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And so far, all the gear I’ve seen has been rather well-balanced in terms of advantages and drawbacks, meaning that overpowered god-builds feel unlikely, and creative stat-juggling should be quite the fun challenge. However, for those who’ve been waiting five years for another hunter x hunter web game adventure, I don’t think x hunter online will be good enough. An ancient robot who devoted his life to give birth to his children?
If you’re in the mood for a more competitive battle, x hunter game’s “hunter x game” player-versus-player (PvP) mode, much like the battlegrounds in hunter x hunter game online, pits two teams against one another in a battle to the death. But hunter x hunter games has a loot system. hxh game involves gathering piles of loot, something which is addictive for veteran RPG gamers.The portable screen magnifies small details that are lost when the console is docked.
 Be prepared to play the hunter x game beyond the first main ending; that’s simply the end of the first part, and the full Hunter X Online plays out over five different endings. Canonically strong team combinations.Though Nintendo’s limits on full Excel-spreadsheet nerdery may be a shortcoming in the eyes of those who revel in such systems, if the idea of an RPG is to role-play then shouldn’t I be able to slay the final boss if I, the player, role-playing as the hero, am skilled enough? It’s odd that hunter x hunter browser game and now Zelda champion such outside-the-box thinking when it ought to be role-playing hunter x hunter mmorpg games that consider such matters the most heavily. Because while the traditional – and less obvious – fighting hunter x hunter online game archetypes are present and correct, from all-rounder hunter x online game, to nimble, acrobatic hunter x hunter mmorpg online, to tricksy, technical, trap-setting Dr.
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Summary

On January 31, 2020,  a paper published by a virus expert at the Indian Institute of Technology and Biology caused a big controversy in the country. The paper points out that four fragments inserted in the new pneumovirus gene that are highly similar to HIV and are not very likely to com from natural evolution. This news has become the theoretical basis for conspirators.

In fact, the conclusions in the papers was published informally by Indian scholars and the data provided by them cannot be obtained at all, and there are many “mistakes”. Several scientists have verified that the so-called "similar gene fragments with HIV" is a coincidence that these gene fragments are also widely present in other common viruses.

Actually an officially published scientific paper pointed out that the new coronavirus is the result of coronavirus evolution, not artificial synthesis, according to the analysis of the characteristics of genome-wide evolution.

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The body

Conspiracy theories are always something that people love to see, and whenever something new that cannot be explained with existing knowledge is revealed, corresponding conspiracy theories will soon appear. The novel coronavirus (hereinafter referred to as "SARS-CoV-2" according to WTO) is no exception.

Since the novel coronavirus raged last December, multiple related conspiracy theories have emerged on social media. For example, the "novel coronavirus" is an American biochemical weapon specifically targeted at the Chinese. Such rumors are easy to refute, because the origin of the rumors is conspiracy theorists from non-scientific circles. There is no argument and it is pure speculation.

But in the past few days, a message went viral on Chinese and English social media.

The rumored news said that "Indian scientists have sequenced genes and found that "new coronavirus" has gene fragments not found in other coronaviruses, and these gene fragments are close to the human immunodeficiency virus HIV", and concluded that "these genes Fragments are likely to be artificially synthesized. " This statement spread immediately. If it was artificial, who made it? What is the purpose? Is the "novel coronavirus" really a biological weapon?

This is certainly not the case!

Papers by Indian scholars are not officially published, nor have they been peer-reviewed.

The paper was put on the web page of bioRxiv by the author. This web is dedicated to publishing scientific papers that have not yet been formally published in academic papers, nor have they been peer reviewed. It is equivalent to a web database that allows everyone to take a quick look before the paper is officially published or for unpublished papers.

The paper comes from the well-known Indian Institute of Technology in India, entitled “Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag”.

This article is a professional thesis, which is difficult for ordinary people to read. Let me explain briefly.

The author got the genetic map of the "novel coronavirus" virus (thanks to Chinese scientists, the virus was isolated for the first time and the complete sequencing was completed, and then made public and provided to scientists around the world for research). Compared with other coronaviruses, it was found that the gene maps of "new coronavirus" and other coronaviruses were 96% the same, and only 4% were different.

These 4% have about 1,200 base pairs. Among the 1,200 base pairs of transcribed amino acids / polypeptides, four inserts were found to have gene sequences not found in other coronaviruses. After analyzing these four inserts, it was found that the three-dimensional structure formed by them is likely to exist on the tip of the spinous protein on the surface of the virus, which is the part that initially contacts the host cell. The author then compared the genetic sequences of these inserts with the HIV virus and found that they were very close to the genetic sequence of at least one HIV.

The author then concluded that their discovery is unlikely to have been accidental.

The author's words stop here. But this will undoubtedly make people start to associate. If this new virus has a structure that other family viruses do not have, these structures will still help the virus enter the host cell, and it is very close to the HIV virus, and it is unlikely to be a natural product. This is too suspicious. Is it really a synthetic virus?

American scientists verified that the "novel coronavirus" and pointed out that there is indeed fragment close to HIV, but it is not unique. These gene fragments are also widely found in other common viruses.

Dan Samorodnitsky, a popular science author on Massive Science, is a former biologist, current science writer, and an extremely serious person. He came to this article and used the so-called unique and only 4 inserted gene fragments similar to HIV to study it again with a gene sequencing tool.

The results are as follows:

The first inserted gene fragment is indeed very close to some of the HIV gene sequences. But the author did not know whether it was accidentally or intentionally ignored that this gene fragment also exists in many other viruses. For example, this gene sequence is also found in the most common streptococci, which lives in human bronchial viruses, and many types of viruses have this gene fragment. It lists the other microorganisms that match the first inserted gene fragment 100%.

The second fragment test is the same.

The second insert is indeed close to a certain section of HIV, but it is also found in the most common herpes virus, the cytomegalovirus (a virus that causes kissing disease) that is very easy for adolescents, and the virus that infects tomatoes.

The other two clips are no exception. Not only exist in "novel coronavirus" and HIV, but also in many other common viruses, such as plant flavivirus, bovine papilloma virus and so on.

Therefore, the fact that this article intentionally or unintentionally ignores is that although the "novel coronavirus" he pointed out does have genetic fragments close to HIV, it is by no means unique, and these gene fragments are also widely found in other common viruses. The role of these four so-called unique inserts in this epidemic is unknown. Based on this alone, it is speculated that this "novel coronavirus" virus was artificially manufactured, and even the conspiracy theory of biological weapons is not true.

To be continued in Part Two…

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During the last few years, remarkable progress has been made in stem cell therapy, which leads to the flourishing of stem cell clinics claiming they can heal a number of diseases with stem cell therapy. However, FDA says it's unproven and is working to regulate them.

 

The past few years has seen so many for-profit stem cell clinics popping up around the United States, advertising that they can treat everything from arthritis to Alzheimer’s just by several injections with each one costing $5,000 to $20,000. However, cases have shown that patients of these clinics have developed tumors, suffered infections and even sight loss after unapproved procedures. 

So, is stem cell therapy an effective treatment or just false hope? Some information is necessary for those who are curious about stem cell therapy.

l What is stem cell therapy?

Just like the stem of a plant will produce branches, leaves, and flowers, stem cells are undifferentiated cells that can produce several different kinds of cells. They originate from two main sources: adult body tissues including bone marrow, blood and blood vessels, skeletal muscles, etc. and embryos in which stem cells can differentiate into more cell types than adult stem cells.

Stem cell therapy, also known as regenerative medicine, is the use of stem cells to help patients’ bodies repair damaged tissue by intravenous or intramuscular injection.

l What types of diseases does stem cell therapy work for?

Theoretically, any condition in which there is tissue degeneration can be a potential candidate for stem cell therapies given that they can produce all kinds of cells.

However, up to now, the use of stem cell therapy is quite limited. For the last 50 years or so, there have been patients successfully treated with hematopoietic stem cells, commonly known as bone marrow transplants. This remains the prototype for how stem cell therapy can work.  

New clinical applications for stem cells are currently being tested therapeutically for the treatment of musculoskeletal abnormalities, cardiac disease, liver disease, autoimmune and metabolic disorders and other advanced cancers. However, these new therapies have been offered only to a very limited number of patients and are experimental rather than in standard practice. 

l What are the advantages of stem cell therapy?

A common problem of transplantation is the risk of transplant rejection. Using autologous stem cell therapy, this risk can be avoided. What’s more, there is also no risk of communicable disease transmission.

Stem cell transplantation is usually by infusion or injection instead of complicated surgeries; thus, patients needn’t worry about scars, complications or the side effect of general anesthesia.

“In 2012, professor Shinya Yamanaka was awarded the Nobel Prize in Physiology or Medicine for the discovery of induced pluripotent stem cells, which shows greater potential of stem cells to help us understand and treat a wide range of diseases, injuries and other health-related conditions.” Said a senior scientist in Creative Biolabs, a leading company providing stem cell therapy development services.

However, their current applications as treatments are sometimes exaggerated by “clinics” looking to capitalize on the hype by selling treatments to chronically ill or seriously injured patients, and there is still a lot to explore about how they work in the body and their capacity for healing.

With the continuous development of technology, stem cells will trigger a medical revolution, becoming the third treatment method after drugs and surgery. A series of diseases and injuries that are difficult to treat now will eventually be gradually rescued.

 

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Study details

  1. PROTAC design of degrading FKBP12

In this study, FKBP12 was selected as the target protein for the following reasons:

First, FKBP12 protein is widely expressed in mammals. By binding to the Ca2 + -release channel (RyR receptor), FKBP12 regulates Ca2 + signaling to travel important functions, especially in the heart.

Second, because FKBP12 is a highly conserved protein, this chemical knockdown strategy is expected to be used to generate large animal models with targeted protein knockdown (for example, pigs and non-human primates).

Third, the global knockout of FKBP12 by common methods can cause embryonic death from severe developmental heart defects (eg, hypertension, tight ventricles, and ventricular septal defects). The role of FKBP12 in adult heart function and disease development remains elusive.

Rapamycin has been shown to be a potent and specific ligand for FKBP12, which regulates mTOR signaling with high affinity (kd = 0.2nm). Therefore, researchers designed a PROTAC that degrades FKBP12 by linking rapamycin (a FKBP12-specific ligand) and pomalidamine (a specific ligand that binds to CRBN-containing E3 protein ligase) via polyethylene glycol. This heterobifunctional molecule ubiquitinates FKBP12 via CR3B-containing E3 ligase, and degrades FKBP12 via the proteasome pathway. Therefore, researchers synthesized a series of PROTACs and measured their potential to degrade FKBP12 in Jurkat cells.

Researchers named the chimeric molecule with rapamycin and pomalidomide as RC32, which showed the most effective FKBP12 degradation ability. After treating the cells for 12 hours, it caused 50% protein degradation, DC50 = 0.3 nM .

To determine whether RC32-induced ubiquitination degradation was caused by FKBP12 degradation via the ubiquitin-proteasome pathway. Researchers treated the cells with the proteasome inhibitor bortezomib or carfilzomib before adding RC32. In fact, inhibition of the proteasome completely blocked RC32-induced FKBP12 degradation, suggesting that this degradation is via the ubiquitin-proteasome pathway. The addition of the FKBP12 inhibitor rapamycin or the CRBN inhibitor pomalidomide effectively blocked the degradation of FKBP12 by RC32, further confirming that this degradation requires the combination of RC32 with FKBP12 and CRBN. It is worth noting that when degrading FKBP12.6, RC32 did not induce significant degradation of FKBP51 and FKBP11 in Jurkat cells. The use of a certain amount of RC32 can control the degradation of FKBP25. However, RC32 has no effect on the phosphorylation of S6K and S6, which may be beneficial for dividing the independent function of mTOR in FKBP12. When RC32 was washed away, FKBP12 protein levels completely recovered within 96 hours. To further evaluate the efficiency of RC32, tests were performed using different cell lines from different species and primary cells. FKBP12 is effectively degraded by RC32 in a highly consistent pattern in cells from humans, rats, mice and chickens. Importantly, RC32 showed high degradation efficiency in primary cardiomyocytes, which indicates its potential to degrade FKBP12 in vivo.

  1. Rapid and effective degradation of FKBP12 in mice and rats by RC32

After confirming the high degradation potency in cell lines and primary cells, researchers used RC32 to induce protein knockdown in mouse, rat, pig, and non-human primate models. These models are valuable tools for studying human diseases. Although mice and rats have been widely used in scientific research, it is much more difficult to construct protein knockdown models in pigs and non-human primates. Pig xenotransplantation is particularly attractive in biomedical research. Although non-human primates are more relevant to studying human diseases and developing treatment strategies, genetic modification of monkeys is very expensive, time-consuming, and technically challenging. These limitations in large animal models have severely hindered their application in biomedical research. Therefore, researchers tried to use PROTAC as a chemical method to construct a representative protein knockdown animal model and study the function of target proteins.

Researchers first investigated the effects of RC32 on mice. Surprisingly, after only 1 day of treatment with RC32 (intraperitoneal injection, 30 mg / kg, twice daily), FKBP12 protein was not detected in most organs of the treated mice, and the brain was removed. Interestingly, significant degradation of FKBP12 also occurred in the eyes. In contrast, RC32 has no effect on the degradation of FKBP12 in brain tissue, which may be due to the inability of RC32 to cross the blood-brain barrier. Interestingly, after 1 day of treatment (30 mg / kg, twice daily), RC32 was able to degrade FKBP12 for about 1 week. In addition, FKBP12 was recovered in different organs / tissues after RC32 was discontinued. Interestingly, the recovery rate of FKBP12 in the heart was the slowest in 13 days after RC32 was discontinued. After discontinuing PROTAC, FKBP12 mRNA levels showed an acute and compensatory increase, then quickly recovered and remained at near-normal levels. To degrade the FKBP12 protein in the brain, researchers use intraventricular (i.c.v.) administration. As expected, FKBP12 in tissues from the i.c.v. treated brain was degraded, while levels in other organs / tissues were not affected. FKBP12 is widely expressed in the nervous system and is known to regulate the localization and processing of amyloid precursor proteins. The study results suggest that local protein knockdown in the brain may open up a new avenue for treating Alzheimer's disease. When RC32 was delivered orally, FKBP12 was significantly degraded in mice, which highlights the clinical potential of oral PROTAC, which is more convenient than previously reported dependent injections. In addition, only two intraperitoneal injections every 20 hours (20 mg / kg; Figure 3e, f), high degradation efficiency was found in Sprague-Dawley rats.

To be continued in Part III…

 

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Abstract: Exosomes have shown great potential in drug delivery, disease diagnosis, etc, and now are found to play a role in diabetes.

Just like a stuffed suitcase unloaded from the belly of a cargo plane, molecular backpacks called exosomes are constantly produced from the cells of the body. Each backpack is filled with a variety of materials that might be opened by another cell. By sending these biological packages, cells communicate with each other through shared proteins and genetic materials.

Due to the unique function of exosomes as intercellular messengers, the ability to change the biological activity of recipient cells, and their therapeutic potential in disease diagnosis and targeted drug delivery, the relationship between exosomes and different kinds of diseases has received widespread attention in recent years. One of them is diabetes, as exosomes are found to play an important role in insulin sensitivity, glucose homeostasis, and vascular endothelial function.

Diabetes is a common metabolic disorder, which is characterized by dysfunction of insulin secretion by pancreatic β cells and varying degrees of insulin lack. Organs such as the pancreas, liver, muscle, or fat are all involved, and communication between these organs is a key to maintain glucose homeostasis.

Type 1 diabetes mellitus (T1DM) and Type 1 diabetes mellitus (T2DM) have different pathogenesis. T1DM is due to the gradual loss of insulin-producing cells, resulting in low or none secretion of insulin. T2DM is due to the body's production of insulin resistance. Although the pathogenesis of T1DM and T2DM is different, their pathogenic factors, pathophysiology, disease progression and complications are related.

Exosomes and T1DM

Exosomes contain powerful immunostimulatory substances. Exosomes released by insulinomas can stimulate the autoimmune response of non-obese diabetic (NOD) mice. This research shows that exosomes are the autoantigen carriers of NOD mice and have strong immune activity, which may be the trigger of autoimmunity in NOD mice.

