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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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Anyone who knows me personally knows that I love being barefoot. You will never catch me wearing slippers or shoes in the house and I often will go out on my deck and on my lawn barefoot. Ah the pleasures of walking barefoot on the grass! Naturally, when I came across the minimalist shoe, I simply had to give it a try! It was a few weeks ago that I went out and purchased myself a nice pair of Five Fingers Minimalist Shoes. Like their name says, these shoes actually have five fingers: a small pocket for each toe, making them much like toe socks.

After a few weeks of getting used to walking in them around the house, it was time to take them out for a real walk. I eagerly slipped them on and stepped out onto my front porch. Wow! I could feel everything under my feet. I have to admit that I was very surprised at how much I could feel through the thin sole. I got in the car and made my way to the trail where I usually walk. It's a gravel trail through a small natural park. Again it was amazing to me that I could feel each little rock under my feet without risking an injury from those same rocks. My feet were literally able to adapt to every little bump in the terrain as I walked along the path. However, my first few power walk steps were a rough reminder that I was not wearing my regular shoes and that I cannot be striking my heel into the ground the same way as I do with my regular sports shoes. It did not take long to adjust my stride and complete my 40 minute walk. I am not a runner and I don't think I will ever take up running as a passion, but I absolutely loved walking with my Five Fingers and look forward to using them again.

Pros of Five Finger Shoes

When your feet are able to feel the terrain as you move, your body is better able to adapt its movements and keep you in proper balance and alignment. Essentially, your body is working the way nature intended it to and your joints are aligning and moving the way they were always meant to.
Your toes are able to move individually as opposed to being confined into one space. The fact that your toes are able to spread as you walk allows you to have a stronger, more stable and aligned push off as you take each step. This is true whether you are walking or running.
Cons of  Five Fingers

There is an adaptation period required for you to be able to walk and run in your Five Fingers without running a high risk of injury. The fact that you simply cannot slip these shoes on and go running like you would with your regular running shoes, can be enough to deter you from the whole idea of the minimalist shoe.
If you have had your feet in a certain type of shoe (running shoes, heels, flip flops, etc.) for most of your waking hours, minimalist shoes may take you a very long time to adapt to. Over time, your body adapts to the imbalances that are imposed on it. For instance, a woman who walks on her three inch heels all day, every day will not be able to quickly transition to a flat shoe without running the risk of altering her body mechanics. Chances are some aches and pains will arise as you transition into your Five Fingers.
Overall, my experience with the Five Fingers shoes was very positive and I greatly enjoy wearing them for my fitness activities. I have taken the necessary time to ease into them by wearing them around the house a few hours at a time while I do my house work and still wear them only for short periods of time while I am exercising and when I am engaging in particular activities that call for their use.

At the end of the day, the Five Fingers Minimalist Shoes truly do serve their purpose. The allow the wearer to keep their feet protected from the harsh terrain while still being able to feel the terrain and adapt their stride the way it was always intended by nature: as if they were barefoot. Once the gradual adaptation period has been passed, these minimalist shoes offer both comfort and stability while performing various activities. Although they have not been around for as long as your traditional running shoe, they will probably increase in popularity as athletes and sports enthusiasts begin to see the benefits these shoes can offer.

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Tumor Cells: What You Need to Know?

What is the composition of tumor cells?

Tumor cell parenchyma is a tumor. The tumor tissue consists of two parts: the parenchyma and the interstitial. Tumor consists of tumor cells, which is the main component of the tumor and has tissue-specific specificity. Tumor is an oncology term. It determines the biological characteristics of the tumor and the specificity of each tumor. Generally, the tissue origin of various tumors is identified according to the parenchymal morphology of the tumor, and the classification, naming and histological diagnosis of the tumor are performed, and the benign and malignant tumors and the malignant degree of the tumor are determined according to the degree of differentiation and the degree of heterotypic formation.

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What are the characteristics of tumor cells?

Tumor cells have three distinct basic features: immortality, mobility, and loss of contact inhibition. In addition, tumor cells have many physiological, biochemical, and morphological features that are different from normal cells. Similar to other diseases, the diagnosis of tumors is based on medical history and physical examination as the most basic and important diagnostic tools. Physical examination includes X-ray examination, ultrasound examination, endoscopy, histological biopsy, blood examination, etc. Here we mainly talk about tumor markers.

What is a tumor marker?

A tumor marker refers to a chemical substance produced by a tumor tissue that reflects the presence of the tumor itself. It can be used for the diagnosis, prognosis and therapeutic observation of tumors.

 

They are classified as the following categories:

  • Oncofetal proteins, such as alpha-fetoprotein, carcinoembryonic antigen;
  • Tumor-associated antigens, (such as CA19-9, CA125);
  • Enzymes, such as dehydrogenation of lactate Enzyme, neuron-specific enolase, prostatic acid phosphatase;
  • Special plasma proteins (such as β2-macroglobulin, this week protein);
  • Hormones (hormone), such as calcitonin, chorionic gonadotropin, adrenocorticotropic hormone;

In addition, proto-oncogenes, tumor suppressor genes and their products are also increasingly used as tumor markers. The use of tumor markers for the determination of tumors has been clinically used for many years and has served many roles in clinical diagnosis and therapeutic observation. In order to improve the accuracy of diagnosis, several related markers are often combined into a combined marker group in the clinic, and a certain tumor is detected at the same time.

How to treat tumors?

There are two viewpoints for treating tumors. One is to remove all or all of the tumor cells in the patient, so that the tumor does not recur in the survival period; the second is to change the characteristics of the cancer cells, so that the course is slowed down or even Stop completely. The conventional methods of tumor treatment include surgery, radiotherapy and chemotherapy.

