, but the topic cannot be completed unless it is done with big data. - With the development of contemporary computer technology and the establishment of genomics and other omics, we may be able to understand the relationship between these data at the overall network level, and then find the theoretically highly clustered or highly correlated objects and their roles according to a certain classification, and then we can verify these possible relationships at the experimental level, so as to establish a macroscopic understanding of life. It seems to be reversed from the current model, this is the dry experiment of biological information, and in the end, it still needs to be verified by the current wet experiment, and a certain equilibrium may be formed in the end. After all, being too biased is incomplete. -

Discuss:

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From our current point of view, the reductionist idea of biological research, that is, the decomposition into pathways and even protein-gene interactions, is actually a continuation of the calculus of Newton's time: the idea of treating biology as a complex function, and decomposing it into small enough pieces, theoretically infinitesimal quantities, can then be infinitely summed, that is, integrals, and can be continuously ascended in this way to construct a sufficiently high-dimensional structure. Theoretically, it is possible to continuously approximate the real biological network through this method, because we can assume that the network is a sufficiently high-dimensional structure, and based on our previous life experience, there is nothing that cannot be explained in the higher perspective dimension, and if there is, then rise to a higher dimension. If we could, we could think of Hilbert space as a kind of modeling of the network, because infinite dimensions can kill everything, of course, this is a mathematical ideal, and the amount of computation is desperately large, so we need to develop various algorithms to simplify. And there is a big monster of Gödel's law of incompleteness, and yes, we think that the construction of Hilbert's formal system is the process of building a model of a network. (I don't comment on the truth or falsity of this inference/brain hole).

This scientific paradigm can be said to have laid the foundation for the brilliant achievements of our modern science, and this reductionist way of thinking does not exclude the holistic mode of thinking, after all, the whole is a sufficiently high-dimensional structure that can be constructed by integral traversal ascending. In the study of biology, we found that it uses a methodology between absolute reductionism and absolute holism: through the influence of the underlying molecules, observe their changes at different levels, such as cells, tissues and organs, etc., and finally build a certain relationship with macroscopic biological processes such as diseases. From our current point of view, the construction of this pathway is a probabilistic behavior, that is, only a certain proportion of macroscopic large-scale expressions will appear in a certain proportion of specific patterns, which may partly explain why the reproducibility of biological papers is still relatively low.

In other words, we believe that the construction of the network is actually a probabilistic connection of various basic modules at different levels. The combination of these modules is a Bayesian process, even if the probability of the overall pathway increasing until a certain threshold is exceeded. And then, haha, an article came out. This can be explained by the emergence of networks, and of course the annealing of metals is also a good explanation angle.

The application of various existing mathematical knowledge, probability theory, graph theory, information theory and other system theory is an attempt to explain this structure. At present, we tend to use sequence analysis, a branch of systems biology, bioinformatics to model the network structure. We use sequences as the basic units to deal with the properties of networks, like the infinitesimal quantities of calculus. Of course, this is just an idea at present, with the limited computer knowledge I have learned so far, we still need to define the sequence and its operation, after all, genomics can use large-scale sequencing ACTG sequences to do various operations to build different levels of databases, so as to extract high-dimensional information such as protein structure. There is a preliminary theoretical reflection on this, as shown in Appendix 1. If true, we will be able to explore the various properties of the network through various operations on the sequence. Of course, at the moment it seems to be just a pipe dream. But if we can really construct this kind of data structure, we can imitate the various equivalence transformations of logical operations to construct new sequences (different concentrations of proteins of different genes) and thus add various operations, and the current ideas are that game theory and equilibrium achievement can be used as a mathematical construct for sequence operations. The path formed by the connection between the sequences is a high-dimensional structure, like the formation of a feedback module.

At the same time, various discoveries in network science, such as the power-law distribution of Barabír, and the small-world model are also very helpful for the construction of networks. Of course, the application of various statistics of mathematics is also necessary, because the emergence of various situations in the network can be distributed to a certain extent. That means we need to make full use of all kinds of big data, which means that we also need to use all kinds of mathematical knowledge, especially various statistical tools. According to the current trend, we still need to learn various programming languages such as C language and have basic programming skills.