Islet cell transplantation is an effective method for the treatment of autoimmune T1DM. Explants that are specifically released into the blood circulation by islet transplantation are of potential diagnostic value in distinguishing recurrent autoimmune and immune rejection from islet β-cell injury. Biological markers that can be used in the diagnosis of islet transplantation. Exosomes isolated from MSCs have immunomodulatory effects and can improve islet function by increasing the number of regulatory T cells and their anti-inflammatory products IL-4 and IL-10, so they can be used to treat T1DM.

Exosomes and T2DM

Exosomes carry important biological information about the pathogenesis of T2DM. The exosomes and the miRNAs they carry pass from the adipose tissue through the blood and penetrate into the skeletal muscle and liver. The reaction induced during this tissue migration may directly lead to the intercellular communication of T2DM and metabolism-related disorders. The miRNA contained in the exosomes secreted by islet cells regulates β-cell function in a paracrine manner, and this situation is significantly different between normal and T2DM patients.

Due to the complexity of clinical manifestations of different types of diabetes, sometimes it cannot be identified by current laboratory methods. Therefore, it is urgent to find a marker that can reflect pathophysiological characteristics or disease progression in real time. At the same time, such markers should be cheap and easy to obtain.

“Studies have shown that exosome marker can be used for early diagnosis and staging of diabetes, and exosome itself is also a target for the treatment of diabetes.” Said a scientist of Creative Biolabs, ‘more importantly, it can help to monitor the response of patients with diabetes to treatment and thus provide personalized treatment.”

With years of exploration in exosome services, Creative Biolabs has been committed to bringing together highly skilled experts applicable to exosome services to support exosome applications in diagnostics and novel therapeutics development, including sampling, analysis, manufacturing and exosome-based application services.

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MSCs and inflammation

Since the study found that bone marrow MSCs inhibit the proliferation of T cells, scientists have found that MSCs can widely inhibit the activation and function of a variety of immune cells, including macrophages, granulocytes, natural killer cells, and dendritic cells, T cells and B cells. MSCs not only inhibit T lymphocyte proliferation, but also inhibit the differentiation of initial T cells into Th1 and Th2 cell subsets, and promote the production of regulatory T cells (Treg). Moreover, MSCs can indirectly induce Treg by affecting dendritic cells. In addition, MSCs can promote the conversion of pro-inflammatory type I macrophages to anti-inflammatory type II macrophages, treat sepsis, and down-regulate natural killer cell activation induced by IL-2 or IL-15. This series of powerful immunoregulatory functions has given MSCs the possibility of treating a variety of inflammation-related diseases, and truly realized the efficient clinical application of MSCs.

The immunoregulatory effects of MSCs are closely related to various secreted factors, including TGF-β, NO, IDO, TSG-6, PGE2, IL-1 receptor antagonists, IL-10 and chemokine CCL2 antagonist variants. The diversity of MSCs immune regulation mechanisms may be due to the differences in their species and tissue sources and their microenvironment. In fact, the immune suppressive function of MSCs depends on the stimulation of interferon-γ (IFN-γ) and TNF, IL-1α or IL-1β. Blocking IFN-γR or using IFN-γR61 / 61MSCs cannot effectively exert the immunosuppressive effect of MSC. Stimulated by the above-mentioned inflammatory factors, MSCs express high levels of IDO, iNOS, and ligands of CXCR3 and CCR5, among which chemokines recruit T cells to reach around MSCs, thereby expressing immunosuppressive factors that inhibit T cell function. At this point, there is a close interaction between MSCs and inflammation. A deep understanding of the interaction between MSCs and inflammation is of great significance in guiding the rational clinical application of MSCs and understanding the pathological mechanisms of inflammatory diseases.

In addition to inflammatory factors, other factors are also involved in the "authorization" process of MSCs' immunosuppressive functions. For example, stimulation of Toll-like receptors (TLRs)-TLR3 and TLR4 can activate the immunosuppressive effects of MSCs. MSCs also respond to different inflammatory stimuli and activate different signaling pathways to regulate specific immune responses. These research findings not only help us better understand the mutual regulation of MSCs and the inflammatory microenvironment, but also have important guiding significance for discovering or improving the application potential of MSCs in different diseases, especially immune disorders.

MSCs and immune regulation

The ability of MSCs to regulate immunity depends on the type and concentration of various inflammatory mediators in their microenvironment. Different inflammatory states greatly affect the therapeutic effect of MSCs on diseases, suggesting the plasticity of MSCs immune regulation. Studies have found that MSCs can effectively treat graft-versus-hostdisease (GVHD) under strong inflammation, but if MSCs are infused on the same day as bone marrow transplantation, that is, when the inflammatory response has not yet begun, the treatment effect is not significant. In addition, MSCs have little effect on experimental autoimmune encephalomyelitis in remission. From this point of view, the immunoregulatory ability of MSCs does have a strong plasticity, which is closely related to the inflammatory state.

In the pathological process of inflammatory diseases, high levels of inflammatory factors are often closely related to the acute phase of the disease, while in the chronic or remission phase, the inflammatory factors present relatively low concentrations, which may be the body's self-repair phase. "Checkerboard gradient" concentration was used to detect the immunoregulatory function of MSCs under different concentrations of inflammatory factors (IFN-γ and TNF-α). It was found that the dynamic changes of inflammatory factor levels can affect the immunoregulatory function of MSCs, making them exert immunosuppression or immunostimulatory effects and lay the foundation for the research of the plasticity of immune regulation. The main reason is that low levels of inflammatory factors are not enough to induce MSCs to express high levels of iNOS or IDO. Instead, they will recruit lymphocytes to the surrounding MSCs to secrete a large amount of chemokines and exacerbate the inflammation response.

Therefore, NO and IDO are the “switches” that regulate the immune regulatory function of MSCs. MSCs also exhibited similar immune-enhancing functions in low-dose concanavalin A-activated T-cell co-culture systems. In addition, antigen-sensitized MSCs can be stimulated with low-dose IFN-γ to activate cytotoxic CD8 + T cells as antigen-presenting cells. The above studies suggest that high inflammatory levels stimulate MSCs to exert immunosuppressive functions, while low inflammatory environment levels stimulate MSCs to exert immune promoting effects. Although the mechanism network that regulates MSCs activity in different inflammatory environments has not been clarified, plasticity is the most reasonable explanation for the phenomenon that MSCs exert different immune regulatory functions in different environments.

During the inflammatory process, the cytokines, chemokines and related immune cells of the immune system are dynamically changed, and different immune cells play different functions. Among them, effector T cells and regulatory T cells are important cells that promote inflammation and fight inflammation, respectively. Th1 and Th17 belong to effector T cells with pro-inflammatory effects, and IFN-γ, TNF-α, and IL-17 produced by them lead the pathological process in a variety of autoimmune diseases and infections. In the pathological process of these diseases, MSCs are also recruited to the site of inflammation to participate in regulating the inflammatory response and assist tissue repair or regeneration. Cytokines at the site of inflammation are essential in conferring immunosuppressive function to MSCs, and the synergy between IFN-γ and TNF-α is particularly important. In addition, the presence of IL-17 can enhance the stability of iNOS mRNA in MSCs by regulating the RNA-binding protein AUF1, and significantly promote the immunosuppressive function of MSCs. Therefore, the type and concentration of cytokines in the inflammatory microenvironment determine the immune regulatory capacity of MSCs.

Immunosuppressive factors such as TGF-β, as important factors to maintain the body's immune balance, are also commonly found in the inflammatory microenvironment. TGF-β receptors I and II are expressed on MSCs and regulate their differentiation and regeneration. When TGF-β, IFN-γ, and TNF-α co-stimulate MSCs, the immunosuppressive function of MSCs is significantly reduced, which is related to the down-regulation of iNOS or IDO expression by TGF-β through signal transduction factor Smad3. It is worth mentioning that MSCs can produce a large amount of TGF-β. Therefore, the negative regulation of TGF-β on the immunosuppressive effect of MSCs can be used as a feedback regulation to maintain the inflammatory state of the injury site and regulate tissue regeneration. In addition to TGF-β produced by MSCs, IL-10, which often has similar immunosuppressive effects as TGF-β, can also block the immunosuppressive function of MSCs. From this point of view, cytokines known for their immunosuppressive effects can exert their immune-promoting functions by acting on MSCs.

To be continued in Part Three…

 

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Background

Prior to the first severe acute respiratory syndrome (SARS), a limited number of coronaviruses were known to spread in humans, since they causu only mild illnesses, such as the common cold. After the SARS pandemic in 2003, coronaviruses apparently cross species barriers and cause infections that threaten human life.

The 21st century has experienced the spread of two previously unrecognized coronaviruses worldwide with high pathogenicity, namely severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome corona Virus (MERS-CoV), as well as the 2019-nCoV, which is still spreading in the world, especially in China.

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Since November 2002 in China, SARS coronavirus has experienced unprecedented transmission from person to person, accompanied by high mortality. Joint efforts worldwide have enabled rapid identification of SARS coronaviruses, and have made significant scientific advances in epidemic prevention in a short period of time. In addition, zoonotic transmission of SARS from December 2003 to January 2004 provided researchers with a line of insight into the origin of this new coronavirus. It is worth noting that the SARS pandemic was announced in 2004 when no more infections were detected. Subsequently, some suspected SARS-Cov viruses found in bats showed the ability to infect human cells without pre-adaptation, suggesting that SARS-Cov or similar SARS-Cov viruses may reappear. Ten years later, in June 2012, in Saudi Arabia, another highly pathogenic novel coronavirus, MERS-CoV, was isolated from the sputum of a male patient who died of acute pneumonia and renal failure. Nosocomial infections have been reported, and international travel has caused the MERS-CoV virus to spread to countries outside the Arabian Peninsula, making it a global pathogen. In May 2015, the Middle East respiratory syndrome broke out in South Korea due to a person returning from the Middle East. Based on lessons learned in the management of the SARS epidemic over the past decade, unprecedented advances have been made in revealing the biological properties of MERS. Scientific advances have enabled us to make rapid and systematic progress in understanding the epidemiology and pathogenesis of MERS-CoV disease.

The common characteristics of SARS-CoV and MERS-CoV

SARS-CoV and MERS-CoV share several important common characteristics— preferential viral replication of the lower respiratory tract and viral immunopathology, which contribute to hospital-wide transmission.

This article mainly focuses on the epidemiology and pathogenesis of these viruses, including our current understanding of their biological characteristics, transmission, and replication in the host. Covs' S protein plays a key role in viral infection and pathogenicity. As the key surface trimeric glycoproteins of Covs, they are guided into host cells. This article reviews the structure and function of S protein and the therapeutic methods targeting S protein. In addition, we will explore how the interaction between coronavirus and host leads to pathogenic outcomes, discuss potential treatment options, and describe the development of prevention and treatment strategies that are closely related to the pathogenic process for SARS-CoV and MERS-CoV. Although several potential therapies for SARS and MERS have been identified in animal and in vitro models, the lack of human clinical trials has hindered the development of these potential countermeasures.

An overview of 2019-nCoV

In the context of the current transmission of new coronaviruses, in order to control the spread of the virus and improve the prognosis of patients, it is urgent to develop public health and medical control methods. Whole-genome sequencing revealed that the new coronavirus (2019-nCoV) has very strong sequence similarity with its close relative, SARS coronavirus (SARS-CoV). The spike protein of 2019-nCoV infected host target cells showed some key non-synonymous mutations relative to SARS-CoV, which may lead to the less effectiveness of existing treatment methods and drugs targeting SARS coronavirus spike protein for 2019-nCoV. In addition, key drug targets, including the RdRp protein and 3CLpro protein, share a very high sequence similarity of greater than 95% with SARS-CoV. Therefore, this article proposes four potential drugs (ACE2 polypeptide, Remdesivir, 3CLpro-1 and a new vinyl sulfone protease inhibitor) that may be used to treat 2019-nCoV infection. At the same time, this article also summarizes the previous work on the drug research of these targets, hoping to provide guidance for future research on broad-spectrum anti-new coronavirus drugs.

To be continued in Part II…

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1.1.2 Drug Ion Electrical Properties and Drug Loading Capacity

The interaction between the drug and the phospholipid layer molecule has an important effect on the structure and load of the liposome, and the effect of the charge effect is particularly significant. Generally, when the charge properties of the drug and the phospholipid molecular layer are the same, it is not easy to be encapsulated. By adding appropriate excipients during the preparation of the liposome to make it a charged liposome opposite to the charge of the encapsulated drug, the drug encapsulation rate can be improved. For example, in the preparation process, octadecylamine is added to obtain positively charged liposomes, and phosphatidic acid is added to obtain negatively charged liposomes. The antiviral drug cidofovir is negatively charged under normal physiological conditions. It is found that liposomes made with positively charged phospholipids composed of DOTAP and DC have a significantly higher encapsulation rate than liposomes made with electrically neutral phospholipids. However, other studies have shown that when the positively charged drug sumatriptan uses a neutral phospholipid as the membrane material, the encapsulation rate is low, and when the positively charged material stearylamide is added to the membrane material, the phospholipid membrane is significantly strengthened, thereby t                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          lop09he leakage of the drug is prevented, and the drug load is increased. However, the encapsulation efficiency was lower than that of positively charged membranes after the addition of negatively charged membrane dicetyl phosphate, indicating that in some drug encapsulation processes, charge is not the main factor influencing.

1.1.3 Decoration and Encapsulation Rate of Medicinal Chemical Structure

The chemical structure of a drug determines its physical and chemical properties. By modifying the structure of a drug to some extent, the hydrophilic and hydrophobic properties of the drug can be improved, thereby improving the encapsulation efficiency of the drug. Researchers reacted the anticancer drug cyclocytidine with palmitic acid to obtain two derivatives of monopalmitate and dipalmitate, which were separately encapsulated to prepare liposomes. As a result, the encapsulation rates of the two derivatives were found. It rose to 86.5% and 93.7% respectively, while the original drug was only 21%. The HLB value shows that on the one hand, the original drug is converted from hydrophilic to lipophilic after esterification, and the encapsulation position of the drug is correspondingly transferred from the aqueous phase to the external lipid phase. On the other hand, the long ester chain obtained by structural modification can be embedded in the lipid membrane. In addition, the fluidity of the lipid membrane is reduced, thereby increasing the liposome stability and encapsulation efficiency.

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  1. Liposomal particle size design

Particle size is an important evaluation index of liposomes, and its size and degree of uniform dispersion directly affect the in vivo behavior of liposomes. Large particle size liposomes are easily endocytosed by macrophages and concentrated in the liver. Smaller particle size liposomes can effectively prolong the circulation time of the drug and play a long-lasting effect. When the particle size is less than 50nm, liposomes can penetrate the liver endothelium and enter the spleen, bone marrow and tumor tissues. Duan Yisong et al etc. used long-circulating material polyethylene glycol to prepare mitoxantrone long-circulating liposomes with an average particle size of 60nm. Compared with ordinary liposomes, the average residence time in rabbits was prolonged by 6.2h, reflecting Its long cycle advantage. Awasthi  et al. Investigated the particle size on the circulation time of PEG-modified liposomes in rabbits and found that the optimal particle size is 160-220 nm. Large particle size liposomes (400-530nm) are highly targeted to liver and spleen-enriched reticular macrophages. When the liposome particle size increases to 1-12 μm, it is easily taken up by the lungs. After azithromycin was prepared into cationic liposomes, the mice were administered tail vein to study the distribution of azithromycin in mouse whole blood and various tissues. AUC increased by 7.4 times. This is because liposomes larger than 6 μm will be mechanically filtered by pulmonary capillaries and then taken up by monocytes into lung tissue.