 

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Synonymous codon substitutions affect the mRNA coding sequence, but the encoded amino acid sequence remains unchanged. Therefore, ostensibly these substitutions do not affect the phenotype and are often ignored in the study of human genetic variation. However, a variety of studies have shown that protein levels, translational accuracy, secretory efficiency, final folding structure and post-translational modifications are regulated by multiple mechanisms. Synonymous codon action has gradually emerged, and the precise mechanism has yet to be discovered. Studies on the interference of synonymous codon substitution on the co-translational folding mechanism often lack in vivo evidence, and usually, rare synonymous codons tend to translate more slowly than ordinary synonymous codons. In addition, rare synonymous codons tend to appear in clusters, many of which are preserved during evolutionary history. The folding rates of many protein secondary and tertiary structures are similar to their synthesis rates, and subtle changes in elongation may also alter the folding mechanism. Theoretically, synonymous rare codon substitutions reduce translational elongation and can provide more time for the N-terminal portion of the nascent protein to form a stable tertiary structure before the C-terminal portion emerges from the ribosome exit tunnel. Is the extra time good or bad for efficient folding? Cells contain a chaperone network to facilitate protein folding. It is unclear whether altered elongation and co-translational folding mechanisms of synonymous codons interfere with chaperone function. 

 

Recently, Ian M. Walsha and colleagues from the University of Notre Dame published an article in PNAS "Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness." They show that synonymous codon changes in the coding sequence of enzymes essential for E. coli growth have a significant impact on cell growth. The researchers tested various mechanisms of this growth defect, including changes in folded protein structure, expression levels, enzyme activity, mRNA abundance, and/or cellular stress responses. The findings are compatible with synonymous substitutions that alter the translation elongation pattern, the rationale being that altered co-translational folding mechanisms lead to more degradable structures. These results suggest that synonymous codon alterations can affect protein folding in vivo and affect the function of chaperones in protein homeostasis networks. Thus, the clever use of synonymous codons can have implications for protein design and the interpretation of synonymous mutations associated with disease.

 

Most of the current understanding of protein folding mechanisms comes from studies of small proteins that fold reversibly upon dilution from chemical denaturants. However, only a small number of proteins can be stably refolded in vitro and remain in a stable state. This suggests that the conformation adopted early in the folding process is essential for successful folding and supports the formation of an early folding intermediate that is different from the conformation formed after dilution from the denaturant. Indeed, there is substantial evidence that chaperones are essential for the successful folding of many proteins in vivo. Although it has been hypothesized that synonymous codon changes can alter the extension rate and alter the folding mechanism, to date, no evidence supporting the hypothesis has been found experimentally in vivo, possibly due to the auxiliary role provided by chaperones. The results presented here indicate that synonymous codon-induced translational elongation affects folding during the synthesis of nascent CAT polypeptide chains. Although the nascent chain generated using different synonymous codons is still stable and CAT has a trimeric structure, the CAT protein generated by translation using the synonymous Shuf1 mRNA sequence is more susceptible to degradation by the cellular protease ClpXP, which leads to severe cell growth defects. Assuming that the ClpXP ssrA degradation tag is attached to the C-terminus of CAT, most of the degradation may occur only after the release of the translated CAT nascent chain from the nucleosome. Notably, even the native Shuf1-CATssrA protein is more susceptible to degradation than native CATssrA, suggesting that codon-induced perturbations persist for some time after translation and folding are complete. Thus, the buffering effect of the cellular proteostasis network is not sufficient to twist the effect of Shuf1-CAT folding defects on cell growth.

 

The ssrA tag approach developed here reveals such interferences in other coding sequences, even though these interferences do not lead to eventual changes in protein structure. Recent in vitro single-molecule force unfolding experiments have shown that some small, ribosome-bound native folding domains can fold at the ribosome by a similar mechanism. However, as these studies point out, the results measured by molecular tweezers do not capture the transient folding of nascent chains during synthesis, and therefore, in these experiments it is the effect of close proximity of the ribosome surface, rather than co-translational folding of the ribosome surface, that is measured. The folding behavior of a reversible folding model may indeed result in indistinguishable folding behavior during translation. However, most of the model proteins selected for these studies were small, whereas synonymous codon-derived co-translational folds were much larger. The in vitro folding mechanism of proteins larger than 175 aa that are retained during co-translational folding is not known. Thus, synonymous codon-derived regulation of elongation rates can play a broad role in efficient folding of larger, and more complex proteins.

 

CAT results indicate that synonymous changes in mRNA coding sequences can perturb the folding of protein sequences even in the presence of chaperone molecules, suggesting that mRNA sequences may have evolved with chaperones, thus effectively supporting the folding of the resulting protein structure. Although knowledge of the co-translational folding machinery is still in its infancy, these results suggest that for large or otherwise complex proteins it should be possible to rationally design mRNA coding sequences to improve in vivo folding yields and identify disease-associated synonymous codon substitutions that are most likely to adversely affect protein co-translation.

 

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2. RNC-Seq

Translating mRNA full-length sequencing (RNC-SEQ) shows a unique advantage that effectively addresses this issue. The cell lysate was loaded on a 30% sucrose pad and all the ribosomal-related translated mRNA and free mRNA and other cellular components were separated by ultracentrifugation. Ultracentrifugation can deposit RNC. RNC-mRNA can be recovered from the granulated RNC, effectively avoiding the interference of high concentration of sucrose, which is beneficial to downstream research. Next-generation RNC-mRNA sequencing technology reveals the full-length information of translated mRNAs, including the abundance and type of mRNAs. By optimizing the centrifugation and sucrose buffer, the recovery of RNC can reach 90%. Under the condition of appropriate buffer, RNC still maintains translation activity. The technical difficulty of RNC-SEQ lies in the separation of complete RNC. The fragility of RNC leads to the dissociation of ribosomes and the breakage / degradation of mRNA, which leads to the biased analysis of RNC-mRNAs.