Appendix 1: Today's article is about Shinya Yamanaka's 12th Nobel Prize-winning article. The logic is as clear as the literature I have read in the past, and it gives people a beautiful feeling, especially when you see so many documents cited in the introduction and discussion, you can't help but feel that scientific research is a great project that relies on close cooperation. Only by basing our work on the work of our predecessors like them can we expand the boundaries of human cognition, which is a great inspiration to me, after all, I have a good impression of the system theory of holism, and I prefer the network theory in it, and I have always tried to use the perspective of the network to interpret the world. If you remember my earlier modeling of love, it was based on a sequence (and also a good definition) matching, and the sequence is an object of operation in the network. But this is all relatively abstract thinking, and I have to go down to the ground to fulfill my ideal of network theory. This article embodies the philosophy of complexity that I want, the mechanism by which four transcription factors can produce pluripotent stem cells, understood from the perspective of the network, is that for a specific central node, it is affected by the transfer and expression of exogenous genes, resulting in a change in the overall network topology, which can be activated to the original pluripotency state of stem cells, like an energy level transition. The above is a bit too abstract, I'll try to simplify it a bit. Then imitate the process of building theories in mathematics. So first of all, the definition, we define genes at different expression levels as certain sequences, such as Oct3/4, -Sox2, -c-Myc, -Klf4, p53, Nanog, Eras, βactin, which can be understood. Theoretically, we can map all the cells in the world to a specific sequence as long as the defined sequence is long enough. Although this is impossible to do, it is theoretically feasible but it is too difficult to operate in practice. The second is to define operations based on sequence-sufficient objects, such as addition, subtraction, multiplication, and division. As a mathematical structure, according to the previous knowledge of bioinformatics, we define the operation of sequence as matching. The pattern of expression levels of the sequences is similar and can be approximated as an equivalence relationship. In this article, we will confirm the pluripotency of iPS cells by measuring various key gene proteins. At the same time, since I have always been fond of game theory in economics, we can idealize this sequence matching process as a sequence of competitive games. The competition between sequences and the achievement of equilibrium are the basis for the formation of important network paths. The coupling of this sequence can form a complex relationship, that is, we need a certain coupling pair to play a special effect, that is, it can form a certain high-dimensional space due to the competitive game of the effect, and its equilibrium is the expression of the specific Markov sequence, which determines the specific expression of reality. One of the key applications is the concept of fixed points, where sequential games can form a certain equilibrium point, i.e., a fixed point. This is reflected in the various marker gene proteins that we commonly use, and we are able to determine the high-dimensional structure by measuring specific fixed points. This leads me to my beloved theory of networking. The network is a high-dimensional structure formed by sequence competition game, and its selective expression is the formation of specific sequences, that is, it can be regarded as the differentiation of various types of cells. The way of thinking about the network is not one-way, but a multi-directional comprehensive expression. For example, the antiarrhythmic drug Tambocor's mechanism of action is to inhibit premature beats, thereby reducing the occurrence of heart disease, but this drug may eventually cause tens of thousands of deaths. The view of the network is not only to suppress or activate specific objects, but to selectively activate and suppress sequences, that is, to have different effects on different objects. In this article, the selective processing of different sequences is necessary to enable the emergence of the final pattern, i.e., the generation of iPS cells, such as Klf4 activating p21-CIP1, thereby inhibiting cell proliferation. This can be understood as the convergence of the Taylor series caused by the first and second orders of the series. To take a very popular example, the chain of suspicion: I know, you know I know, I know you know, you know I know, you know I know, you know I know...... Theoretically, it can be carried out indefinitely, but the reality is that everyone stops after a few cycles, which is convergence. Of course, I found that smart people can do it a few more times, and there is a certain distribution. The third is the extension of various concepts based on definitions. Based on the known knowledge, we know that the expression levels of various genes in cells are dynamic, and that there is a specific housekeeper that the expression level of genes is stable, so we can construct certain sequences based on the measured data and discover various patterns through sequence alignment. What we can do now is to process the significant up-regulation and downregulation of a limited number of genes, and our future direction is to synthesize these data to discover patterns such as transduction of specific combinations of transcription factors that can lead to the formation of stem cells, and specific tumor formations, and so on. The pattern found in this paper is that the high expression of Oct3/4, -Sox2, -c-Myc, and -Klf4 can make the sequence expression of cells close to that of embryonic stem cells, so that it can be approximated that this is equivalent.

These are my own observations and reflections in the laboratory, and even if they are full of reckless and naïve conjectures, they are also experiences that I should cherish.