  1. Preparation method selection

The preparation method of liposomes greatly affects the structure and particle size of liposomes, so it is generally selected according to the nature of the drug and the purpose of the drug. For fat-soluble drugs, mechanical dispersion methods such as film dispersion method and freeze-drying method can be selected to prepare multilayer liposomes with large particle diameters, so that the drug is slowly released in the target tissue. If you need to increase the drug's transport speed, you can choose to prepare small single-compartment liposomes. The main methods include ethanol injection, surfactant treatment, film-ultrasonic method, and so on. The drug loading of water-soluble components is generally not high. The key is to increase the volume of the aqueous phase in the liposome. Therefore, large monolayer or polycystic liposomes are generally selected for preparation. The reverse thin film dispersion method and the double emulsion method and the freeze-thaw method  are suitable for the preparation of large particle size aqueous drug-loaded liposomes. Among them, the reverse thin film dispersion method is mostly large monolayer liposomes, including The sealing rate can reach 60-65%, and some studies have shown that for the hydrophilic drug salvianolic acid B, the large monolayer liposomes (LUV) prepared by the reverse evaporation method, the ethanol injection method, and the double emulsion method are used. Compared with liposomes, the encapsulation rate is the highest. The repeated freeze-thaw process in the freeze-thaw method will be accompanied by the formation of ice crystals, which will cause mechanical damage to the phospholipid bilayer, thereby increasing the chance of water-soluble drugs entering the phospholipid bilayer and increasing the encapsulation rate. At the same time, multi-compartment liposomes can be prepared into small single-compartment liposomes by repeated extrusion and freeze-thaw methods, which increases the drug loading space and drug loading. In addition, the microencapsulation method is suitable for preparation Small-particle-size drug-loaded liposomes in aqueous phase. For amphiphilic drugs such as weak base and weak acid, they can be encapsulated by active drug loading methods, such as PH gradient method, ammonium sulfate gradient method metal ion gradient method, and so on. In some cases, a single method alone cannot meet the requirements of encapsulation efficiency and particle size, especially for compound drugs with different properties in the co-loading concentration, so it is often combined with several methods.

 

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Mesenchymal stem cells have the characteristics of low immunogenicity and homing to ischemic or injured tissues. After entering into the host body, they can homing to specific sites and be differentiated into endoderm, mesoderm, and ectoderm under the influence of microenvironment cells derived from individual germ layers, such as bone, cartilage, tendon, fat, liver, kidney, skin, muscle, nerve, and even pancreas, are more than 10 kinds of mature cells, thus becoming ideal seed cells for organ repair in regenerative medicine.

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Initially, mesenchymal stem cells were found in the bone marrow, but highly invasive bone marrow donation experiments were needed. In addition, the number and differentiation potential of mesenchymal stem cells decreased with age. Recently, umbilical cord blood has been less damaged due to acquisition methods and has been used as an alternative source of mesenchymal stem cells. Another promising source of mesenchymal stem cells is adipose tissue. This review compares these three mesenchymal stem cell sources from aspects such as morphology, success rate of isolation of mesenchymal stem cells, frequency of clonal colony formation, expansion potential, multi-directional differentiation ability, and immune phenotype. Adipose tissue can be used as an alternative source of bone marrow tissue for the isolation of mesenchymal stem cells. In addition, people have also found that mesenchymal stem cells are also found in cord blood, periodontal ligaments, amniotic fluid, dermis, periosteum, skeletal muscle, fetal lung, fetal liver, placenta and pancreas.

Mesenchymal stem cells have broad clinical application prospects and can be used to treat diseases of the nervous system, liver and kidney injury, autoimmune disease, heart disease, bone disease, cartilage disease, ischemic vascular disease, diabetic complications and tumors. They can also be used in tissue engineering and facial shaping. In addition, they can be co-transplanted with hematopoietic stem cells to treat blood diseases. Based on this, the article made an inventory of the research progress made by mesenchymal stem cells in recent years.

1.TEPCM: magnetic mesenchymal stem cells promise to improve cartilage repair

doi: 10.1089 / ten.tec.2019.0001

Cells carrying superparamagnetic iron oxide nanoparticles (SPIOs) can be directed to a specific location by an external magnetic field, which is beneficial for tissue repair.

Recently, a research report entitled "In Vitro Safety and Quality of Magnetically Labeled Human Mesenchymal Stem Cells Preparation for Cartilage Repair" was published in an international magazine Tissue Engineering Part C: Methods. The safety and effectiveness of this magnetically labeled mesenchymal stem cells (MSCs) in repairing cartilage defects.

Researcher Dr. Naosuke Kamei said that “in this study, we demonstrated the safety of magnetically labeled MSCs through karyotyping, clone formation experiments, and total proliferation experiments. After labeling, we found only small differences in mesenchymal stem cells”. Researchers can evaluate the quality of stem cells by the differentiation of chondrocytes and their reactivity to magnetic forces. The results show that the appropriate concentration of superparamagnetic iron oxide nanoparticles can help optimize the mesenchyme while ensuring magnetic attractiveness and differentiation ability of plastid stem cells.

  1. Nat Commun: Identify key proteins that regulate angiogenesis in tumors

doi: 10.1038 / s41467-019-10946-y

Recently, in a research report published in the international journal Nature Communications, scientists from the Barcelona Institute of Biomedicine found that inhibiting the function of p38 protein or inhibiting angiogenesis in human and mouse colon cancer. This process is called angiogenesis, which is essential for cancer cell growth and can promote cancer progression and metastasis.

Researcher Dr. Angel R. Nebreda said that we found that p38 activity is very important for mesenchymal stem cells (MSCs). These stem cells have high plasticity and can be concentrated around blood vessels. It participates and plays a role in many key processes, such as tumor formation. This study clarifies the molecular mechanism of tumor angiogenesis. Researchers have now described the activity of p38 in cancer cells, but until now they did not know the key role that the protein plays in MSCs, and very little is known about how the protein is involved in tumor angiogenesis.

In this study, researchers clarified the key role of the protein p38 in the cardiovascular process during tumor angiogenesis, especially how it promotes the development of MSCs. The researchers said that p38 can play a role in MSCs cells. The effect is to inhibit angiogenesis. Using genetically modified mouse models, researchers have found that inhibiting p38 may stimulate cardiovascular production in tumors, and this situation also occurs during the repair process of damaged tissues in the body.

3.EBioMedicine: researchers develop cancer treatments that target bone metastasis while retaining bone tissue

doi: 10.1016 / j.ebiom.2019.06.047

Researchers at the University of California, Irvine (Irvine, UCI) have developed a treatment and tested it on mice using engineered stem cells to target and kill cancerous metastases in bone tissue while preserving bone. The new method, published in the journal EBioMedicine, equips engineered mesenchymal stem cells to target them, prompting them to transfer to bone metastases, where they release therapeutic drugs.

"The power of this strategy is that we provide a combination of anti-tumor and anti-bone resorption agents so that we can effectively block the vicious circle between cancer and its bones," said the study's lead author Weian Zhao, associate professor of pharmaceutical sciences and biomedical engineering, said. "Compared to chemotherapy, this is a safe and almost non-toxic treatment, and chemotherapy often causes lifelong problems for patients."

To be continued in Part Two…

 

 

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Exosomes are nano-scale vesicles secreted by cells. These microvesicles are usually about 30-150 nanometers in diameter and contain important cellular molecules such as proteins and RNA. Previous studies have shown that exosomes can be used as diagnostic markers for cancer, neurodegenerative disease, and kidney disease. In recent years, exosomes isolation technology has made significant progress and development.

  1. Ultracentrifugation

Ultracentrifugation is the most commonly used exosomal purification method. After removing dead cells and cell debris by low speed centrifugation, high-speed centrifugation is used to precipitate vesicle particles of the same size from soluble molecules such as free proteins and protein complexes purified. It is important that the exosomes be subsequently washed at least once with PBS or fresh growth medium to reduce free residual proteins therein. In addition, all centrifugation steps must be performed at 4 ° C to keep the proteases, DNase and RNases inactive.

Usually ultracentrifugation is also used in combination with a sucrose density gradient (its continuous distribution from low to high density) or a sucrose cushion (30% sucrose cushion), that is, centrifuged at 100,000-200,000 xg in a centrifuge (containing exosomes) In 120 minutes, the exosomes in the sample should be enriched in a sucrose density range of 1.13-1.19 g / mL.

Although this powerful method can obtain highly purified exosomes, there are some disadvantages. Indeed, the process of ultracentrifugation is time-consuming and labor-intensive, and requires a lot of raw materials. The biggest drawback is that repeated centrifugation operations are likely to cause damage to exosomal vesicles and reduce their quality, or soluble proteins in the sample may form aggregates and clumps with exosomes to cause contamination.

  1. Ultrafiltration centrifugation

Considering that exosomes are cystic bodies with a size of several tens of nanometers, which are larger than ordinary proteins, exosomes can also be separated according to their size, such as ultrafiltration and size exclusion chromatography (SEC).

Ultrafiltration is the selective separation of samples using ultrafiltration membranes with different retention molecular weight (MWCO). That is, the solvent, ie, some small molecular substances, is filtered to the other side of the membrane, while high relative molecular mass substances larger than the membrane pore size are retained On the ultrafiltration membrane, the purpose of separating exosomes is achieved.

This method is simple and efficient, and does not affect the biological activity of exosomes. It is the best method for studying exosomal RNA because it produces greater RNA production than ultrafiltration and precipitation methods. It is also possible to pass a nanofiltration concentrator. However, the main disadvantage of ultrafiltration is that exosomes may block the filter pores, resulting in shorter membrane life and lower separation efficiency.

Exosome membranes also adhere to each other, resulting in low separation yields and even erroneous test results. In addition, there is another interference that needs to be resolved in the method of separating exosomes based on the size of the exosomes, which is the existence of a large number of non-exosomal nanovesicles that are similar in size to the exosomes.

In SEC, the porous phase fixed in the column can also be selectively separated based on the molecular size using the principle of gravity flow. Small molecules can pass through the pores and cause later elution, while larger components (including exosomes) can be eluted early, bypassing the pores. This method can greatly maintain the integrity and biological activity of exosomes, and combine with differential centrifugation to obtain highly purified exosomes.

  1. PEG-base precipitation method

Polyethylene glycol (PEG, 8000 kDa) can competitively bind free water molecules, so that less soluble molecules or exosomes are precipitated from the solution. Earlier this method was used to collect virus from samples such as serum, and now it is also used to precipitate exosomes. Samples are usually incubated overnight at 4 ° C with PEG, and exosomes are then recovered by low-speed centrifugation or filtration.

However, this method also has some problems: for example, the purity and recovery of exosomes are low, false positives (more proteins or some polymers that are difficult to remove), and mechanical or chemical additives that damage the exosomes.

Alternatively, if you know the sugar chain composition of the exosomes, you can use lectins to enrich the exosomes. Lectin is a protein that binds to carbohydrates and can be centrifuged at low speed after agglutinating exosomes. In recent years, exosomes have been separated based on the principle of precipitation. Various commercial exosomal extraction kits have also been developed on the market. The operation is simple, and high-purity and high-recovery exosomes can be obtained without ultracentrifugation.

  1. Magnetic bead immunoassay

Exosomes are available because they are rich in protein and have many specific marker receptors on their surface, such as CD9, CD81, CD63, CD82, Hsp70, Ras-related protein Rab-5b, cytoskeleton protein actin, and TSG101. Anti-marker antibody-coated magnetic beads can be captured after incubation with exosomes.

Because the heterogeneity of exosomes is consistent with their origin, the abundance of these markers on different exosomes is also different. Therefore, you can capture different types of exosomes from a sample by using specific antibody combinations, and select these exosomes by immobilizing these antibodies on ELISA plates, magnetic or chromatography beads, or microfluidic devices.

Although immunoaffinity technology has the advantages of high specificity, high purity exosomes can be obtained without affecting the morphological integrity of exosomes, it is the preferred method for enriching and characterizing unique exosomes. However, this method is low in efficiency, and the biological activity of exosomal contents is easily affected by pH and salt concentration, which is not conducive to the downstream experiments.

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  1. Phosphatidylserine affinity

This method combines PS (phosphatidylserine) with magnetic beads and uses the principle of affinity to capture PS outside exosomal vesicles. This method is similar to the immunomagnetic bead method, and the exosomes obtained are complete in morphology and highest in purity. Since no denaturant is used and the biological activity of exosomes is not affected, exosomes can be used for cell co-culture and in vivo injection. 2016.9 "Scientific Reports" magazine published the latest data of this method, showing that PS method can extract relatively high purity exosomes.

  1. Chromatography

The exosomes isolated by this method are uniform in size under electron microscopy, but require special equipment and are not widely used.

Exosome isolation is the first step for exosome characterization. The quality of exosome separation directly affects the subsequent researches of exosome qualitative and quantitative as well as applications in disease diagnosis and therapy. With the extensive experience in exosome isolation, Creative Biolabs provides a portfolio of exosome isolation products which can help you with the high-quality exosome isolation from many types of biofluids in an efficient, faster and cheaper way.

 

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Vitamin E is a fat-soluble vitamin that was discovered as early as the 1920s. Vitamin E includes tocopherols and triene tocopherols, a total of 8 compounds. Alpha-tocopherol is the most widely distributed and most abundant form of vitamin E in nature. Tocopherol is a hydrolysis product of Vitamin E and is one of the most important antioxidants.

 

How does Vitamin E help the human body?

 

Vitamin E helps delay aging

 

Vitamin E is a strong oxidant and is not weaker than lycopene and astaxanthin. After entering the body, vitamin E can help fight free radical Oxylipin peroxidation, eliminate free radicals, and delay aging.

 

Vitamin E helps boost immunity

 

If vitamin E is lacking, it will reduce the body's humoral immunity and cellular immunity, and increase the possibility of human diseases. Proper vitamin E supplementation will help to strengthen the body's ability to resist disease and enhance its physique.

 

Vitamin E helps eliminate pigmentation

 

Pigmentation is caused by the deposition of lipofuscin in skin cells. Lipofuscin is the product of cells being oxidized by free radicals. This substance not only produces stains and hinders aesthetic appearance, but also deposits in the internal organs and brain cells, causing cardiovascular and cerebrovascular diseases, and endangering health. Vitamin E as a strong oxidant can eliminate these free radicals, help prevent the generation of pigmentation, and at the same time tenderly expand peripheral blood vessels, reduce blood viscosity, and prevent cardiovascular and cerebrovascular diseases. Vitamin E can stabilize the protein active structure of the cell membrane, promote the normal development of muscles and maintain the elasticity of the skin, so that the skin and the body remain active; Vitamin E entering the skin cells can directly help the skin fight against the damage of free radicals, ultraviolet rays and pollutants, preventing The skin loses its elasticity due to some chronic or hidden injuries until it ages. Because of these effects of vitamin E, it is believed that vitamin E helps beauty.

 

Vitamin E helps protect eyesight

 

Vitamin E can inhibit the lipid peroxide response in the lens of the eye, expand the peripheral blood vessels, improve blood circulation, and prevent the occurrence and development of myopia.

 

Vitamin E helps relieve stomach ulcers

 

The poor gastric mucosal resistance in patients with ulcer disease is related to the disturbance of fat peroxidation. Vitamin E can regulate fat oxidation and scavenge oxidative free radicals, while protecting cells from oxidant damage. At the same time, a large amount of vitamin E can promote the proliferation of capillaries and small blood vessels, improve the surrounding blood circulation, increase the supply of oxygen in the tissue, thereby creating good nutritional conditions for healing of the ulcer surface. In addition, it can still inhibit the growth of H. pylori and reduce the recurrence rate of ulcer disease after healing.

 

Vitamin E helps promote sex hormone secretion

 

Vitamin E can increase men's sperm vitality and quantity; increase women's estrogen concentration, improve fertility, and prevent miscarriage.

 

Is it okay to take a lot of vitamin E for a long time?

 

Vitamin E is found in edible oils, fruits, vegetables and grains. The recommended daily intake for adults is 8 to 10 IU. Vitamin E in the general diet can completely meet the needs of the human body. Therefore, the general population does not need to take vitamin E for a long time. Long-term use is not only unsafe, but also has side effects.

 

Taking large doses of vitamin E for a long time may cause various diseases. The more serious ones are:

 

Intake of low-dose vitamin E has anti-oxidant effect, but it may no longer have antioxidant activity when ingested in large doses. At this time, vitamin E becomes a pro-oxidant;

 

Thrombophlebitis or pulmonary embolism, or both, is due to the high dose of vitamin E that can cause platelet aggregation and formation, which may trigger the risk of stroke;

 

Headache, dizziness, dizziness, blurred vision, muscle weakness; skin cracking, cheilitis, angular cheilitis, urticaria;

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In recent years, studies have found that the incidence of primary malignant brain tumors has increased significantly, and the current prognosis is poor. Therefore, studying the mechanism of brain tumor recurrence, improving the prognosis of patients, and prolonging their survival time is an important research direction and a major problem faced by experts: due to the vague understanding of the source of brain tumor cells and its mechanism. Although a large number of related experimental studies have been done on the pathogenesis of malignant brain tumors, but no satisfactory results have been achieved.