3. Ribo-Seq

Ribosome map (Ribo-seq), first published by Science in 2009 in Ingolia et al, which studied translation from another perspective. Treating cell lysates with low concentrations of ribonuclease (RNase) degrades mRNA, except for ribosome-protected RNA fragments. Next-generation sequencing (NGS) was used to analyze 22-35 nt mRNA fragments (ie, ribosome footprints: RFPs), which correspond to ribosome-protected fragments (RPFS) to reveal the location and density of ribosomes. Based on positional information, the distribution and density of ribosomes on each transcript, information such as the start codon position (including non-ATG start), codon usage bias, upstream ORFs (uORFs), and translation pause landscape can be inferred. These aspects cannot be studied by other translation methods. In 2016, an optimized Ribo-seq method-super-resolution ribosome profiling was developed, which was able to observe a strong global 3nt periodicity in a single transcript. In addition, this method improves Ribo-seq's ability to reveal small ORFs (sORFs) and unannotated new coding regions, possibly encoding proteins from annotated non-coding RNA and pseudogenes. The ribonuclease used in the ribosome map was also taken into account. It was found that ribonuclease T1 is the enzyme that can best maintain the integrity of the ribosome, and also can convert the multimer into a single nucleosome. A variant of Ribo-seq is translation complex profile sequencing (TCP-seq). Fast-cooled yeast cells were cross-linked with formaldehyde to cause their translation complexes with mRNA to stagnate in their natural positions, and then subjected to RNase digestion. Then sucrose gradient ultracentrifugation was used to separate whole ribosomes and small subunits (SSU), and RNA fragments of up to 250 nt in these fragments were sequenced. Natural distribution maps were obtained at the beginning, extension and termination stages of translation. This method can observe the SSU footprint on the 5 'untranslated region (UTR) of mRNA and capture the position of any type of ribosome-mRNA complex at various stages of translation.

However, Ribo-seq experiments are complicated and expensive. Compared to RNC-SEQ, it requires a large number of cells as a starting material. Since Ribo-seq of all species requires depletion of hybrid-based rRNA, Ribo-seq is usually limited to several model organisms. For many other species, especially the wide variety of bacteria, Ribo-seq is difficult to perform due to the lack of rRNA probes, and expensive customization of rRNA probes seems to be the only option. Many other factors affect the number of RFPs, such as pseudo RPF. Since Ribo-seq mainly analyzes coding sequences (CDS), where ribosomes bind to mRNA. Untranslated regions (UTRs) highly related to translation regulation cannot be effectively analyzed. In addition, Ribo-seq often generates many "RFPs" that are aligned to non-coding RNA, indicating a significant false positive rate.

Another disadvantage is the short RFP length (24-26 nt for prokaryotes and 28-30 nt for eukaryotes), which is limited by the size of the ribosome and cannot be extended further. In order to obtain sufficient coverage of moderately abundant mRNAs, the amount of sequencing needs to be expanded (usually more than 100 million reads per sample), which means that sequencing and computational costs are high. Nevertheless, many translation events, especially the splice junctions of splice variants and circular RNAs, remain difficult to cover. The stitching alignment algorithm performs poorly at detecting connection points in these short reads. In contrast, the full-length RNC-seq sequence is the sequence of the entire mRNA; therefore, longer read lengths are suitable. Longer reads result in almost complete coverage of most translated mRNAs, including low-abundance mRNAs. This allows efficient detection and quantification of ligation, for example, translation of various splice variants of BDP1 and BRF1 and translation of circular RNA CircLINC-PINT, which are almost impractical using Ribo-seq.

It is worth emphasizing that the density of RFP does not represent translation activity. The RFP density is directly proportional to the translation initiation rate and inversely proportional to the elongation. If translation is completely stalled on a certain mRNA, RFP will be highly enriched in that mRNA, but translation activity will be zero.

4. TRAP-Seq

Inada et al. reported ribosome affinity purification (RAP) or translating RAP (translating RAP: TRAP). The large ribosomal subunit protein Rp25p produced under the control of a tissue-specific promoter and a fusion affinity tag (such as polyhistidine, green fluorescent protein (GFP), etc.) is used at the C-terminus. These ribosomes are then affinity purified (beads or columns) and isolated from ribosomes of other cell types. TRAP-SEQ specifically enriches RNC-mRNA in difficult-to-separate samples isolated ribosomes cannot be contaminated with non-ribosomal mRNPs co-precipitated with ribosomes because TRAP-SEQ does not use ultracentrifugation. TRAP-SEQ has its unique advantages in isolating translated mRNA from specific cell types in complex tissues. However TRAP-seq requires a stably transfected cell line to produce labeled ribosomal proteins. When applied to plants and animals, it is inevitable to build stable transgenic organisms. This is time-consuming and expensive, and it is not suitable for those that have not yet been established Species that stabilize transformation methods. In addition, overproduction of labeled ribosomal proteins has the potential to alter the structure and properties of these ribosomes. As a result, the system is no longer under physiological conditions; careful evaluation should be performed before all conclusions are applied to general scenarios.

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Yvonne Chen, an associate professor at the University of California, Los Angeles and a member of the Johnson Comprehensive Cancer Center, genetically modified immune cells such as T cells to attack the most evasive enemy: cancer. New cancer immunotherapies produce immune cells that can effectively kill blood cancers, but it is difficult to kill solid tumors. She is designing ways to allow immune cells to "beat" solid tumors. She presented her research at the 64th Annual Meeting of the Society for Biophysics, San Diego, California, USA, on February 18, 2020.

 

T cells are white blood cells that patrol and attack invaders in our bodies, but they also need to avoid attacking our own cells, a way in which cancer can evade immune system surveillance. Solid tumors are cancers that form tumor masses in the body, accounting for 90% of cancer cases and can even inactivate immune cells. These tumors can be surrounded by a protein called transforming growth factor beta (TGF-β). The protein TGF-β can inhibit the activity of T cells in the tumor environment. Chen discovered a way to help T cells overcome TGF-β inhibition to resist tumor cells.

 

T cells are genetically engineered to express a receptor called the chimeric antigen receptor (CAR), which is designed to recognize tumor-associated proteins, the tumor antigens. Once a cell presenting a target antigen is encountered, CAR-T cells can bind the target cell and kill it. Given that CD19 is an antigen found on B cells, T cells engineered to express a CAR that targets CD19 (CD19 CAR-T) have been approved by the US Food and Drug Administration (FDA) for the treatment of B-cell leukemia and lymphoma.

 

Although CD19 CAR-T cell therapy has shown encouraging clinical results, sometimes a cancer cell population without CD19 appears throughout the treatment. "Clinical trials have shown that 50% of lymphoma patients treated with CD19 CAR-T cells relapse within 6 months, and many of these cases involve tumor cells that no longer express CD19," Chen said. To avoid this, Chen designed T cells targeting CD19 and CD20 to reduce the likelihood of any cancer cells escaping therapy. This bispecific CAR-T cell therapy is currently being tested in a phase I clinical trial carried out at UCLA.

 

Besides hematological cancers, solid tumors have been the focus of many ongoing research efforts in Chen's laboratory. "Immunotherapy works well for hematological tumors, but not for solid tumors, in part because of TGF-β-induced immunosuppression," Chen said. Her new approach to using CAR that has been genetically engineered to target TGF-β is a good start for solid tumors. "TGF-β CAR has shown the potential to safely and effectively enhance the antitumor efficacy of T cell therapy."

 

Chen and colleagues constructed CARs that could respond to TGF-β by enhancing defense. "T cells expressing TGF-β responsive CAR are not inhibited in response to TGF-β, but are ready to meet and attack tumor cells when exposed to high concentrations of TGF-β," Chen said. Chen and colleagues are developing TGF-β CAR-T cells that also target another tumor-specific marker in order to develop next generation T cell therapies that can be effective against immunosuppressive solid tumors.

 

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But after the passion faded, the problem gradually emerged. Among them, a major problem that researchers cannot solve is how to start the RNAi process only in the cells that are needed. To make things worse, many people were in hurry to undergo a series of therapies that have just rushed into human trials with only preliminary verification in the laboratory.

"Many early clinical trials are very unwise, and many people want to be the first to do clinical trials," said Professor Mark Kay, a gene therapy expert at Stanford University. He knew these tests will not succeed.

The seeds of disaster, which were buried from the beginning, quickly bear bad fruit. Soon after, RNAi therapies in some studies showed unexpectedly dangerous side effects in the human body. These therapies are either ineffective or harmful to the body because they cannot be delivered to the correct cells in the body.

The entire RNAi field fell to the bottom in an instant, and many biopharmaceutical companies including Roche, Pfizer, and Merck have decided to exit. In 2014, Merck sold its RNAi technology company Sirna at a discount. It was bought by a biotechnology company called Alnylam.

The rise of bright star

Alnylam was founded in 2002, right at the midpoint of RNAi's scientific breakthrough (1998) to the Nobel Prize (2006). Its name is a bit difficult to read, but there is an interesting story behind it-it is derived from the word "Alnilam".

Like the stars, more than 10 years ago, cutting-edge companies developing RNAi technology can be seen everywhere, and Alnylam seems to be no different from them. But when RNAi therapy fell into a trough, Alnylam was one of the few lucky ones to survive. This does not mean that it has never experienced pain. On the company's official website, Alnylam wrote that in the early days of its establishment, it had also encountered many challenges: the departure of its partners and the loss of confidence in the technology by the outside world had brought a big blow to Alnylam. Only within the company can we see the belief and optimism that make RNAi therapy a reality.

Of course, countless cases prove that faith and optimism alone are not enough. What really drives Alnylam forward is a key technology he invented during the "darkest moment" of RNAi therapy. In 2010, the company published a paper that impacted the entire field of RNAi therapy-they found that using ligand-based technology, people could finally deliver targeted RNAi therapies. Yokohama's biggest obstacle to scientists' progress was removed. In front of them is a bright road to the approval of first RNAi therapy.

First RNAi therapy

After finding the key to solving the problem, Alnylam quickly established a series of research and development pipelines to address a variety of rare genetic diseases. Among them, its leading RNAi therapy, patisiran, treats a disease called hATTR amyloidosis. The root cause of this disease is mutations in the gene encoding thyroxine transporter, which causes the abnormal accumulation of amyloid in the human body and causes damage to organs and tissues. It is a severe and fatal rare disease. The life expectancy of a patient is only 2-15 years from the onset of symptoms.

Patisiran can exert the "silencing" effect of RNAi on genes. By inhibiting the expression of specific mRNAs, this therapy can effectively prevent the generation of mutated thyroxin, clear the amyloid deposits in tissues, and restore tissue function.

In September 2017, Alnylam and his partner Sanofi announced the positive top-line results of patisiran in a phase 3 clinical trial. Studies have shown that the new drug has reached the primary clinical endpoint, as well as all secondary clinical endpoints. At the 18-month node, patisiran significantly reduced patients' neurological lesions and improved their quality of life compared to placebo.

Two months later, Alnylam submitted a rolling listing application to reduce the time required for listing. The US FDA has also granted patisiran breakthrough therapy designation and orphan drug status, accelerating its introduction. On August 3, the UK granted patisiran "Early Access" status, allowing patients to get treatment before the treatment is officially launched. Today, humans finally ushered in the approval of the first RNAi therapy.

Some personal opinions

As we know, the  even research and development of new drugs is not easy, even with Nobel Prize support. Even if they can finally leave the laboratory and come to the patient's bed, these new therapies often go through a long research and development journey. Monoclonal antibodies, for example, have gone through a long journey, as are RNAi therapies today.

And perhaps only the bravest fighters can open up a path of benefit for the patients from the thorns of doubt.

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The background

The outbreak caused by the new/novel coronavirus (2019-nCov, SARS-CoV-2, or COVID-19) has attracted the attention of people throughout the world. Among them, there is a question that has drawn widen attention: why the number of confirmed cases are increasing rapidly since January 18, 2020? How can the novel coronavirus be detected quickly?

In this article, some questions are discussed and explained.

The pathogen of this unexplained case of viral pneumonia was initially determined to be "novel coronavirus". Whether it can be detected quickly and accurately becomes the key to prevention and control.

Prior to the emergence of specific diagnostic methods, suspected cases could be found based on patient signs. Unfortunately, the specificity of this new type of coronavirus pneumonia is not strong. Compared with SARS, the onset of pneumonia was not urgent, and some patients did not even have a high fever. In the flu season, it is very difficult to distinguish patients with new coronaviruses from.

On January 16, the first batch of PCR kits for the new coronavirus was delivered to provincial CDCs, which was the direct cause of the rapid increase in the number of confirmed cases in recent days.