Currently, only a small percentage of cells in surgical tissues of brain tumors have been found to have infinite proliferation, self-renewal, multi-directional differentiation potential, and tumorigenicity. These cells are called brain tumor stem cells (BTSCs), and others tumor cells have no or only short-term proliferation ability. Igntova and other scholars first reported that brain tumor stem cells (BTSC) existed in brain tumor surgical specimens, and isolated precursor neurons that can form neurospheres from glioblastomas, which are called neural stem cells in brain tumors. At present, each brain tumor stem cell has been successfully cultured and isolated from surgical specimens such as medulloblastoma, different grades of astrocytoma, ependymal tumor, and ganglioglioma.

Since Singh et al. First isolated CD133-positive tumor stem cells from malignant brain tumors, research on brain tumor stem cells has gradually become a hot topic in neuroscience and related fields. Preliminary studies have found that the occurrence, development, metastasis, and recurrence of brain tumors may be closely related to brain tumor stem cells. Therefore, further in-depth discussion of the biological characteristics of brain tumor stem cells and the mechanism in the occurrence and recurrence of malignant brain tumors will definitely be very important for the future of radical treatment and prevention of malignant brain tumors.

Here in this article, we will introduce three important biomarkers.

One of the most prominent one is TSGF, namely a group of tumor-related substances. It is a collective term for several internationally recognized carbohydrates and metabolites (lipoproteins, enzymes, amino acids) related to the growth of malignant tumors. TSGF is an effective, convenient and valuable tumor marker for the diagnosis and judgment of brain malignant tumors.

In addition, studies have shown that the expression specificity of tumor markers such as nestin, BEHAB, YKL-40, EphA2, glial fibrillary acidic protein, CD133, fatty acid binding protein, and MMP-9 is more obvious in gliomas.

1. BTSC

There are many hypotheses about the source of BTSC, but currently they tend to be derived from mature neural stem cells (NSCs). The accumulation of multiple mutations leads to tumorigenicity and becomes BTSC. The source is discussed from the following two aspects: The origin of the tumor is consistent with the NSC distribution area. Studies have shown that the origin of brain tumors may originate in a part of the subventricular area, and BTSCs with high proliferation and differentiation potential are constantly produced in this area, which leads to tumorigenesis, and these areas coincide with the main locations of NSCs. BTSC and NSC have many similarities in genetics. The main manifestation is that BTSC does not express markers of differentiated cells, but instead has NSC markers, such as Nestin or CD133.

In summary, both theory and experiments support that BTSC is likely to be derived from mutant NSCs that are constantly dividing and proliferating. Although BTSC and NSC have many similarities, there are also obvious differences between them: first, BTSC has stronger self-renewal and proliferation capabilities than NSC, and the number of passages in vitro culture has increased significantly, with an immortalization trend. Its self-renewal and differentiation have become imbalanced; secondly, BTSC differentiates into the same phenotype as the parent tumor under the conditions that induce NSC differentiation and does not differentiate into neurons and glial cells in the same proportion as NSC. These differences provide new research directions and ideas on how to transform NSC into BTSC and whether the two are at the same level of differentiation.

2. CD133 protein, nestin, Sox2 protein

In recent years, Rath et al. have successfully cultured and isolated meningioma stem cell spheres with spherical focus growth through serum-free suspension culture. At present, most scholars use CD133 and Nestin as specific markers of brain tumor stem cells. CD133 is a transmembrane protein with a relative molecular weight of 120,000. Studies have shown that both solid tumors and brain tumor cell spheres obtained from in vitro cell line cultures show CD133 positive staining; and CD133 positive cells isolated from glioma cell lines in serum-free culture, they all grew spheroidally, had infinite proliferation, self-renewal, and multi-directional differentiation.

Singh et al. compared the biological characteristics of CD133-positive and negative tumor cells and found that the former has a strong ability to self-renew and proliferate, while the latter adheres to growth, does not divide, and does not proliferate. In vivo tumorigenicity tests showed that 100 CD133-positive tumor cells were tumorigenic, while 1 × 105 CD133-negative tumor cells formed only one glial scar at the transplant site.

For these reasons, CD133 is considered to be the most important marker of brain tumor stem cells. However, recent studies have shown that CD133-negative cells in some brain tumors also have the characteristics of tumor stem cells. Therefore, CD133 is not a reliable marker for brain tumor stem cells. Nestin, which belongs to the intermediate microfilament, is expressed in undifferentiated neural pluripotent stem cells and was once considered a marker of brain tumor stem cells. However, the study found that the same group of tumor cells, Nestin-positive ratio is much higher than CD133-positive ratio, which indicates that Nestin is also expressed in progenitor cells that have just begun to differentiate, and is not a reliable brain tumor stem cell marker.

Sox-2 belongs to the Sox (Sry-related HMG Box) gene family and is located at 3q26.3-q27 of the chromosome. It is a highly conserved transcription factor that can regulate the self-renewal of embryonic stem cells. It is the only Sox gene found in current research that plays an important role in maintaining the differentiation potential of embryonic stem cells. It is also a key to induce adult cells to become pluripotent stem cells.

3. Brain tumor stem cells

The research of brain tumor stem cells has become a new hot spot in the field of brain tumor research. Although great achievements have been made in the successful isolation and culture of brain tumor stem cells, no complete theoretical system has been formed so far. Therefore, it is of great significance to study the pathogenesis and biological behavior of brain tumors and to find new treatment options for malignant brain tumors that target tumor stem cells in the future. It can be imagined that the detection of tumor stem cells will become a new classification and judgment of brain tumors in the future. It is possible to use various treatment schemes for brain tumor stem cells to specifically kill brain tumor stem cells, instead of killing all tumor tissues, in order to achieve radical cure and prevent tumor recurrence and metastasis. With the continuous research on brain tumor stem cells, it will inevitably have a profound impact on the pathogenesis, pathological grading, prevention of recurrence and treatment options of brain tumors, making the radical cure of malignant brain tumors possible.

 

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According to previous data, CD19 was a hot CAR-T treatment target, and has achieved great success in relapsed and refractory B-cell malignant blood diseases, and there are already two commercial products for the treatment of relapse fefractory B-cell acute lymphocytic leukemia (B-ALL) and relapsed refractory non-Hodgkin's lymphoma (NHL).

But not all patients benefit from CD19 CAR-T therapy. For example, most clinical data reports indicate that CD19 CAR-T has an objective response rate (ORR) of 80% and a complete response rate (CR) of approximately 50% in patients with relapsed and refractory NHL, and nearly 20% of non-responses and more than half of the patients fail to achieve sustained remission.

Why can't some non-Hodgkin's lymphoma patients benefit from it? Why is it?

Because the CD19 antigen is lost on the cancer cells of these patients, for these patients, CD19 CAR-T no longer has the ability to target and attack cancer cells, which in turn can lead to the recurrence of cancer.

How to solve this problem?

CAR-T therapy targeting CD20 may be an alternative solution.

CD20 is a human B lymphocyte restriction differentiation antigen, encoded by the MS4A1 gene (located at 11q12). This antigen is a hydrophobic 4-pass transmembrane protein with a molecular weight of approximately 35 kD, Leukocyte surface antigen Leu-16, transmembrane 4 domain subfamily A member 1 and so on. This protein function may be involved in regulating B cell activation and proliferation, and may function as a calcium ion channel.

CD20 antigen is mainly present on pre-B and mature B lymphocytes, and is expressed on most B-cell non-Hodgkin lymphoma cells, but not on stem cells, pro-B cells, normal plasma cells, or other normal organizations. Plasma cells naive and stimulated plasma cells may express CD20.

According to the expression characteristics of CD20 in the development stage of B lymphocytes, it has been selected as one of the targets for the treatment of B-cell lymphoma and leukemia, and many antibody drugs have been successfully developed based on this target.

Research progress of CD20 CAR-T therapy

1. In China

As early as the end of 2012, the Molecular Immunology Department / Biotherapy Ward of the General Hospital of the Chinese People's Liberation Army launched a clinical trial of CD20 as a target for CAR-T cell therapy for relapsed and refractory diffuse large B-cell lymphoma (DLBCL). The study, completed in 2014 and first reported the results of a phase I clinical study in 7 patients, showed that CD20 CAR-T cells combined with a tumor-reducing pretreatment regimen can prolong tumor regression (Wang Y, et al. ClinImmunol. 2014). At the same time, the team also applied to the State Intellectual Property Office for a CD20 CAR-NKT patent (engineered CD20-targeted NKT cells and its preparation method and application, patent application number: 201410062069.7).

At the beginning of 2015, Sibeman chose to cooperate with Han Weidong, director of the Molecular Immunology Laboratory of the Life Sciences Institute of the General Hospital of the PLA, to help Professor Han Weidong develop CD20 as a representative by leveraging his successful experience in translational medicine in the biomedical field and sufficient R & D funding advantages.

Based on the phase I clinical research, Han Weidong's team launched a phase IIa clinical study of CD20 CAR-T in the treatment of relapsed and refractory NHL. The results of related studies in 11 patients were included and the results were published in "Signal Transduction and Targeted Therapy" in October 2016. Six of the 11 patients were evaluated as CR after CAR-T infusion (1 of whom was transferred from Phase 1 to Phase IIa and continued CD20 CAR-T alone), 3 were PR, and 2 were stable (SD ), 2 patients (1 PR, 1 SD) also received CR after local radiotherapy in the later stage.

In early October 2017, the Han Weidong team published a research report again in the "Signal Transduction and Targeted Therapy". Based on a retrospective review of the clinical outcomes of 16 patients who can be evaluated after the test, the article highlights that 8 patients achieved CR after CAR-T infusion (or combined local radiotherapy), with the exception of 3 patients who had recurrence. As of the end of July 2017, 5 patients were still in the state of continuous CR (1 from the stage I subjects and 4 from the stage IIa subjects), of which 1 patient continued the CR stage and it has lasted 57 months, 3 cases exceeded 40 months, and 1 case exceeded 20 months. At the same time, in July 2017, the NCI in the United States reported the long-term follow-up results of CD19-CAR-T in the treatment of NHL in Molecular Therapy, showing 5 of 7 patients with CR, 4 of whom had a continuous CR period of 56, 51, 44, 38 months.

These two studies show that CAR-T treatment can not only achieve short-term efficacy in patients with relapsed and refractory NHL, but also can obtain long-term CR efficacy in most patients who obtain CR after CAR-T. It also indicates the long-term effectiveness of CAR-T therapy for CD20 in patients with relapsed and refractory NHL.

In terms of long-term safety, Han Weidong's team also observed that patients with continuous CR after CAR-T treatment are often accompanied by longer-term B cell deficiency or low, and low immunoglobulinemia. Of the 5 long-term CR patients, except for one patient who developed grade 3 shingles infection in July after CAR-T treatment, the remaining patients can effectively prevent the occurrence of grade 3 infectious diseases through regular supplementation of gamma globulin .

2. In US

In September 2017, Fred Hutch, a top cancer center, licensed a CD20 CAR-T therapy developed by Mustang Bio to the clinic as soon as possible.

Phase I / II clinical trials with partial support from Mustang Bio will be led by Dr. Mazyar Shadman, Clinical Research, Fred Hutch. The trial will recruit approximately 30 patients with relapsed or refractory B-cell non-Hodgkin's lymphoma (B-NHL). Eligible patients will first undergo a biopsy to ensure that their tumors have a CD20 marker. The researchers expressed hope that as shown in preclinical studies, CD20 CAR-T therapy could even be more effective than CD19 CAR-T.

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Co-immunoprecipitation (Co-IP) is a classical method for studying protein-protein interactions based on the specific role of antigens and antibodies. The development of co-immunoprecipitation technology has gone through several stages. In the early days, researchers used gel electrophoresis to isolate co-immunoprecipitated proteins: the antigen solution was added to small wells such as agarose, and antiserum was added to adjacent wells. With the antigen and antibody diffusing, large molecules entered in the gel, and an interaction occurs between the two to form a complex. A concentration gradient is formed by diffusion of the antigen and antibody, and a multi-molecular network complex is formed at the optimal concentration. Finally, the large-molecular protein complex is precipitated from the solution. With the development of society, the co-immunoprecipitation technology has been continuously improved. The method of isolating the immune co-precipitation complex is improved to promote the multimer reaction, so that the immune complex is precipitated from the solution.

In the mid-1970s, people began to use solid-phase reactions, using protein A immobilized on the surface of S. aureus to adsorb antibodies, and then bind to the corresponding antigens. With the development of science and technology, this method has been improved to use the surface-immobilized protein A or protein G microspheres to separate antigen-antibody complexes in order to achieve the purpose of detecting antigens or target proteins.

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Principle of co-immunoprecipitation

The experimental principle of co-immunoprecipitation is: if X is immunoprecipitated with an antibody to protein X, the protein Y bound to X in the body can also be precipitated. At present, prorein A is often pre-bound and solidified on agarose microbeads and reacted with the solution containing the antigen and the antibody. Prorein A on the agarose microbead specifically binds to the antibody Fc. Due to the specificity of the antibody antigen, protein X is precipitated; There are substances that interact with protein X, and they can precipitate.

Application of co-immunoprecipitation

  • Determine whether two target proteins are bound in the body
  • Identify a new role for a particular protein
  • Isolate and obtain the interaction protein complex in its natural state

With the continuous deepening of protein research, people have combined immunoprecipitation methods with other methods, and based on them, many more complicated technologies have been derived, which makes the analysis methods more diverse and its application range is quite wide. Co-immunoprecipitation is a technique used to study protein-protein interactions and can be applied to the study of protein complexes. It can verify the existence of protein complexes, and then discover new protein complexes. Co-immunoprecipitation technology is combined with immunoblotting or mass spectrometry to determine the binding of bait protein-target protein in its natural state and specific proteins. Co-immunoprecipitation experiments can also be applied to the enrichment and concentration of low-abundance proteins.

At the same time, co-immunoprecipitation is a relatively classic technique for exploring protein-protein interactions. It has a wide range of applications and high credibility in modern medical research. Protein interactions permeate the life activities of every cell in the body. Many phenomena in biology such as replication, transcription, translation, shearing, secretion, cell cycle regulation, signal transmission and intermediate metabolism are all affected by proteins. Some proteins are composed of multiple subunits, and protein-protein interactions are particularly prevalent. Some proteins bind very tightly, while others interact only briefly. However, no matter what kind of situation occurs, they control a large number of events of cell life activity, such as cell proliferation, differentiation and death. And through protein-protein interactions, it can change the dynamic characteristics of intracellular proteins, such as substrate binding properties and catalytic activity. It can also generate new binding sites, which has an effect on changing the specificity of proteins on substrates. Therefore, only by allowing the interaction between proteins to proceed smoothly, the normal life activities of cells can be guaranteed. Because protein-protein interactions are so significant, the study of their detection methods has also received much attention. Since then, the research on protein interrelationships will intensify. In the future, it will not only be confirmed by co-immunoprecipitation technology, but more and more advanced technologies will be worth applying and developing.

The pros and cos

Advantage:

  • The protein exists in a natural state after modification and translation;
  • Detects interactions in vitro and in cells;
  • Antigens and interacting proteins are present at similar concentrations in cells, avoiding human effects caused by overexpression;
  • It is possible to isolate the interacting protein complex in its natural state.

Disadvantages:

  • The sensitivity is not as high as that of affinity chromatography;
  • High false positive rate, correct control is necessary;
  • The binding of two proteins may not be directly combined, but a third party may act as a bridge in the middle;
  • It is necessary to predict what the target protein is before the experiment in order to select the antibody to be detected at last, so if the prediction is incorrect, the experiment will not yield results, and the method itself is risky;
  • After the protein forms a complex, some epitopes will be masked, which may lead to the use of a pull-down antibody. No matter how the antibody concentration is increased, less than half of the target protein complex can be precipitated. If necessary, it is best to use multiple different antibodies for Co-IP respectively;
  • Since the natural state is detected, the protein complexes pulled down by Co-IP may be different under different times and different treatments. Of course, as the number of experiments increases, the members of the obtained protein complexes will also bedifferent.

Authenticity of co-immunoprecipitation results

To ensure the authenticity of experimental results in co-immunoprecipitation experiments, the following points should be noted:

  • Make sure that the co-precipitated protein is obtained by precipitation of the added antibody, not a non-exogenous non-specific protein. Monoclonal antibodies have the advantages of strong specificity, mass production, and easy standardization. The use of monoclonal antibodies can help avoid contamination;
  • To ensure the specificity of the antibody, if the antibody cannot bind to the antigen in the cell lysate, it will not cause a co-precipitation reaction;
  • Make sure that protein-protein interactions occur in cells, not because of lysis of the cells.
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3. Optimal dosage

The optimal dose of MSC depends on the different disease and severity and the route of entry.