The Coronavirus Detection PCR Kit works roughly by extracting RNA from patient samples, performing reverse transcription-polymerase chain reaction (RT-PCR), and amplifying trace amounts of virus information in the samples by amplification reactions, and finally read the signal fluorescently. If the signal is positive after PCR, it can be said that the virus is present (infected) in the sample, otherwise it is not infected.

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  1. How long does a nucleic acid test take?

It takes about 16 hours at the beginning (the data in this article is estimated based on the normal laboratory operation time and does not represent the actual time).

The new coronavirus that caused this pneumonia, like its close relatives SARS and MERS, its genetic information is composed of single-stranded RNA. To complete its detection, at least two steps are required to extract viral RNA and reverse transcription PCR (RT-PCR). Extracting viral RNA itself also involves multiple steps such as lysing the sample and purifying the RNA, which can take several hours. RT-PCR generally takes at least three or four hours to complete. If the entire process line is operated, it will take about one working day, that is, about 6-8 hours. The test results need to be reviewed, and repeated experiments will take twice the time.

Based on this, some organizations have developed a simplified version of the kit, which can reduce the time of a single test to about 3 hours.

  1. How to develop and produce kits?

Some people might wonder why did it take so long to develop and produce a kit after a month or so from the discovery of the first patient to the delivery of the kit?

Let's take a look at the development process of the kit.

Pathogen isolation and investigation: The pathogen that caused the outbreak is a brand new coronavirus. Before confirming that this is a pathogen that has never been seen, we need to exclude all known pathogens: including all types of influenza viruses (influenza A, avian flu, etc.), adenoviruses, rhinoviruses and Coronaviruses that cause pneumonia (SARS, MERS), Chlamydia, Mycoplasma, etc.

Design primers: This is a very critical step. Only by designing appropriate primers can the PCR reaction for virus detection be performed. Scientists need to perform genome-wide sequencing and bioinformatics analysis on this newly found virus, design primers (that is, a DNA sequence), verify its specificity and sensitivity, and ensure that it can only recognize new coronavirus genes.

Industrialized production: It can be said that all components in all viral nucleic acid detection kits are the same except for primers, so the synthesis of a large number of appropriate primers is the key to industrialized production kits.

Pathogen isolation and screening takes at least a week, and sequencing and bioinformatics analysis take about three days. Probe design and specificity checks take another two or three days. In other words, it takes almost two weeks to put the designed primers into industrial production when everything is going well.

In addition to nucleic acid testing, protein testing is also on the way.

In addition to the current nucleic acid detection methods used in health systems, detection techniques based on viral proteins can also play a significant role in the rapid detection of pathogens.

In addition to the genetic information carried by DNA / RNA, the protein shell of the virus also carries a large amount of information about the structure and pathogenic mechanism of the virus. It can also determine whether the patient is infected with the virus and whether it is immune to the virus.

Nucleic acid detection relies on primers, and detection of viral proteins mainly depends on antibodies. As long as an antibody with sufficient affinity and specificity is found, the tedious steps of extracting viral RNA can be omitted and the protein immunoassay can be directly performed using the serum or sputum of the patient at the onset of disease (generally enzyme-linked immunosorbent assay, ELISA). The results will get in 3-5 hours. If a new-generation immune reactor based on a microfluidic platform is used, the total detection time can be shortened to 30 minutes under the condition of ensuring detection sensitivity, and real-time diagnosis can be truly achieved.

The difficulty in the development of protein-based virus detection lies in the production of antibody screening. The production of experimental antibodies is highly dependent on animals (generally produced by animals such as mice, rabbits, and sheep). A few days after the injection of the antigen, the animal's immune cells need to be screened to find lymphocytes that can be used to produce highly specific antibodies and expand them. Even at full speed, it will take 2-3 weeks to get the initial available monoclonal antibodies. Compared to RNA-based PCR kits, protein detection kits require longer development time and higher production costs.

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Abstract: Scientists have made progress in research of childhood cancer and the latest treatment will also cover mental health assessment.

February 15th is International Childhood Cancer Day, reminding us that childhood cancer deserves more attention since more than 250,000 children are diagnosed with cancer and about 90,000 children die from cancer every year.

In recent years, the incidence of childhood cancer has been increasing, but children suffering from cancer have not received enough attention from all walks of life. How to make breakthroughs in the field of childhood cancer treatment and find more accurate and effective treatments is a problem that scientists worldwide have been exploring. Recent research on childhood cancer has made some new progress.

Precision medicine of childhood cancer: JAMA Network Open published a research result of the CHU Sainte-Justine TRICEPS team that genomic profiling will help clinical treatment of childhood cancer. Until April 2019, the team had enrolled 84 patients. In 87% of patients, the study identified genomic anomalies that allowed for better patient management. The therapeutic alternatives are personalized because the proposed actions will be different for each patient. The discoveries can lead to a "targeted" therapy because it specifically seeks to block (or bypass) the action of genes that cause cancer progression.

Gene technology for Ewing sarcoma: Ewing sarcoma is a childhood cancer driven by EWS-ETS transcription factor fusion oncoproteins. The majority of tumors express wild-type TP53, and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for TP53 wild-type Ewing sarcoma, scientists used a genome-scale CRISPR-Cas9 screening approach and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies.

New target for treating childhood cancer: SWI/SNF is a multi-component protein complex that plays an important role in chromatin remodeling. It is also likely an important tumor suppressor, as indicated by the fact that approximately 20% of human cancers carry a mutation in one or more SWI/SNF protein components. This SWI/SNF component protein is mutated in a number of cancers, including malignant rhabdoid tumor (MRT), a highly aggressive, nearly uniformly fatal childhood cancer. William Tansey and his laboratory found that cancers bearing an SNF5 mutation may be susceptible to therapy by MYC inhibition, providing yet another reason why the discovery of clinically viable MYC inhibitors is of utmost importance. 

The purpose of modern childhood cancer treatment is not only for the survival rate and the extension of survival time, but also to improve the quality of life. The principle of treatment of pediatric tumors has been raised to the integration of eradication of tumor, functional maintenance and mental health.