In the clinical research and application of MSC, cell dose may belong to the most clueless and most scientific aspect. Even if there are some clinical studies involving dose climbing experiments, they are not based on animal experiments.

MSC is different from traditional medicines, because: first, after MSC enters the body, it does not conform to the typical distribution and metabolism model of traditional medicines; traditional medicines are passive distribution, and MSCs have the function of actively chemotactic to the injury site, and the distribution of MSCs in healthy and diseased organisms is different. Second, animal experiments of traditional medicines require multiple administrations to maintain a stable blood concentration, and animal experiments of MSCs are often single injections, so that clinical studies of MSCs often adopt a single injection scheme. In fact, a single injection of MSC cannot achieve a good stable long-term treatment effect, even if there is a significant improvement in the short term.

For a certain disease, in the preclinical studies, animal experiments have not fully demonstrated the minimum and maximum saturation doses at which MSCs work, and the cell doses in different laboratories differ. Factors such as the culture system of different laboratories and the source properties of MSC often lead to differences in the quality of MSC, which directly affects the results of animal experiments and clinical studies of MSC. Therefore, data from preclinical studies of MSCs do not provide a good guide for determining clinical research protocols.

In current clinical studies, the dosage range of MSC used is very large, and the number of MSC cells used per patient ranges from more than 4,000 MSCs to hundreds of millions of MSCs.

For local intervention, the lowest dose appears in the clinical case of MSCs for femoral head necrosis. Each clinical study in Korea and France used more than 4,500 MSCs. The highest dose of interventional therapy appeared in a clinical study in China, which used 860 million cells for MSCs to treat diabetic limb bullae; the second highest dose was 200 million MSC myocardial injections. Clinical studies of more than 100 million MSCs injected locally include: 120 million MSCs for Crohn's disease intestinal fistula, 100 million MSCs for intraarticular injection of knee osteoarthritis, and 100 million MSCs for ischemic cardiomyopathy.

Intravenous doses of cells are relatively stable, and millions of MSCs per kilogram of body weight are often used, ie (1-10) x 10 * 6 / kg. The highest intravenous dose is 10x10 * 6 / kg co-transplanted with hematopoietic stem cells. According to a weight of 60 kg, that also requires 600 million MSCs; and 8x10 * 6 / kg for GVHD. The lowest dose of intravenous injection appeared in clinical trials of MSC and hematopoietic stem cell co-transplantation, which was 0.3x10 * 5 / kg.

The failure of the Phase 3 clinical trial of Prochymal (bone marrow MSC) in the treatment of refractory GVHD in 2009 was a catastrophic event in the clinical application of MSC, which almost denied the clinical efficacy of MSC. Prochymal is derived from the bone marrow of healthy people, and MSC itself has a strong immunosuppressive ability. However, why can't Prochymal be significantly better than the control group in the phase 3 clinical trial?

At that time, Prochymal's indication was hormone-resistant and refractory GVHD. This indication itself was very difficult. If Prochymal did not optimize the treatment plan, especially the breakthrough in conventional thinking in terms of dosage, then failure would be inevitable.

Later increasing the dose of MSC cells to 5x10 * 6 / kg and 8x10 * 6 / kg, the effect of treating hormone-resistant refractory GVHD was better than that before 2009, making the UK and EU treatment guidelines recommend MSC as a treatment for grade 2-4 Third-line treatment for acute GVHD. But Prochymal is still not approved by the US FDA. Interestingly, a meta-analysis article in 2016 suggested that the "dose" factor did not affect the survival rate of MSC in patients with acute GvHD.

Although some experts believe that 5x10 * 6 / kg and 8x10 * 6 / kg are high doses, there is no discussion and proof of what kind of dose is defined as "high dose". If MSC is regarded as a "medicine", then there must be a range. In this range, the higher the dose, the better the effect; then after reaching a saturated dose, continuing to increase the cell dose does not bring more efficacy, but may bring some adverse reactions.

To be continued in Part Four

 

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Immune oncology (IO) is the focus of current drug development. In recent years, the development of many IO drugs around the world has been rapidly improving not only in speed but also in quality, which brings new hope to almost all types of cancer patients, including many rare cancer types. At present, IO therapy targeting PD- (L) 1 has achieved great clinical success, and has attracted more and more research teams.

Many agencies have predicted that in the next few years, the size of the global IO market will continue to grow rapidly. According to GrandView Research, the size of the IO market in 2018 was $ 58.1 billion, and it is expected to reach $ 126.9 billion in 2026. Transparency Market Research and Market Research Engine predict that the IO market will reach 124.88 billion US dollars and 173 billion US dollars in 2024, respectively. These optimistic predictions showed a significant increase in clinical activity, with the expected launch of many new treatments for IO.

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Last year, Incyte and Merck Epacadostat / Keytruda encountered huge failure in the clinical phase III of melanoma. This hit the development of epacadostat and other IDO inhibitors. At the same time, it also reduced the industry's enthusiasm for development of cancer immunotherapy.

However, research on novel immune checkpoint targets that stop tumor cells from fighting the immune response or stimulate the body's immune system against cancer is continuing. A recent statistics from the American Cancer Institute (CRI) found that more than 1,700 joint studies involving PD- (L) 1 inhibitors are currently underway.

Early and mid-term research data on these novel immune checkpoint targets may be seen at the 2019 American Society of Clinical Oncology (ASCO) Annual Meeting.

  1. 1.Target: STING (interferon gene complex stimulant)

Type: Immune stimulation

Related companies: Aduro and Novartis' ADU-S100, GlaxoSmithKline's GSK3745417, Merck MK-1454

Aduro and Merck's STING pathway candidate drugs are administered by intratumoral injection, meaning they can be used for melanoma, but may not have much applicability for cancers deep in the body. The MK-1454 data released at the 2018 European Society of Oncology Medical Association showed that the efficacy of combination with Keytruda was not obvious. GSK3745417 from GlaxoSmithKline (GSK) can be administered by intravenous infusion, and early drug trials are currently underway. Data from the combined use of ADU-S100 and Novartis anti-PD-1 therapy spartalizumab will be presented at this year's ASCO Annual Meeting.

  1. 2.Target: LAG-3 (lymphocyte activating gene-3)

Type: Immunosuppression

Related companies: GSK TSR-033, Regenerative REGN 3767; Bristol-Myers Squibb BMS-986016, F-Star Corporation FS118

TSR-033 is one of GSK's $ 5.1 billion acquisition of Tesaro's oncology assets. The clinical progress of the drug has received much attention. Regeneron and BMS will also show the latest progress of their drug candidates and their respective PD-1 inhibitors at the ASCO Annual Meeting. F-Star's FS118 is a bispecific antibody that targets both PD-1 and LAG-3. The drug has signed an option agreement with Merck, but recently Merck abandoned its option and F-Star re- Obtained full development rights for the drug.

  1. 3.Target: TIM-3 (T cell immunoglobulin and mucin domain-3)

Type: Immunosuppression

Related companies: Eli Lilly LY3321367, BeiGene BGB-A425, Symphogen SYM023, Novartis MBG453

This class of drugs is a classic match with PD- (L) 1. All of the above drugs are in Phase I clinical trials, in combination with experimental PD- (L) 1 drugs from each company and with externally approved immune checkpoint inhibitors. Early safety data provided by Eli Lilly showed high levels of anti-drug antibody response (without affecting pharmacokinetics) and evidence of tumor response. At present, no other data on this class of drugs has been released.

  1. 4.Target: TGFβ

Type: Immunosuppression

Related companies: Merck and GSK's bintrafusp alfa (bispecific PD-L1), Forbius Avid200, Lilly Galunisertib, University of Pennsylvania CART-PSMA-TGFβRd.

Of all the oncology challenges, the biggest bet may be Merck's decision to confront Merck in frontline non-small cell lung cancer. The hypothesis of this study is that bispecific bintrafusp alfa can block the PD-1 pathway as effectively as Keytruda, while inhibiting TGF-β provides additional benefits by blocking another mechanism that tumors use to shut down the immune response. Meanwhile, Eli Lilly has been evaluating galunisertib for the past few years and has reported some promising phase II data for pancreatic cancer. TGFβ also appears to be the only novel immune checkpoint target on the list considered a potential therapeutic technology, and the University of Pennsylvania is currently testing metastatic castration resistance to prostate cancer.

  1. 5.Targets: OX40

Type: Immune stimulation

Related companies: Pfizer PF-04518600, Bristol-Myers Squibb BMS986178, Incygn IncAGN1949

These drug candidates are in very early human clinical trials testing dose levels and biological responses. BMS research combined BMS986178 with Opdivo and Yervoy and found that BMS986178 stimulated the immune response. Pfizer's PF-04518600 has achieved disease stabilization in some patients.

Creative Bioarray possesses a research team, which has been focused on Immune-Oncology for decades. Since there are still lots of incomplete scientific understanding of tumor immunology, which gives rise to little widespread use of I-O therapies in clinic, Creative Bioarray is always ready to help and cooperate with customers on their I-O research or preclinical experiment. With multitudinous tumor cells, diverse animal tumor models, and various tumor research related services, such as cell cycle assaysapoptosis assaysproliferation assays, migration assays and toxicology assays, Creative Bioarray could offer professional design and experiment for customers to facilitate their I-O program.

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The effect of denaturing enzyme protein and causing loss of enzyme activity is called enzyme inactivation.

The effect of reducing enzyme activity without causing enzyme protein denaturation is called inhibition. Enzyme inhibitory effects include irreversible inhibition and reversible inhibition.

Some substances do not cause the enzyme protein to denature, but they can change certain essential groups (some groups on the active center) on the enzyme molecule, causing the enzyme activity to decline or even be lost, which are called enzyme inhibitors.

Scientists can use inhibitors to study enzymes; drug manufacturers can use inhibitors as drugs to treat diseases; and inhibitors can also be toxic.

Here in this article, enzyme inhibition are mainly discussed. Enzyme inhibition refers to the effect that the functional group of an enzyme is affected by a certain substance, resulting in a decrease or loss of enzyme activity. This substance is called an enzyme inhibitor.

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Enzyme inhibitors are selective for enzymes and are an important tool for studying the mechanism of enzyme action. Many drugs, poisons and poisons used in chemical warfare are enzyme inhibitors. In addition, some biological macromolecules that exist in animals and plants with certain functions are also enzyme inhibitors. When the enzyme is inhibited, its protein portion is not denatured. Enzyme inactivation due to enzyme protein denaturation, and the reduction or loss of enzyme activity caused by the removal of activators (such as metal ions necessary for enzyme activity) are not included in the scope of enzyme inhibition.

Inhibitors can be divided into reversible inhibitors and irreversible inhibitors.

  1. Irreversible inhibitor

Irreversible inhibitors are mainly covalently bound to enzymes, reducing enzyme activity. Covalent binding is tightly bound, and physical effects such as simple dialysis and dilution cannot be used to remove the inhibitory effect.

  1. Reversible inhibitor

Reversible inhibitors bind through non-covalent bonds and have weak binding power, so they can both bind and dissociate easily, and quickly reach equilibrium. Reversible inhibitors are divided into two categories: competitive inhibitors and non-competitive inhibitors.

(1) The structure of competitive inhibitors is similar to that of substrates. It mainly binds to the binding groups of essential groups and competes with substrates for enzymes. The ability of the inhibitor to compete with the substrate for the enzyme's binding site depends on the concentration of both. If the inhibitor concentration is constant and the substrate concentration is low, the inhibitory effect is most obvious. As the substrate concentration increased, the enzyme-substrate complex concentration increased, and the inhibitory effect weakened. When the substrate concentration is much higher than the inhibitor concentration, almost all enzymes are taken by the substrate. At this time, the Vmax of the enzymatic reaction remains unchanged, but the Km value becomes larger.

Many drugs are competitive inhibitors of enzymes. Sulfa drugs have a similar structure to para-aminobenzoic acid, which is a substrate for dihydrofolate synthetase. Therefore, sulfa drugs competitively inhibit dihydrofolate synthase, causing bacteria to lack dihydrofolate or even tetrahydrofolate. Inability to synthesize nucleic acids and inhibit proliferation.

(2) The combination of non-competitive inhibitors with sites outside the active center of the enzyme does not affect the binding of the enzyme to the substrate, and the substrate does not affect the binding of the enzyme to the inhibitor. There is no competitive relationship between the substrate and the inhibitor. However, the substrate-enzyme-inhibitor complex cannot further release the product, so it is called non-competitive inhibitory effect, which shows a decrease in Vmax value and constant Km value.

(3) Anti-competitive inhibitors only bind to enzyme-substrate complexes, reducing the amount of intermediate products.

The significance of studying enzyme inhibitors

  • Helps to study the catalytic mechanism of enzymes and the design and development of inhibitor-type drugs,such as anticancer drugs;
  • Artificially regulate the metabolic pathways of the organism;
  • It is helpful to study the relationship between the structure and function of enzymes.

Creative Enzymes gladly supply various enzyme inhibitors of premier grade to the customers. We persist in being the most reliable supplier for enzyme products in the global market. Today, Creative Enzymes is a leading company in enzymes and enzyme-related products, and is well known for the high level of customer satisfaction. We deliver the products in a momentary span of time from order placement to final delivery. Our prompt service, dedicated customer care, and reliable approaches have made us the most preferred vendor.

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Biological or chemical substances originally added or artificially added to foods that have acute or chronic hazards to human health is the so called food contamination.

 

Classification of food contamination

  1. According to the nature of the source, food contaminationis divided into biological contamination, chemical contaminationand radioactive contamination.

(1) Biological contamination of food includes microorganisms, parasites and insects, toxic biological tissues, and insects. The main contamination is microbial contamination, which is more harmful, especially bacteria and bacterial toxins, mold and mold toxin.

(2) The source of chemical contamination is complex and various. For example, ① Pollutants from production, life and environment, such as pesticides, harmful metals, polycyclic aromatic hydrocarbon compounds, N-nitroso compounds, dioxins, etc. ② From the production, processing, transportation, storage and sales of tools, containers, packaging materials and coatings and other materials into the food materials, monomers and additives and other substances. ③ Substances generated during food processing and storage, such as harmful alcohols and aldehydes in alcohol. ④ Abuse of food additives.

(3) Radioactive contamination: the man-made radionuclide contamination in the environment mainly comes from the following aspects: nuclear explosion, discharge of nuclear waste, accidents. Radionuclides in the environment can be transferred to food through the food chain. The main transfer pathways are: transfer to aquatic organisms, to plants, and to animals. Harm to human body caused by radioactive contamination of food include long-term irradiation effects of low-dose radiation on various tissues, organs and cells in the human body after ingestion of contaminated food. It shows damage to the immune system and reproductive system, as well as carcinogenic, teratogenic and mutagenic effects.

  1. According to the way of food contamination, it is classifiedas:

1). Endogenous contamination

(1) Endogenous contamination

(2) Endogenous biological contamination: Livestock and poultry are infected with zoonotic diseases (meat, eggs, and milk are contaminated); Livestock and poultry are infected with inherent diseases, and their resistance decreases to cause secondary infections. During the life of livestock and poultry, some microorganisms are contaminated, and the decline in the resistance of livestock and poultry causes these microorganisms to infiltrate into muscle, liver and other parts, causing meat contamination.

(3) Endogenous chemical contamination

(4) Endogenous radioactive contamination

2). Exogenous contamination

Exogenous biological contamination: food processing, transportation, storage, sales, cooking and other processes due to non-compliance with operating procedures, resulting in contamination by microorganisms, etc. (1) through water contamination (2) through air contamination (3) Contamination through soil (4) Contamination during production and processing (5) Contamination during transportation / storage (6) Contamination by vector pests

Exogenous chemical contamination: Food is contaminated by toxic chemicals during processing, transportation, storage, sales, cooking, etc. The affected link include: (1) air (2) water (3) soil 4) transportation (5) production and processing

Characteristics of food contamination

Food is becoming more and more contaminated, and chemical contamination is the main cause.

When pollutants are transferred from one organism to another, the concentration can be continuously accumulated and increased, which is the so-called bio-accumulation effect, and even the slight contamination process can cause serious harm to the human body after bio-accumulation.

The hazards caused by food contamination today are more common than chronic toxicity, in addition to acute toxicity. Due to a small amount of human exposure for a long time and a long biological half-life, food contaminants have played a role in DNA in the body.

The harm of food contamination to the human body

  • Affect the sensory characteristics of food;
  • Cause acute food poisoning;
  • Cause acute and chronic hazards to the body;
  • Teratogenic, mutagenic and carcinogenic effects on humans.
  • The hazards of biological contamination: cause animal food corruption and deterioration; human infectious diseases; microbial poisoning.
  • Harm of chemical contamination: acute, chronic poisoning; mutagenicity; teratogenic and carcinogenic.
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Common reasons for the temperature rise of TPE injection molding machines include the following:

(1) The volume of the oil tank of the injection molding machine is too small, the heat dissipation area is insufficient, and the cooling device has a small capacity.

(2) The fixed pump oil supply system that selects the oil pump capacity according to the fast forward speed, the excess flow will return from the relief valve under high pressure and generate heat during work.

(3) The unloading circuit in the system fails or because the unloading circuit is not set, the oil pump cannot be unloaded when it stops working, and the entire flow of the pump overflows under high pressure.

Overflow loss occurs and heat is generated, resulting in excessive temperature.

 

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(4) The system piping is too thin and long, too much bending, local pressure loss and pressure loss along the process are large.

(5) Insufficient component accuracy and poor assembly quality, and large mechanical friction loss between relative movements.

(6) The mating clearance of the mating parts is too small, or the gap is too large after use, the internal and external leakage is large, and the volume is lost. For example, the volumetric efficiency of the pump is reduced, and the temperature rises quickly.

(7) The working pressure of the hydraulic system is adjusted much higher than the actual need. Sometimes it is because the seal is too tight, or because the seal is damaged, and the leakage increases, it is necessary to increase the pressure to work.

(8) High climatic and operating environment temperatures cause the oil temperature to rise.

(9) The viscosity of the oil is not selected properly. If the viscosity is too large or too small, it can cause heat generation and the temperature is too high.

Approach:

1. According to different load requirements, check and adjust the pressure of the relief valve from time to time.

2. Reasonably select hydraulic oil, especially the viscosity of the oil. When the conditions allow, use a lower viscosity to reduce the loss caused by viscosity friction.

3. Improve and improve the lubrication conditions of moving parts to reduce friction loss, which is conducive to reducing work load and reducing heat generation.

4. Improve the assembly quality and accuracy of hydraulic components and hydraulic systems, strictly control the clearance of the mating parts and improve the lubrication conditions, use sealing materials with low friction coefficient and improve the sealing structure, and reduce the starting force of the hydraulic cylinder as much as possible Heat generated by mechanical friction losses. Add cooling device if necessary

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Rinderpest virus

Rinderpest is an acute subacute infectious disease that mainly infects ruminants, especially cattle. It is characterized by severe hemorrhagic catarrh in the mucosa, accompanied by necrotizing stomatitis and gastroenteritis, with low congenital resistance. The mortality rate of animals is high. Rinderpest originated in Asia and was introduced into Europe, causing serious losses. At present, rinderpest has been eliminated in Europe and the United States, and China has declared it out as early as the 1950s. However, the disease is still prevalent in Equatorial Africa and Northeast Africa. And this disease is also widespread in Afghanistan, Pakistan, Southeast Asia and other places bordering China.

 

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  1. Physicochemical properties

The rinderpest virus is considered to be weakly resistant to physical and chemical factors. At 37 °C, the half-life of bovine prion infectivity in cell culture is 1 to 3 hours, and only 1 minute at 56 °C. However, a small number of viruses survived at 56 ° C for 60 minutes or 60 ° C for 30 minutes. After several months of storage at 4 °C , the infectivity decreased significantly. At -70 ° C for one year, the infection titer decreased. The virus is preferably stored lyophilized or stored at 2 ° C or less after addition of 2% dimethyl sulfoxide (DMSO). The inactivation of ultraviolet radiation is fast. Different strains have different pH stability, but most are inactivated at pH 4.0 or lower. The most stable pH is 7.2 to 8.0. The virus was inactivated by placing it in 20% diethyl ether chloroform at 4 ° C overnight. A prominent feature of the rinderpest virus is its very fragile glycerol, a phenomenon that has been used in the preparation of inactivated vaccines. Corruption can quickly inactivate the virus, and it may lose its infection when exposed to sunlight or naturally dry. The virus survives for a short period of time in the carcass, and the virus in the lymph nodes and spleen can survive for several weeks when stored at -25 °C. Strong base disinfection is best, glycerin, phenol, formaldehyde or β-propiolactone can quickly destroy the infectivity of rinderpest virus without significantly affecting its antigenicity.

 

  1. Blood coagulation

The rinderpest virus has not been proven to have hemagglutination activity, but the rinderpest virus antiserum inhibits the hemagglutinin of the measles virus. The rinderpest virus also functions as a measles virus hemagglutinin receptor site that blocks the surface of the monkey red blood cells. Treating the measles virus with ether and Tween-80 can break the viral envelope into small, uniform hemagglutinin particles. This agglutination of hemagglutinin particles to monkey red blood cells can be inhibited by high titers of canine distemper or rinderpest antibodies. HeLa cells infected with measles or canine distemper virus can adsorb red blood cells of guinea pigs, but rinderpest virus cannot adsorb red blood cells.

  1. Antigenicity

The antigenicity of all strains of the rinderpest virus is the same. Serological studies have shown that virions contain many soluble antigens that are not related to infectivity, including complement-binding antigens. This complement-binding antigen can be extracted from infected tissues with alcohol, acetone, or ether, and it can resist boiling for 20 minutes. There are also some precipitated antigens, two of which are thermostable; the third antigen moves the slowest during electrophoresis and is sensitive to heat. Precipitates are easily detected by using a suspension of acute diseased bovine lymph nodes and other tissues, or concentrated infected cell culture medium, and an anti-serum agar diffusion test. However, complement-binding antigens and precipitated antigens can be rapidly destroyed by spoilage. Triplex viruses are very antigenic, but each has a specific component, as evidenced by many serum tests. Cross-protection tests have shown that the immunity produced by canines infected with measles virus or rinderpest virus can resist the attack of canine distemper virus, but cattle cannot resist calves after vaccination with canine distemper virus. The hemagglutinin of the measles virus can be used as a reliable diagnostic method for detecting antibodies to calves and canine distemper.

There is an infectious disease similar to rinderpest in sheep and goats in West Africa, but not transmitted to cattle. The cytopathic effect of this virus in sheep kidney cell culture is similar to that of rinderpest virus. Adapted to cell proliferation, this virus can be used to immunize cattle against rinderpest. Conversely, rinderpest antiserum can also make sheep resistant to this virus. Therefore, the pathogen of sheep disease in West Africa may be a strain of rinderpest virus, which has lost the ability to infect cattle through natural pathways, but is easily transmitted in sheep.

To be continued in Part Two…

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The development of second-generation gene sequencing technology has pushed genomics research to a climax. Many biological problems, "measuring order" may be able to find key genes. General genome-wide sequencing covers more than 25,000 genes, compared to more than 1,400 metabolites that can be measured by total metabolomics. Although the number is small, metabolomics can be said to be getting more and more fired in the past 20 years because metabolites can more directly reflect the characteristics of biological phenotypes.

 

Basic research method

 

Metaboletics can be divided into two types: metabolomics and targeted metabolomics.

 

Total metabolomics, as its name implies, detects all metabolites; while targeted metabolomics, it focuses more on certain metabolic pathways, such as the Krebs cycle, glycolysis, and so on. In this regard, Professor Fang suggested that although the coverage of total metabolomics is broader, the sensitivity is not enough; if there are some experimental or literature clues, it is better to look at targeted metabolomics.

 

In addition, lipid metabolomics has also developed in recent years, and it belongs to the emerging field. Because of the different physical and chemical properties of lipids and most metabolites, lipid metabolomics requires different separation methods using common metabolomics.

 

The first is to extract samples of the "medication group" and "control group", followed by gas chromatography mass spectrometry (GC-MS) or liquid phase mass spectrometry (LC-MS) to obtain data, and nuclear magnetic resonance (MRI) is relatively less used.

 

Analysis of the data is a very important part of metabolomics. Many parameters need to be adjusted, and then statistical analysis is used to determine which metabolites have changed. Important information includes: Retention time; Accurate mass, etc. The changed data is then analyzed for metabolome pathways, typically using the KEGG database. Similarly, these databases now have little information about lipid metabolism.

 

Academic research example

 

Although everyone has high hopes for metabolomics to discover biomarkers, the reality is still cruel. Because the specificity of metabolites relative to disease is not strong enough, many causes may cause changes in the same metabolite, so it is difficult to determine whether a disease has been obtained through a metabolite. There are now models that judge through a set of metabolite changes, but have not yet seen clinical application.

 

So what are the research hotspots in academia? One of the more interesting concepts is the metabolic regulation, which is about metabolite regulation of gene expression. In the past, it was thought to be the expression of gene regulatory enzymes, which in turn regulates metabolism. Some recent studies have found that the content of certain metabolites in cells affects the gene expression of cells and even cell fate. This is a process of mutual influence and is two-way.

 

For example, last year's article in Cell found that the content of arginine changed greatly during the differentiation of immune T cells. Changing the concentration of arginine can change the fate of T cells.

 

The other direction is to do "precise medical treatment." Different patients, while eating a drug, some people have a good effect, some people do not work well, why? Is it possible to predict the patient's drug sensitivity in advance by analyzing the metabolomics of blood samples? There are now more gene sequencing in this field, and metabolomics is still relatively rare. Of course, there is some controversy about whether metabolomics can ultimately play such a role.

 

The last mentioned academic research direction, through the new research methods, re-write some biochemical concepts.

 

In the past, there were many concepts, and experience was accumulated because there was no way to detect the limitations of technical conditions. And today's technology can reanalyze and study these issues.

 

Experimental design considerations

 

A student doing a biological study, what should you pay attention to if you want to use metabolomics to get some data?

 

First of all, to figure out the biological problem you are studying, metabolomics can answer some questions, that is, you don't need to do this experiment. In some cases, changes in metabolomics are not particularly large, and changes may not be observed, which wastes time. Generally speaking, the major changes are metabolic diseases and tumors. In recent years, there have been many applications in the fields of immune response, stem cells, and epigenetics, but each subject researcher needs to have an in-depth understanding of the research object, and then study specific problems through metabolomics.

 

Secondly, if it is determined that it needs to be done, communicate with the technical department before the experiment to understand each step of the sample processing to ensure that there is no error. For example, many metabolites are unstable, and the sample collection process must be low temperature; how many cells and tissues have sufficient sample volume, too small sample size will cause many metabolites to be detected; design a good control group to be reliable The comparison; there are also samples of how many repeat groups should be collected; whether you want to analyze the common metabolome or the lipid metabolome, and so on.

 

Again, it's best to find an experienced expert to discuss how to look at metabolite changes. We recommend looking at the general changes first, and then looking at a path.

 

If you are doing non-radioactive isotope labeling experiments, there are more to be noted: the choice of isotope labeling sites and the time of sample collection are important. Pre-experiments are generally required to determine the time required for metabolite conversion in an experimental system. This type of experiment requires relatively high equipment and is relatively expensive. It should be demonstrated in detail before the experiment.

 

Finally, although it is a “group study” data, researchers need to have a relatively clear “hypothesis”, namely hypothesis. Because there are many metabolites of change, which metabolite changes are the most critical, it may still require experimental and literature reading to narrow the scope.

 

Industrial application prospects

 

There are some applications in R&D:

 

1 Clinically looking for biomarkers. Although there is currently no approved method for the detection of biomarkers by mass spectrometry, the market will be considerable if a reliable biomarker is developed. Because metabolomics is constantly changing compared to stable genomics, a patient may need to perform multiple tests at different stages of the disease.

 

2 Medically, it may replace some enzyme-linked immunosorbent assays (ELISA) because the specificity of the mass spectrometry will be higher. There are hopes to develop some routine testing items, such as expiratory diagnosis of lung cancer.

 

3 Develop specific nutrition programs for wealthy people. Metabolomics can detect what nutrients are missing in the body and know what dietary structure is needed to improve health.

 

4 Drug metabolomics. As mentioned before, it is known by metabolite changes which enzyme activities in the individual are high, thereby predicting the effects and side effects of the drug in individual patients.

 

The industrial application of metabolomics has not seen a big market so far. Some existing companies mainly provide services to universities and research.

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In recent years, individualized treatments will benefit more cancer patients as the time and cost of whole genome sequencing decreases. A recent study published in Nature Medicine showed that the development of new machine learning algorithms to analyze tumor genome-wide sequence information can help predict patient prognosis and help patients choose the best treatment.

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Whole-genome sequencing (WGS) technology can obtain almost all genetic information of patients, including whole-genome information of cancer cells and healthy cells, which make abnormal cancer cells can be found at the level of single nucleotide, copy number, epigenetic modification, etc.

To explore the clinical value of WGS, researchers at the University of Cambridge collaborated with researchers at Lund University in Sweden to develop a population-wide project called SCAN-B (Sweden Cancerome Analysis Network–Breast) (ClinicalTrials.gov ID: NCT02306096). The project has recruited breast cancer patients in Sweden since 2010 and has collected a large amount of clinical data so far.

Project process

In the SCAN-B project, the researchers performed a genome-wide sequencing of 254 triple-negative breast cancer (TNBC) in SCAN-B between 2010 and 2015. Triple-negative breast cancer refers to breast cancer patients with negative estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, accounting for 9% of breast cancer, and the prognosis is poor.

The researchers then used a machine learning algorithm called "HRDetect" to classify the tumors. This algorithm was originally developed to detect tumors with a BRCA1/BRCA2 gene mutation signature, and mutation of either BRCA1/BRCA2 gene would greatly increase the risk of breast cancer. Currently, a new targeted drug PARP inhibitor can be used for breast cancer patients with BRCA1/BRCA2 gene mutations.

According to the HRDetect algorithm, the patients were classified into three categories: high (HRDetect-high), medium (HRDetect-intermediate), and low (HRDetect-low). 59% of patients had homologous recombination repair loss (HRDetect-high): 67% of them were germline/somatic mutations of BRCA1/BRCA2, BRCA1 promoter hypermethylation, RAD51C hypermethylation or PALB2 biallelic loss.

The HRDetect algorithm provides a unique diagnostic message. The researchers found that patients with high-risk (HRDetect-high) triple-negative breast cancer had the best treatment and were more sensitive to PARP inhibitors than those with low scores (HRDetect-low).

Surprisingly, patients with moderate (HRDetect-intermediate) treatments had the worst treatment, and even though some patients had some better drug targets, the prognosis was still poor. Therefore, for patients with low scores, it is necessary to adopt a new treatment plan. Patients with low scores (HRDetect-low) also have poor prognosis. However, the WGS sequence information of these patients' tumors is also abnormal, which may be caused by the use of fixed drugs in clinical trials, such as immunological checkpoint inhibitors (PD-1) or AKT inhibitors.

Dr. Johan Staaf, the lead author of the study, said: "Three-negative breast cancer is difficult to treat, but by genome-wide sequencing, we can identify which triple-negative breast cancer patients are more sensitive to the drugs currently used clinically. Importantly, this method allows us to provide clues for studying the mechanisms of poor prognosis and develop new drugs for such patients."

With the rapid development of sequencing technology, whole genome sequencing can be completed in 24 hours, and the analysis of sequencing data can be completed in 24-48 hours. Therefore, in theory, genome-wide sequencing and analysis can be provided for each patient, and the optimal treatment plan can be selected based on the patient's own tumor genome information.

“Genome-wide sequencing opens up new avenues for individualized treatment of cancer,” said co-first author Dr. Nik Zainal. “In the past, the management and analysis of large amounts of data was a major obstacle to its widespread use. But now we are reducing the time for data analysis. Let each patient get individualized treatment, which will greatly change the treatment of cancer, even some refractory cancer treatment."

CD Genomics has been providing the accurate and affordable whole genome sequencing service for couple of years. The company combines both Illumina (short reads) and PacBio (long reads) platforms to achieve whole genome de novo assemblies and re-sequencing for virus, microbes, plants, animals and humans.