For the first two principles, childhood cancer patients are very different from adults, for they are at the stage of growth, thus the treatment plan must take the impact of treatment on the normal development of children into consideration. For example, the widespread use of radiation therapy in adults has been severely limited in childhood cancer treatment due to the risk of skeletal deformities, gonad damage, and intellectual effects in children. In the future, there will be anti-tumor drugs and treatment being more safe, effective, and less toxic, such as biological treatment, gene therapy, differentiation induction therapy to replace traditional chemotherapy and radiotherapy.

For mental health, on the basis of further improvement of tumor eradication and long-term survival, the mental health of children with tumors will be increasingly valued. Psychological problems, including children's self-experience, psychological effects, concerns about relapses and difficulties in participating in society, should be brought to the attention. Parents should be noticed that organ function and appearance reconstruction are also necessary for those who have suffered growth stagnation, and whose appearance is abnormal due to surgery and radiation therapy.

In short, the childhood cancer treatment is being improved, and under the premise of continuous increase of survival rate, children's long-term psychological and physical health, as well as functional maintenance must be considered when formulating treatment plans.

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Quantum dots, also known as semiconductor nanocrystals, are nanoscale materials composed of a small number of atoms. The number of atoms in a quantum dot is usually between a few and a few hundred, and their size in all three dimensions are less than 100 nm. The movement of carriers in the three dimensions of quantum dots is limited by the size effect. Due to the quantum confinement effect, the energy levels of carriers in quantum dots are similar to those of atoms having a discontinuous energy level structure, so quantum dots are also called artificial atoms. Due to their special energy level structure, quantum dots exhibit unique physical properties. This paper mainly discusses some properties of quantum dots, including quantum confinement effect, quantum size effect, surface effect and luminescence property.

1 Quantum confinement effect

Generally, the smaller the volume, the greater the bandwidth, so the optical and electrical properties of the quantum dots are highly dependent on the size of the material. Generally, when the size of the quantum dot is equal to or smaller than the exciton Bohr radius of the corresponding bulk material, the movement of the carrier electron-hole pair is in a strongly restricted state. When the energy gap increases as the particle size becomes smaller, the semiconductor material is quantified. The energy after quantification of the semiconductor material is: E(R)=Eg+h²π²/2uR²-1.8/εR. In the formula, Eg is the bulk band gap, u is the mass of electrons and holes, ε is the dielectric constant of the quantum dot material, R is the radius of the particle, and E(R) is the lowest excitation energy. The value obtained by subtracting Eg from E(R) is the amount of increase in kinetic energy.

2 Quantum size effect

It can be seen from the above formula that the quantum confinement energy and the coulomb interaction energy are proportional to 1/R2 and 1/R, respectively, the former can increase the band gap energy (blue shift), and the latter can reduce the band gap energy (red shift). When R is small, the quantum confinement can be more sensitive to R. As R decreases, the quantum confinement energy increases more than the Coulomb interaction energy, resulting in a blue shift of the spectrum.

3 Surface effect

Surface effect means that the specific surface area of quantum dots increases with the decrease of particle size, resulting in insufficient coordination of surface atoms and increased number of unsaturated bonds and dangling bonds, thus the atoms on the surface of quantum dots are extremely unstable and easily bind to other atoms. This surface effect gives the quantum dots a large surface energy and high activity, which not only causes changes in the atomic structure of the quantum surface, but also causes changes in the surface electron energy spectrum. Surface defects lead to trapped electrons or electron holes, which in turn affect the luminescent properties of quantum dots, causing nonlinear optical effects.

4 Luminescence property

The principle of luminescence of quantum dots is similar to that of conventional semiconductor luminescence, that is, carriers in a material reach an excited state after receiving external energy, and release energy when carriers return to the ground state, and this energy is usually released in the form of light. Unlike conventional luminescent materials, the luminescent materials of quantum dots have the following characteristics.

4.1 Adjustable emission spectrum

Semiconductor quantum dots are mainly composed of elements in IIB-VIA, IIIA-VA or IVA-VIA group. The luminescence spectra of quantum dots of different sizes or materials are in different bands. For example, the luminescence spectra of ZnS quantum dots covers the ultraviolet region, and the luminescence spectra of CdSe quantum dots covers the visible region, while the luminescence spectra of PbSe quantum dots covers the infrared region. Even for the same quantum dot material, the luminescence spectrum is different if the size is different.

4.2 Wide excitation spectrum and narrow emission spectrum

The range of the spectrum that triggers the quantum dot to reach the excited state is wide, and the quantum dot can be excited as long as the excitation light energy is higher than the threshold value. Regardless of the wavelength of the excitation light, as long as the material and size of the quantum dots are not changed, the emission spectrum of the quantum dots is fixed, and the emission spectrum range is narrow and symmetrical.

4.3 Large stokes movement

The peak of the emission spectrum of a quantum dot material is usually red-shifted relative to the peak of the absorption spectrum. The difference between the peak of the emission and absorption spectrum is called the Stokes shift. The Stokes shift of quantum dots is larger than conventional materials. Stokes shift is widely used in the detection of fluorescence spectral signals.

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“automatic litter box”的图片搜索结果

Changing the cat litter has got to be one of the most despised chores, at least at my house. It's like drawing the short straw and ending up with the chore no body wants. If you hate changing cat litter, you may be interested in how we solved this dilemma and stopped changing cat litter. No, we didn't get rid of our beloved cats or just let it go (that would be stinky!). We invested in an automatic litter box.

An automatic litter box is self cleaning unit that makes quick work of cat box cleanup. Typically, they work around the clock on a timer and scoop and store waste for later disposal. Some models even go further and wash and disinfect themselves. This is great because waste doesn't have time to sit and develop the bacterial growth that gives it that funky smell.

There are quite a few different brands and types of automatic litter boxes. Some even bear quite the resemblance to a human potty, believe it or not. When choosing a self cleaning cat litter box, you should consider the type of litter the box uses. Many units require that you purchase special litter while others use ordinary clumping litter. Still others operate with preferment granules that never need changing. Be careful when you select a box because the type of litter can have a major impact on the overall lifetime cost of running the unit.