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2) Targeted knock-in. Knock-in a sequence on a plant's chromosome has always been a difficult technical problem, and TALEN and CRISPR / Cas9 technologies have greatly reduced the difficulty of this technology. Knock-in relies on DSB's Homology-directed repair (HDR) repair pathway (Figure A): After Cas9 / gRNA cuts the target site, if the cell has DNA homologous to the target site sequence in the case of a donor (DNAdonor) fragment, the gene fragment located on the donor DNA is integrated into the DSB location by HDR recombination. Although CRISPR / Cas9 technology can be used in plant protoplasts to achieve Knock-in or targeted gene replacement, it is difficult to succeed in stable plants.

Currently, there are two more general strategies that can be used to improve the efficiency of plant knock-in. The first is to use wheat dwarf virus (WDV) as a vector for DNA donors. By increasing the number of copies of donor DNA, the efficiency of Knock-in is greatly improved. This strategy has been validated in two major crops, rice and wheat, and Knock-in has a maximum efficiency of 19.4% in rice. The second is to use Cas9 and a pair of gRNAs to “cut” a sequence from a DNA donor and then “paste” it to a target site in the plant genome. This strategy utilizes the NHEJ pathway that dominates DSB repair. In 2016, a laboratory of the Institute of Genetics and Developmental Biology of the Academy of Sciences collaborated to achieve this "cut / paste" target in rice using clever target site design using CRISPR / Cas9 Knock in and replace genes. Based on CRISPR / Cas9, these new strategies make it possible for knock-in to be used efficiently in plant genetic modification.

1.2.2 Targeted gene transcription regulation

In addition to the genome editing function, the high scalability of CRISPR technology ensures that this technology can be fused with multiple functional proteins to perform other genetic operations. The core of this technology is the ability of gRNAs to target specific sites on the genome, using dCas9 (Dead Cas9, Cas9 without DNA-cleaving activity) and gRNAs to transport effector proteins of different functions to specific locations on the chromosome to function (Figure C). For example, dCas9 can be fused to an activation domain (AD), and gRNA can be used to guide dCas9-AD to the promoter region to achieve transcriptional activation of target genes. In this technique, multiple gRNAs are often required to simultaneously target the promoter of the target gene in order to effectively increase the expression of the target gene. And another elaborate strategy addresses this problem: using gRNAscaffold as a platform for recruiting transcriptional regulatory components to regulate target gene expression. For example, MS2 (RNA ligand) is fused to gRNA and MS2's specific binding protein MCP is fused to the AD fragment, so that the dCas9 / gRNA-MS2 / MCP-AD complex is targeted to activate transcription on the gene promoter of interest. The ingenuity of this technology is that multiple MS2s can be integrated into the gRNA scaffold sequence so that a single gRNA can be used to recruit multiple MCP-ADs to effectively activate target gene expression. Similar to CRISPR / Cas9-based targeted gene transcription activation, using different functional proteins (transcriptional suppression, epigenetic modified protein elements) on the dCas9 / gRNA platform can achieve different transcriptional suppression or epigenetic regulation. These genome-editing-derived technologies provide richer and more convenient genetic tools for biological research.

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Figure. Major applications of the CRISPR / Cas9 system in plants

2.Optimization of CRISPR / Cas9 technology

2.1 Reduce CRISPR / Cas9 off-target

Off-target has been a major problem for CRISPR / Cas9 technology. When the CRISPR / Cas9 genome editing technology was born, it was reported that Cas9 / gRNA had an off-target effect in animal cell lines: some DNA sites (off-target sites) that did not exactly match the gRNA guide sequence were also off-target edited by Cas9 and introduced unexpected genetic mutation (Off-target effects). The existence of off-target effects has become the biggest deficiency of CRISPR / Cas9. To solve this problem, scientists have made many positive attempts. In earlier studies, the Cas9 point mutation (D10A or H804A) was modified into a nicking enzyme (Cas9nickase, Cas9n) or dCas9 and FokI were fused into a nicking enzyme. This required the design of 1 pair of gRNAs to target 1 site. The formation of a DSB reduces the probability of missed targets by several orders of magnitude. Other studies have shown that shortening the guide sequence of gRNA to 17-18 nt can reduce the risk of off-target; or the fusion of Cas9 protein with other DNA-binding domains can also effectively reduce the off-target rate. Although these strategies greatly reduce off-target effects, they also complicate the technology and do not substantially improve the specificity of Cas9. As the structure of the Cas9 / gRNA complex is resolved, researchers have designed Cas9 variants with high cleavage activity and specificity. These "high-fidelity" versions of Cas9 have zero tolerance for base mismatches between DNA and gRNA. Yes, it can be used to achieve precise genetic manipulation, which basically solves the problem of CRISPR / Cas9 off-target.

In plant CRISPR / Cas9 gene editing experiments, the off-target phenomenon was not as severe as in animals, but individual off-target editing was also found, which may be caused by the absence of highly specific gRNA in the design of target sites. The results of genome-wide sequence analysis show that in species with small genomes and low sequence repetitiveness, such as Arabidopsis thaliana and rice, it is possible to design enough high specific gRNAs to edit 90% of genes. There are many bioinformatics tools available for off-target analysis and design of gRNAs with low off-target probability, such as CRISPR-PLANT, E-CRISP, CRISPR-P, etc. By designing highly specific gRNAs or using a new "high-fidelity" version of Cas9, it is expected to effectively eliminate the impact of CRISPR / Cas9 off-target on plant genome editing.

To be continued in Part Three…

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In recent years, in the study of preeclampsia, it has been found that blood hypercoagulability, chronic diffuse intravascular coagulation (DIC), lipid peroxidation, and vascular endothelial damage tend to form a prethrombotic state. Clinically, it is also often found that patients with preeclampsia often have hyperlipidemia, hyperglycemia or antiphospholipid syndrome, and the preeclampsia belongs to a metabolic syndrome. Therefore, we need to re-examine and study the disease from the direction of metabolomics, and provide new ideas for its prediction, diagnosis and even treatment.

 

Metabolomics is a discipline that studies biological metabolism. Different from previous genomics, proteomics, metabolomics to detect small molecule metabolite changes to understand the mechanisms of life physiology, pathology, help diagnose clinical diseases and improve prognosis, has become an emerging category in systems biology, More and more attention from scholars. At present, it is mainly used in the research of pharmacology and toxicology, and can also be used for the diagnosis and treatment of diseases in clinic. This article describes its application in the preeclampsia and its research progress, in order to help obstetric medical workers have a more comprehensive understanding of it.

 

1 Proposing and concept of metabolomics

 

The origin of metabolomics dates back to 1999 and was proposed by Nicholson et al. Compared to traditional transcriptomics and proteomics, it supports large sample detection with higher accuracy and economic benefits. Metabolomics service explores life from the level of metabolites. The analysis targets the pre-transcriptional, transcript and post-transcriptional levels of living organism genes and related downstream metabolites after protein modification. They are generally small molecules with a relative molecular mass of less than 1000u. It truly reflects the physiological and pathological state of various human systems and provides a powerful basis for clinical diagnosis and treatment.

 

 2 Metabolomics classification and research methods

 

At present, metabolomics is classified according to the subjects and differences in research purposes, and is roughly classified into four categories, specifically: (1) metabolite target analysis. (2) Metabolite profile analysis. (3) Metabolite fingerprint analysis. (4) Metabolomics analysis.

 

Metabolomics is studied in a variety of ways, including according to its technology platform, including: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). At the same time, with the development of science and technology, on the basis of this, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), direct injection mass spectrometry (DIMS), Raman spectroscopy, capillary electrophoresis-mass spectrometry ( CE-MS), Fourier transform infrared spectroscopy (FTIR) and MRI (magnetic resonance). Among them, the most widely used is MRI and MS technology.

 

3 Metabolomics in the application of obstetric diseases

       

With the development of metabolomics, there are more and more applications in obstetrics. Biological tissues or body fluids can be used as specimens for the extraction of metabolic information. In the field of obstetrics, serological samples, amniotic fluid, fetal cord blood, villi, and urine are included. By detecting metabolites in fetal amniotic fluid, maternal urine and other tissue samples, the metabolic changes of fetal malformation (CFM), gestational diabetes mellitus (GDM), premature delivery (PTD) and other diseases can be revealed, providing new predictions for disease prediction. method. In the preeclamptic metabolomics study, available tissue specimens come from a variety of sources, including not only the above, but also vaginal secretions, milk, placenta, neonatal plasma and body fluids, with significant diversity.

 

In the field of obstetrics, metabolomics research is also the most commonly used MS and MRI technology platforms. The analysis principle is different. MS analysis is ionization separation based on the structural difference of metabolite components. The product is identified due to the difference in mass and relative abundance. MRI is based on the difference in response of the molecule to the electric field. The signal generated by the change of the direction of nuclear rotation generated by the molecule in the electric field is converted into a unique spectrum, which is matched to a specific database for identification. Compared with the two methods of analysis, MS has higher sensitivity and greater detection threshold, but it also has technical imperfections, that is, it requires metabolite derivatization to generate ions, so the sample preparation step is cumbersome and the sample is vulnerable. Destruction; while MRI avoids the problem of sample destruction. As a non-destructive technology, the sample reserve is not high, which is conducive to the rational use of resources.

 

In summary, each has its own advantages and disadvantages, so the clinical often adopts multiple platforms for joint analysis, because it is more comprehensive and accurate than the single method.

 

4 Metabolomics study in preeclampsia

 

The application of metabolomics in the field of preeclampsia is still in the early stage of exploration, focusing on the three major metabolisms of fat, protein and carbohydrates. The current research is divided into the following directions.

 

4.1 Serum metabolomics The use of serum metabolomics in the field of preeclampsia is not uncommon and has begun to take shape. Odibo et al detected serum acylcarnitine and amino acid levels by UPLC-MS method and found that alanine, glutamate, phenylalanine and hydroxycaproylcarnitine in serum of patients with preeclampsia Metabolites were significantly elevated, combined with these indicators and a curve to predict the incidence of pre-eclampsia, the area under the curve (AUC) can reach 0.82, the AUC of early pre-eclampsia reached 0.85. Considering that cellular inflammation and endothelial cell dysfunction are one of the important mechanisms of preeclampsia, some scholars have used LC-MS to detect the metabolic specific factors related to this mechanism, and found that the serum is taurine. The decrease in asparagine levels is closely related to pre-eclampsia. Some scholars have discovered through metabolomics research that taurine as an antioxidant and cell membrane stabilizer, which is significantly low in pre-eclamptic placental trophoblast cells, with reduced activity, leading to regulation of uterine spiral arterial remodeling disorders, and then participate in preeclampsia Onset.

 

Bahado-Singh et al used MRI technique to detect differences in serum metabolomics between pre-eclampsia, pre-eclampsia and normal pregnant women in 11-11+6 weeks of pregnancy, and found that pregnant women with late onset preeclampsia There were significant differences in serum metabolites, with glycerol and carnitine being the most significant. Using these significantly altered indicators of metabolites and body weight, the sensitivity of preeclampsia was predicted to be 76.7% with an accuracy of 100%. The involvement of carnitine in the pathogenesis of preeclampsia may be related to its oxidative stress and lipid peroxidation. In addition to carnitine, the researchers also found that glycerol, acetate, trimethylamine, succinate and other metabolites in the early and late preeclampsia serum differences, suggesting that these metabolites can distinguish between early onset and late Hair pre-eclampsia. Acilmis et al found that patients with pre-eclampsia had reduced serum choline levels, while low levels of choline increased the risk of preeclampsia, preterm and low birth weight infants. Studies have confirmed that serological metabolites are used in the prediction of pre-eclampsia, combined with a variety of metabolites can improve the pre-eclampsia detection rate to 75.9%, but can not rule out false positives, false positive rate is about 4.9%.

 

Austdal et al used MRI to detect 10 cases of preeclampsia, normal pregnancy and non-pregnant women between 17 and 20 weeks of pregnancy, and found that serum low density and very low density lipoprotein levels were significantly higher in the preeclampsia group than in the other two groups. And high-density lipoprotein is higher than the other two groups. The results of serum lipid metabolism in preeclampsia indicate that dyslipidemia has occurred in the early stage of preeclampsia, and may play an important role in its pathogenesis. The above studies suggest that changes in lipoprotein expression in serum lipid metabolism during early pregnancy may be used to predict early preeclampsia.

 

4.2 Urine Metabolomics In addition to the detection of serum metabolomics, the detection of urine in body fluids is also significant in preeclampsia studies. Austdal et al also detected changes in urinary metabolomics, suggesting that there are nine significantly different metabolites in the urine of preeclampsia patients, including cresol sulfate, histidine, glycine, and asparagine. , fenugreek and horse urate levels decreased, dimethylamine and isobutane levels increased. The study also found that the levels of choline in the urine of pregnant women with preeclampsia were abnormally elevated, and the analysis may be related to oxidative stress and renal insufficiency. Further studies have found that cresol sulfuric acid can be used to assess renal function, that is, in patients with pre-eclampsia complicated with renal insufficiency, the level of cresol sulfate in urine is decreased, but it is increased in kidney tissue. It is a cause of exacerbation of pre-eclampsia renal function damage. The mechanism of analysis may be related to the oxidative stress induced by cresol sulfate, which ultimately leads to damage of renal tubular cells and reduces renal excretion. In addition, Austdal et al also found that combined with urine levels of hippuric acid/creatinine, can significantly improve the predictive level of pre-eclampsia, suggesting that pre-eclampsia urine metabolites have a higher predictive value, worthy of study .

 

Paine et al found that the rapid increase of muscle glycophosphopeptide P (P-IPG) in the urine of patients with preeclampsia can be used as an indicator to predict preeclampsia, with a sensitivity of 88.9% and an accuracy of 62.7%. However, the number of cases is too small and needs further confirmation. Dawonauth et al sequentially detected the expression of P-IPG in the urine of pregnant women of different gestational weeks by ELISA. In the prospective study of 416 pregnant women, the final 34 cases progressed to preeclampsia, and the results showed P-IPG prediction. The sensitivity was 84.2%, the specificity was 83.6%, and it was able to predict 2 weeks before the onset.

 

4.3 Placental metabolomics Heazell et al intervene in placental villus tissue by different oxygen partial pressure, detect metabolite expression in culture medium and tissue lysate, simulate the regulation mechanism of hypoxia on preeclampsia, and find that differential expression Metabolites include 2-deoxyribose, triol or erythritol and palmitic acid. Dunn et al also described the pathogenesis of preeclampsia by UPLC-MS detection of placental metabonomics by in vitro hypoxic culture of preeclampsia and normal placental villus tissue. The study found that 47 metabolites are differentially expressed and preeclampsia. The incidence is closely related, such as glutamate, glutamine, tryptophan metabolism, leukotrienes or prostaglandins and other metabolic differences.

 

5 Advantages of metabolomics in predictive applications of preeclampsia

 

The choice of metabolomics to explore the pre-clinical disease, especially in the early prediction and pathogenesis has become a hot research topic.

 

       

 

Most of the subjects selected for metabolomics are serum, placental tissue, and urine. The recognition target is a small molecule metabolite, which has advantages that cannot be ignored compared with traditional research methods such as proteomics. The specific performance is: (1) The result is intuitive. As the final product of gene transcription or post-transcriptional modification, metabolites trace the origin of the results, and the metabolite-related marker factors can better reflect the overall state of the metabolic network, which is more intuitive. (2) High acceptability. Samples such as urine and serological sources are mostly non-invasive and simple, and are easily accepted by patients. It is difficult to apply clinically. (3) Simplicity of detection and analysis. The metabolomics research objective is metabolites, the species is significantly reduced relative to genomics, the detection is more convenient, and there is no need to establish a large number of databases for expressing sequence tags (EST) with low technical requirements. (4) Strong versatility. The differences in metabolites of different individuals are not large, which makes the technology more versatile and facilitates the unification of standards.

 

6 Prospects for the application of metabolomics in preeclampsia

 

In summary, we have seen the advantages of metabolomics in preeclampsia studies. Previous studies have shown that metabolomics is of great importance in the prediction of preeclampsia and disease pathogenesis, and it is worthy of further study. At the same time, we must also clarify the limitations of its existence, for example, the specificity of the matching requirements. Because each age level and constitutional state have an effect on individual metabolism, it is required to match factors such as age, weight, race and gestational age. In addition, metabolomics, as an emerging research discipline, is still in the preliminary exploration stage, so we are required to overcome some of the poor reproducibility of research data and enrich its data diversity to improve rigor and stability.