Another thing to keep in mind about these self cleaning boxes is that it may take kitty some time to adapt to the unit. Some make a certain degree of noise while they are in their cleaning mode which can be a put off to more skittish kitties. Our cats took their time adjusting but after a few days they had no problem using their new box.

If you would like to stop changing cat litter, an automatic cat litter box is a great investment. This once avoided chore now seems not so bad and even the kids don't mind being tasked with it. Now there's a bonus! Another big benefit is that it always stays fresh, even after a long day when nobody is home to monitor the litter box.

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Whether the tactical operation is one for the military, SWAT, or law enforcement, footwear is key to ensuring that the mission - not foot discomfort or inadequate tactical boot performance - is the foremost consideration when mental focus is critical. There are many options in footwear for uniformed officials and with those options come the responsibility of determining which shoes or boots are best suited to specific work situations.

Considerations

When selecting footwear, first consider the type of operations performed. By considering the job duties and exposures to elements and other situations, an overwhelming selection of footwear can be narrowed down to a few prime selections:

• Is the typical work scenario one within a command station or behind a desk, on patrol in vehicles, and involving very little walking? If the answer is affirmative here, low cut shoes are most suited to this work environment. Low cut shoes without ankle support allow more flexing at the ankle, such as when sitting behind a desk or at the wheel of a car for prolonged periods of time. Patrol officers will likely find low-cut shoes suited to their beat, too.

• Does the job involve primarily walking, light running, and sudden movements during situations of physical confrontation? If so, mid-cut footwear provides ankle support, comfort, and help prevent injuries such as sprains.

• Is there off-pavement hiking, running, a high degree of physical confrontation, kicking and other more extreme action? In these instances, high cut boots are optimum, such as those issued to military soldiers. They are not often considered as comfortable as lower cut versions, but really protect the ankles and lower legs when it counts.

As part of missions, uniforms and footwear may come into play during the time required for response, recall or activation. If split-second dressing is required, for example, fumbling with small eyelets and laces may prove cumbersome and possibly even dangerous. Consider the dressing phase and whether arriving at the duty station occurs in a full state of dress, or if there are times when dressing in seconds arise as highly important.

Materials of footwear composition also come into play during tactical missions. Waterproof wear may be required or water-resistant coverage could be considered enough for the work detail. Plastic may melt in extreme heat, such as fires or direct sunlight. Metal may be reflective and give away position or alert subjects to positioning. Metals may also easily tear into laces or other parts of the tactical boots. Non-slip soles may be necessary, although some might make too much noise on polished floors. Ensure soles and heels will endure and are specifically designed to suit the surfaces on which the majority of the job's duties are performed.

On the inside of the shoes or tactical boots, determine whether extra inserts are needed for the expected level of interior wear and tear and to prevent blistering. If padding or extra support is needed, either seek footwear which provides that or seek gel inserts or other optional cushioning to customize fit. Toes may need to be protected by steel to prevent broken bones during combat maneuvers, in the event of physical confrontations, kicking, or items being dropped onto feet.

Finally, because of the importance of foot health during tactical operations, gaining a podiatrist's consultation may help in selection of a tactical boot or service shoe which will prevent or alleviate problems and pain associated with back, knee, foot, or other injuries. When in doubt about the decision to be made, perhaps the best resources for advice are the other officers or soldiers within the unit.

Tactical World Store is a company specializing in selling professional tactical gear. We dedicated to provide the highest quality Tactical Footwear (Mainly including tactical boots, military combat boots and tactical work shoes) for law enforcement, military, security, fire, EMS, postal, public transportation.

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If you're a cat owner, you'll know that above all they are clean animals. And if your cat is healthy, the cat's natural independence means that they require far less tending to than most pets. Undoubtedly the major chore for cat owners is keeping their cat tray clean. There will be the inevitable cat odors lingering in the atmosphere as you near changing time.

So what's the solution? An increasingly popular remedy is the self cleaning litter box. You'll notice immediately that it is larger than average, but it's the hidden functions that make the difference. A motion detector sensor is instantly set off when the cat enters inside. A few minutes later, the cat waste is pulled into a clump by an electronic rake and moved to a secure closed box or bag. This tray will still need changing, but the odor will be low to non existent and the changing time is a lot quicker and less messy. Some automatic cat litter boxes even have hourly cleaning rotations to ensure maximum hygiene.

Let your cat get used to the new tray gradually. Your cat may be initially worried about the cleaning sound that the machine makes. However, it isn't loud, it's more of a humming sound so your cat should soon get used to it. Try to reward your cat for successful use.

Most automatic litter boxes have easy to follow set up instructions. Remember to set it up in a quiet place that your cat is comfortable in. The clay litter that they use is reasonably priced. Or, if you want to be green as well as clean, use eco friendly crystals instead. Either way you'll find that the self cleaning litter box will make your cat's life a lot easier, and yours too.

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According to a recently published report in Nature Communications, scientists from Duke University Medical Center and other institutions revealed how stem cell mutations quietly occur and spread to a wide range of areas in the colon until they eventually dominate and develop into malignant tumors. By using an innovative model system in mice, researchers can visually mark colon cancer mutations by promoting stem cell luminescence, and then they can observe mutations that occur in colon cancer in animals and shed light on the intestinal tract. And it can clarify a process from death to death that occurs in the intestine. One mutation will overcome the other mutation and eventually become the driving force for malignant tumors.

 

Researcher Joshua Snyder said that this study reveals a process we have not observed before: how mutant precancerous stem cells spread in the colon and sow the seeds of cancer. The technology we use lays a solid foundation for testing new therapies, and these therapies will eventually effectively block early, precancerous processes; researchers hope that one day they can target and clear these precancerous cells to effectively prevent cancer.