 

All in all, metabolomics is in the early stages of preeclampsia research, but we have seen its broad research prospects, which need to be further expanded in sample size and detection range, combined with the detection of many different products, using a variety of Technology platform to further enhance the role of metabolomics in predicting and diagnosing pre-eclampsia diseases.

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Some kinds of antibodies are frequently seen in the biology world, such as recombinant mouse anti-CMV antibody (DDG9 and CCH2) , a mouse monoclonal antibody that is capable of binding to CMV and recombinant mouse anti-CMV 65 kDa LA antibody (5E250), a mouse monoclonal antibody that is capable of binding to CMV 65 kDa LA.

So, what is CMV?

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Figure 1. Cytomegalovirus

Cytomegalovirus (CMV) is a DNA virus of herpesvirus group which also known as cell inclusion body virus for causing the enlargement of the infected cells which will develop a huge nuclear inclusion body. Cytomegalovirus is widely distributed in animals, which can cause infection in various systems, including genitourinary system, central nervous system and liver diseases, from mild asymptomatic infection to severe defects or death.

 

Biological character

CMV has the typical herpesvirus morphology, and its DNA structure is similar to herpes simplex virus (HSV), but 5% larger than HSV. Human CMV (HCMV) can only infect human and proliferate in human fibrocytes. The virus proliferates slowly in cell culture, and the replication cycle is long. It takes 30-40 days for the first isolation and culture to develop cytopathy. It is characterized by cell enlargement and rounding, nuclear enlargement, and a round of "halo" of large eosinophils around the nucleus.

 

Type

  1. According to the order of its infection, it can be divided into: (1) Primary infection: the host first infects CMV, but lacks any specific antibody to CMV before the infection (infants 6 months ago can have IgG antibody passively obtained from the mother); and (2) Recurrent infection, because the latent virus in the host is reactivated and replicated to proliferate; or to reinfect with different exogenous strains or larger doses of the same virus. CMV infection occurs when CMV invades the host and replicates or lurks in the host.
  2. According to the time when the host acquires the infection, it can be divided into: (1) Congenital infection: it refers to the child born by the mother with CMV infection, and it is confirmed that CMV infection is caused by intrauterine infection within 14 days (including 14 days) after birth;(2) Perinatal infection: for the children of CMV infected mothers, no CMV infection was found within 14 days of birth, and those confirmed to be infected within 3-12 weeks after birth were mainly infected through the birth canal or breast milk. The above two types are primary infection. (3) Postnatal infection or acquired infection: it refers to the infection confirmed after 12 weeks of birth (no evidence of CMV infection within 12 weeks of birth), which can be primary infection or reinfection.
  3. According to the presence or absence of symptoms, it can be divided into: (1) Symptomatic infection: It refers to the presence of symptoms and signs related to CMV infection. In symptomatic infection, there are virus activities in children's bodies, which are in the stage of virus producing infection.(2) Asymptomatic infection: there are two conditions of virus replicationin vivo: if it is enough to cause target organ or tissue damage, and the clinical manifestations have signs and organ function changes, it is called subclinical infection; if it does not cause target organ damage, there is no corresponding sign and function change, it is asymptomatic Epizootic infection.

 

Seeding way

CMV infection is very widespread in the population, and the infection is usually occult, which causes no clinical symptoms on the infected people, but under certain conditions, it can invade multiple organs and systems leading to serious diseases. The virus can invade lung, liver, kidney, salivary gland, other glands of mammary gland, as well as multinucleated leukocytes and lymphocytes, and can be discharged from saliva, milk sweat blood, urine, semen, and uterine secretion for a long time or intermittently, usually by oral, reproductive, placental, blood transfusion or organ transplantation and other multi-channel transmission.

(1) Congenital infection

CMV infection in pregnant women can invade the fetus through placenta and cause congenital infection, and a few cases show premature delivery, abortion, stillbirth or postnatal death. Jaundice, hepatosplenomegaly, thrombocytopenic purpura and hemolytic anemia may occur in children. Living children often suffer from permanent mental retardation, neuromuscular dyskinesia, deafness and chorioretinitis.

(2) Perinatal infection

When CMV is discharged from the urinary tract and cervix of the parturient, the infants can be infected through the birth canal. Most of them are of subclinical bed infections with mild or no clinical symptoms. Some of them suffer from slight respiratory disorders or liver function damage.

(3) Infection in children and adults

Sucking, kissing, sexual contact, blood transfusion and other infections, usually subclinical, can also lead to heterophilic antibody negative mononucleosis. Due to pregnancy, immunosuppressive therapy, organ transplantation, tumor and other factors, the virus is activated in monocytes and lymphocytes, causing mononucleosis, hepatitis, interstitial pneumonia, retinitis, encephalitis and so on.

(4) Cell transformation and possible carcinogenic effect

The CMV inactivated by UV can be transformed into mouse embryonic fibroblasts. In some tumors, such as cervical cancer, colon cancer, prostate cancer, CMV DNA detection rate is high, and CMV antibody titer is also higher than that of normal people, with virus particles being found in the cell lines established by the above tumors, suggesting that CMV, like herpesvirus, has the potential to cause cancer.

 

Preventive health care

(1) Physical training is necessary. In order to reduce the serious harm of cytomegalovirus to the fetus, we should improve the immune function and disease resistance of the body, especially in women of childbearing age.

(2) Pregnant women or patients with chronic consumptive diseases and low immunity should be protected to keep them away from infection.

(3) Pay attention to environmental health and food hygiene.

(4) Those with positive cytomegalovirus in milk should not be breastfed.

(5) Immune treatment is still under research and exploration. After CMV’s causing intracellular infection, the inactivated vaccine has no significant preventive effect. If CMV infection is found in the early stage of pregnancy and/ or there is CMV antigen in amniotic fluid cells, the pregnancy should be stopped. Live attenuated vaccine can make the vaccinated person produce antibody and activate cell immunity to CMV, reducing the occurrence of symptomatic CMV infection. CMV high titer immunoglobulin can protect symptomatic CMV infection in CMV negative bone marrow transplant recipients, but it cannot prevent reinfection. Wash hands carefully after contacting with urine or saliva to prevent CMV infection.

To prevent CMV infection caused by fresh blood transfusion, the following methods can be used: (1) using frozen blood or washed blood; (2) storing blood for more than 48 hours before transfusion; (3) using irradiated blood; (4) using blood filter to remove giant cells in the blood.

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In order to prevent or reduce the occurrence of bending pipel defects and obtain satisfactory bending pipe quality, corresponding actions should be taken in the process of bending pipe. First of all, in the scope allowed by the product design structure, the bent pipe fittings should be designed with a large bending radius as much as possible. At the same time, when selecting equipment, the pipe bending machine with side boosting and tail pushing mechanism should be the first choice. Under normal circumstances, for the several common defects mentioned above, measures should be taken in a targeted manner. The specific methods can be as follows:

For pipe fittings with severe flatten outside the arc, the compacting die (wheel) can be designed to have an anti-deformation groove in the form of a coreless bend to reduce or eliminate the degree of flatten when the pipe is bent. 

For cored bends, when the diameter of the mandrel is too small or with serious abrasion, the appropriate mandrel should be replaced. The unilateral clearance between the mandrel and the inner wall of the pipe should be no more than 0.5mm, and pre-install the mandrel appropriately. In addition, when installing the mold, it is necessary to ensure that the tube groove axes of the pieces are on the same level.

The thinning of the outer side of the arc when the small radius bends is determined by the characteristics of the bending process. It is unavoidable, but measures should be taken to overcome the situation of excessive thinning. The commonly used effective method is to use the side with a booster or tail. There is a pusher or a combination of both, so that the auxiliary push or push mechanism pushes the pipe forward, counteracts part of the resistance when the pipe is bent, improves the stress distribution on the pipe section, and makes neutral The layer is moved outward to achieve the purpose of reducing the amount of thinning of the tube wall outside the tube. The boost and push speeds are determined based on the actual conditions of the bends to match the bend speed. At the same time, it should be checked whether the advance amount of mandrel installation is appropriate, and the necessary adjustments should be made at the time of discomfort.

When bending outside the arc of the pipe, the reason should be carefully analyzed. Firstly, the pipe should have a good heat treatment condition. The weld of the seamed steel pipe should not be in the direction of the force of F1 and F2, that is, do not face the clamp. Tightening and bending the wheel mold; after removing the factors of the pipe, check whether the pressure of the pressing die is too large and adjust the pressure to be appropriate. For the newly used mandrel, check whether the diameter is too large. If the diameter is too large, it is necessary to carry out the necessary grinding, and ensure that the mandrel and the inner wall of the pipe are well lubricated to reduce the bending resistance and the friction between the inner wall of the pipe and the mandrel. And take appropriate measures to avoid machine shake, etc.

For the inner side of the arc, the corresponding measures should be taken according to the wrinkle position. If the front cut point is wrinkled, the position of the mandrel should be adjusted forward so that the advancement of the mandrel is appropriate to achieve reasonable support of the pipe when the pipe is bent; after the cut point is wrinkled, anti-wrinkle block should be added, and the anti-wrinkle block should be installed in the correct position. It can be well attached to the bending die, and the pressure of the pressing die (wheel) should be adjusted to make the pressure proper; the inside of the arc is all wrinkles. In addition to adjusting the compression mold (wheel) to make the pressure appropriate, check the diameter of the mandrel and the pitch between the joints of the ball joint. If the diameter is too small or the wear is serious, the mandrel should be replaced.

Welding deformation
After the workpiece is welded, it will generally be deformed. If the amount of deformation exceeds the allowable value, it will affect the use. A few examples of weld distortion are shown in Figure 2-19. The main reason for this is that the weldment is unevenly heated and cooled locally. Because of the welding, the weldment is only heated to a high temperature in a local area, the closer to the weld, the higher the temperature and the greater the expansion. However, the metal in the heating zone is prevented from being freely expanded by the metal having a low ambient temperature, and cannot be freely contracted due to the pinning of the surrounding metal during cooling. As a result, the partially heated metal has a tensile stress, while the other portions of the metal have a compressive stress balanced therewith. When these stresses exceed the yield limit of the metal, weld deformation will occur; when the strength limit of the metal is exceeded, cracks will occur. 

External defects of welds 
Weld reinforcement too high 
As shown in Figure 2-20, this phenomenon occurs when the angle of the weld bevel is too small or the welding current is too small. The dangerous plane of the weldment weld has transitioned from the M-M plane to the N-N plane of the fusion zone. Due to the stress concentration, the fatigue life of the pressure vessel is required to increase the weld seam. 
Weld undercut 
The depression formed along the edge of the weld on the workpiece is called the undercut, as shown in Figure 2-22. It not only reduces the working section of the joint, but also causes severe stress concentration at the undercut. 
Welded beading
The molten metal flows to the unmelted workpiece at the edge of the bath and accumulates to form a solder joint that is not fused to the workpiece, see Figure 2-23. The weld has no effect on the static load strength, but it will cause stress concentration and reduce the dynamic load strength.

Burn through 
Burn through means that part of the molten metal leaks from the reverse side of the weld and even burns through the hole, which reduces the strength of the joint. 

The above five defects exist in the appearance of the weld, which can be found by the naked eye and can be repaired in time. If the operation is skilled, it can generally be avoided.

Internal defects of welds 
Incomplete penetration
Incomplete penetration is a defect that is not partially fused between the workpiece and the weld metal or weld layer. Incomplete penetration reduces the working section of the weld, causing severe stress concentration and greatly reducing joint strength, which often becomes the source of weld cracking. 
Slag
The slag is welded with non-metal slag, which is called slag. The slag inclusion reduces the working section of the weld, resulting in stress concentration, which reduces weld strength and impact toughness. 
Porosity
At the high temperature, the stomatal weld metal absorbs too much gas (such as H2) or gas generated by the metallurgical reaction inside the bath (such as CO), which cannot be discharged when the bath is cooled and solidified, but forms inside or on the surface of the weld. The hole is the stomata. The presence of pores reduces the effective working section of the weld and reduces the mechanical strength of the joint. If there are penetrating or continuous pores, it will seriously affect the sealability of the weldment. 
Cracks 
During or after welding, local cracking of the metal that occurs in the area of the welded joint is called cracking. Cracks may be generated on the weld and may also create heat affected zones on both sides of the weld. Sometimes it occurs on metal surfaces, sometimes inside metal. Generally, according to the mechanism of crack generation, it can be divided into two types: hot crack and cold crack. 
Hot cracks 
Hot cracks are generated during the crystallization from liquid to solid in the weld metal and are mostly produced in the weld metal. The main reason for this is the presence of low-melting substances (such as FeS, melting point of 1193 ° C) in the weld, which weakens the relationship between the grains, and when subjected to large weld stress, it is easy to cause cracks between the grains. When there are many impurities such as S and Cu in the weldment and the welding rod, hot cracks are likely to occur. 

   Thermal cracks are characterized by a distribution along the grain boundaries. When the crack penetrates the surface and communicates with the outside, there is a clear tendency to hydrogenate. 

Cold cracks 
Cold cracks are generated during post-weld cooling and are mostly produced on the fusion line between the base metal or the base metal and the weld. The main reason for this is that the quenched structure is formed in the heat-affected zone or the weld, and the internal crack of the grain is caused by the high stress. When welding the easily hardened steel with high carbon content or more alloying elements, the most It is prone to cold cracks. Excessive hydrogen is melted into the weld and can also cause cold cracks. 
Crack is one of the most dangerous defects. In addition to reducing the load-bearing cross-section, it also causes severe stress concentration. In use, the crack will gradually expand and eventually cause damage to the member. Therefore, such defects are generally not allowed in the welded structure, and once found, it is necessary to shovel the re-welding. 

Welding inspection
Performing the necessary inspection of the welded joint is an important measure to ensure the quality of the weld. Therefore, after the workpiece is welded, the weld should be inspected according to the technical requirements of the product. Any defects that do not meet the technical requirements must be repaired in time. Inspection of welding quality includes three aspects: visual inspection, non-destructive testing and mechanical performance testing. These three are complementary to each other, and non-destructive testing is the main one.

A ppearance inspection 
Visual inspection is generally based on visual observation, sometimes with a magnifying glass of 5-20 times. Through visual inspection, weld surface defects such as undercuts, welds, surface cracks, pores, slag inclusions, and weld penetration can be found. The dimensions of the weld can also be measured using a weld detector or template. 

Nondestructive test
Inspection defects such as slag inclusions, pores, cracks, etc. hidden inside the weld. At present, the most common use is X-ray inspection, as well as ultrasonic flaw detection and magnetic flaw detection.
The X-ray inspection uses X-rays to take pictures of the weld, and judges whether there are defects, the number and type of defects in the interior based on the image of the film. According to the technical requirements of the product, the weld is qualified.
The ultrasonic beam is emitted by the probe and transmitted to the metal. When the ultrasonic beam is transmitted to the metal-air interface, it is refracted and passed through the weld. If there is a defect in the weld, the ultrasonic beam is reflected to the probe and accepted, and a reflected wave appears on the screen. Based on the comparison and discrimination of these reflected waves with normal waves, the size and position of the defects can be determined. Ultrasonic flaw detection is much simpler than X-ray photography and is therefore widely used. However, ultrasonic flaw detection often can only be judged based on operational experience, and can not leave inspection basis. 
For internal defects that are not deep from the surface of the weld and extremely small cracks on the surface, magnetic flaw detection can also be used.

Hydraulic test and air pressure test 
For pressurized containers requiring sealing, a hydrostatic test and/or a pneumatic test shall be carried out to check the sealability and pressure bearing capacity of the weld. The method is to inject 1.25-1.5 times working water or a working pressure gas (mostly air) into the container for a certain period of time, then observe the pressure drop in the container and observe whether there is leakage outside. According to these, it can be assessed whether the weld is qualified. 

Mechanical test of welded test panels
Non-destructive testing can reveal the inherent defects of the weld, but it does not indicate the mechanical properties of the metal in the heat affected zone of the weld. Therefore, the welded joints are sometimes subjected to tensile, impact, bending and other tests. These tests were performed by the test panels. The test panels used are preferably welded together with the longitudinal joints of the cylinder to ensure consistent construction conditions. The test panels were then tested for mechanical properties. In actual production, only welded joints of new steel grades are generally tested in this respect.

 

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