 

In the article, the researchers used a molecular staining technique that can mark multiple common colon cancer mutations in stem cells from a single tumor, resulting in a fluorescent barcode. When transferred to mice, the fluorescent stem cells can be effectively tracked, revealing the cellular and molecular dynamics of precancerous events. Researchers have found a key difference in the generation of precancerous mutant cells in the intestinal dwelling tissues of infants and adults. At critical times, newborns are particularly sensitive to the mutational effects of intestinal stem cells, which will unknowingly seed a large number of precancerous mutations in their intestines (a process known as regional canceration), thereby increasing the risk of neonatal disease. These mutated cells will grow and spread, which cannot be detected by current screening techniques. Of course, these cells are generally harmless, but if appropriate, they can develop into cancer soon after adulthood.

 

Researchers have pointed out that certain colon cancer mutations in the patient's body can cause an increase in "fertility" in the surrounding environment of the precancerous area, eventually leading to the rapid spread of cells in the intestine and causing fatal consequences. Certain common mutations caused by external sources (such as injury or environmental exposure) can interfere with the environment surrounding stem cells and cause rapid growth and spread of cells in the precancerous area. These conditions are particularly deadly to adults and occur much faster than previously expected, as if throwing matches in an arid forest.

 

Researcher Snyder said that field cancerization is considered to be the decisive event initiating the growth process of cancer, including breast cancer, skin cancer, and lung cancer. How cells compete and expand in a field, which promises to help develop technologies and novel therapies for early diagnosis. Researchers are currently conducting other studies to use fluorescent barcodes to observe cancerous fields in breast cancer. They are aiming to clarify whether precancerous lesions such as ductal carcinoma in situ are caused by malignant or benign mutations.

 

Creative Biostructure, founded in 2005, is specialized in providing cost-effective contract services to both academia and biotech/pharmaceutical industries in the field of structural biology and membrane protein technologies.

We have developed all-in-one, gene-to-structure pipelines for the structure determination of macromolecules of your interest. With a team of experienced professionals, Creative Biostructure is able to solve the structure of many challenging proteins including GPCRsion channels, transporters, enzymes and viral targets. We also provide a comprehensive list of products and other related services to facilitate your research in structural biology.

Creative Biostructure has also built up a unique and comprehensive Membrane Protein Screening Platform. Aiming at elucidating the fundamentals of membrane protein systems, we provide gene-to-structure services on the purification, crystallization, structure determination and analysis of various membrane proteins.

 

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Nanomaterial is a kind of material with special properties that at least one dimension in three-dimensional space is in the order of nanometer (1~100 nm), or is composed of nanostructure units. It is known as "one of the most important strategic high-tech materials in the 21st century". Due to its special structure and extremely unstable thermodynamics, nanomaterials have many special properties such as small size effect, surface effect, quantum size effect and macroscopic quantum tunnel effect, as well as many physical and chemical properties that traditional materials do not have, such as high chemical activity, strong adsorption, special catalysis, special optical properties, special electromagnetic properties, hydrogen storage properties, etc., so they are widely used in medicine, manufacturing, materials, communications, biology, environment, energy, food and other fields.

 

Characterization and testing technology is the fundamental way to scientifically identify nanomaterials, recognize their diverse structures, and evaluate their special properties. The main purpose of characterization of nanomaterials is to determine some physicochemical properties of nanomaterials such as morphology, size, particle size, chemical composition, crystal structure, band gap, and absorption characteristics.

 

The characterization of nanomaterials can be divided into the following parts:

 

(1) Morphological characterization: TEM, SEM, AFM;

(2) Component analysis: AAS, ICP-AES, XPS, EDS;

(3) Structural characterization: XRD, ED, FT-IR, Raman, DLS;

(4) Characterization of properties: optical, electrical, magnetic, thermal, force.

 

Some common testing methods.

  1. Scanning electron microscopy (SEM)

SEM mainly uses the secondary electron signal imaging to observe the surface morphology of the sample, that is, using a very narrow electron beam to scan the sample and produce various effects through the interaction of the electron beam with the sample. Secondary electrons can produce a magnified image of the sample surface, which is established chronologically as the sample is scanned, i.e., using point-by-point imaging to obtain a magnified image.

 

SEM can acquire the information of various physical and chemical properties of the tested sample itself, such as morphology, composition, crystal structure and electronic structure. SEM is also a conventional instrument for characterizing the morphology, particle size, and size of nanomaterials, which are generally used in the literature of nanomaterials. In addition, SEM is usually equipped with EDS, which is used to analyze the elemental composition and the proportion of the material.

 

  1. Transmission electron microscopy (TEM)

TEM is the projection of an accelerated and aggregated electron beam onto a very thin sample, where electrons collide with atoms in the sample and change direction, resulting in solid angle scattering. The magnitude of the scattering angle is related to the density and thickness of the sample, so that different light and dark images can be formed. TEM with a resolution of 0.1 to 0.2 nm and a magnification of tens of thousands to millions of times was used to observe the ultrastructure, i.e., structures less than 0.2 microns and invisible under a light microscope. TEM is a conventional instrument for characterizing the morphology, particle size, and size of nanomaterials, which are generally used in the literature of nanomaterials.

 

In general, TEM is equipped with High-Resolution TEM, EDX (Energy Dispersive X-ray Spectroscopy) and SAED (Selected Area Electron Diffraction). High-resolution TEM is used to observe the crystal plane parameters of nanomaterials and deduce the crystal form of nanomaterials; EDX is generally used to analyze the elements contained in the sample, as well as the ratio of elements occupied; SAED is used to realize the in situ analysis of the morphological characteristics and crystallographic properties of crystalline samples.

 

  1. Atomic force microscopy (AFM)

AFM is an analytical instrument that can be used to study the surface structure of solid materials, including insulators. It studies the surface structure and properties of a substance by measuring the very weak interatomic force between the surface of the sample to be measured and a micro-force sensitive element.

 

The advantage of AFM is that the sample surface can be observed at high magnification under atmospheric conditions and can be used for almost all samples (there is a certain requirement for surface finish) without the need for other sample preparation to obtain a three-dimensional topography image of the sample surface.

 

  1. X-ray Diffraction (XRD)

XRD is a research method to analyze the diffraction pattern of a material by means of X-ray diffraction and obtain information on the composition of the material, the crystalline structure of the material, the structure or morphology of atoms or molecules inside the material.

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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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