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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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The pathogen that causes new coronary pneumonia is a new type of coronavirus, which is closely related to the previously familiar severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Infected patients will have fever, fatigue, and dry cough as the main clinical manifestations. In severe cases, acute respiratory distress syndrome will progress rapidly, or even die. So far, no specific drugs and vaccines have been approved for marketing.

After the new type of coronavirus (SARS-CoV-2) invades host cells, it begins to replicate in large numbers. The two processes of transcription and replication of the genetic material RNA genome are the core. The transcription of genetic material will finally be translated into the structural constituent protein of the new virus, and its replication will form the RNA genome of the new virus. RNA-dependent RNA polymerases (RdRp), also known as non-structural protein 12 (nsp12), can be assembled with multiple other non-structural proteins to form an efficient RNA synthesis "machine". As the core component of this transcriptional replication machine, RNA polymerase is one of the most important antiviral drug targets. Disrupting its function is expected to prevent virus replication and ultimately achieve the goal of treatment. The research on the drug targets of the new coronavirus, especially RNA polymerase, is very important for the research and development of such targeted drugs and the verification of the pharmacodynamic mechanism.

The structure of the complex analyzed shows that the RNA polymerase of the new coronavirus has the conservative characteristics of other viral RNA polymerases, and contains the NiRAN (Nidovirus RdRp-associated nucleotidyltransferase) characteristic domain of the Nidovirus; at the same time, viral RNA polymerization enzymes and virus non-structural proteins nsp7 and nsp8 constitute the core unit of transcription and replication machinery. Excitingly, the researchers also discovered for the first time a unique "β hairpin" domain at the N-terminus of the RNA polymerase of the new coronavirus. The discovery of this domain is to clarify the biology of the new coronavirus RNA polymerase. Through in-depth analysis of the atomic resolution structure, the research team discovered the key amino acid residues for the function of the new coronavirus RNA polymerase, and through the "hepatitis C virus polymerase ns5b-Sofosbuvir" effect. The structure of the “molecular” complex was compared, and the possible mode of action of the effector molecules (that is, the final product after metabolism) of remdesivir and fapilavir in inhibiting the new coronavirus RNA polymerase was proposed.

This study is the first to finely describe the internal structure of the transcription and replication machinery of the new coronavirus "RdRp-nsp7-nsp8", and shows how the effector molecules of drug candidates such as Radixivir and fapilavir can precisely target and inhibit viral RNA synthesis. Furthermore, a reasonable mechanism explanation was put forward for exerting pharmacodynamic activity, which laid an important theoretical foundation for the in-depth study of the molecular mechanism of the new coronavirus replication, and opened up a new way for the development of specific drugs against new coronary pneumonia.

In addition, the "Anti-Coronavirus Research Alliance" formed by the research team of Shanghai University of Science and Technology and its collaborators also jointly published the important research results of the new coronavirus on Nature "Structure of Mpro from COVID-19 virus and discovery of its inhibitors" "It is the first to successfully analyze the high-resolution three-dimensional structure of the main protease (Mpro), the key drug target of the new coronavirus, and to use three different drug discovery strategies to find inhibitors against the new coronavirus.

In the research strategy designed from scratch, the "Alliance" found that the Michael receptor N3 is a potent inhibitor of the main protease, and was the first to analyze the 2.1Å high-resolution complex structure of the main protease-N3 (and later Increase to 1.7Å), which is also the world's first three-dimensional structure of the new coronavirus protein to be resolved. In order to facilitate relevant scientific and technological workers to develop antiviral drugs targeting this enzyme as soon as possible, the "Alliance" for the key research issue published the research results for the first time and published the structure in the Protein Data Bank (PDB). Since January 26, the team has directly provided data to the laboratories of more than 300 universities, research institutions and enterprises. This structure was selected as the February Molecule of the Month by PDB protein structure database, and was reported by PDB.

Since then, the "Alliance" has continued to jointly use virtual screening and high-throughput screening strategies to screen more than 10,000 old drugs, clinical drugs, and natural active products, and found several species that have significant inhibitory effects on the main protease. Lead drugs, including disulfiram, carmofur, ebselen, shikonin, Tideglusib and PX-12. Subsequent anti-coronavirus experiments showed that both ebselen and N3 can significantly inhibit the replication of the neocoronavirus at the cellular level. It is worth mentioning that ebselen has been used in clinical trials for the treatment of various diseases such as hearing impairment (completed the second clinical phase), and has a good safety performance. The above-mentioned research results have laid an important foundation for the rapid development of anti-coronary pneumonia drugs with clinical potential.

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Abstract: Similar to SARS-CoV, SARS-CoV-2 is also a family member of coronavirus. But why is it leading to such a serious situation and when can we get rid of it? This article will explain the details about the development and future trends of SARS-CoV-2.

 

 

As of July 1, 2020, the number of people diagnosed with COVID-19 worldwide has reached 11,669,259, and the death toll is as high as 539,906. On the frontal battlefield, there are still many doctors and nurses around the world struggling and working hard to save more lives.

You may still remember the SARS epidemic caused by SARS-CoV in 2013. The virus started in Guangdong, China and a total of 5327 clinically diagnosed cases and 349 deaths have been reported in Mainland China. It also belongs to coronavirus, but why is this novel coronavirus caused by SARS-CoV-2 more serious than the similar virus of 2013?

Coronavirus is a kind of pathogen that mainly causes respiratory and intestinal diseases, which mainly comes from domestic animals, poultry and wild animals (such as civet cats, bats). SARS-CoV can cause infection of the lower respiratory tract and induce pneumonia. The onset is rapid and highly contagious, with fever as the first symptom, with chills, headache, muscle aches, general fatigue and diarrhea gradually developed. Patients who are seriously ill may suffer from respiratory distress syndrome.

The novel coronavirus, SARS-CoV-2, will infect the upper and lower respiratory tracts at the same time, which also allows it to spread like flu. A difference is that after the first symptoms of the patient, there are a large amount of viruses in the nose and throat of the upper respiratory tract, and the virus level in the nasal cavity is higher.

At present, the most difficult challenge is those who are infected without symptoms. Although they do not show any symptoms, the virus levels in their bodies are comparable to those of symptomatic patients. Therefore, they not only have hidden characteristics, but also have the ability to be contagious, which makes it difficult to control the epidemic. In addition, SARS-CoV-2 has the characteristics of strong contagion, high population susceptibility, long incubation period, and diversified clinical manifestations, posing a huge threat to people's health and seriously affecting people's normal work and life.

Experts of respiratory diseases said that the pneumonia caused by SARS-CoV-2 is likely to turn into a chronic, long-term human disease like the flu. As SARS-CoV is highly transmissible and pathogenic with the main attack organ the lungs, it is not easy to survive for a long time, and it cannot continue to spread. However, SARS-CoV-2 can also attack the kidneys, heart and other organs, thus is more difficult to cure.

Scientists from Creative Biolabs, a CRO service provider once did a lot of research on SARS-CoV and MERS said, "We still know very little about SARS-CoV-2, but every study is a small piece of this huge virus puzzle."

One thing is clear, protection against infections will most likely not stop the virus from spreading, but it will slow its spread. Many countries are currently conducting many antiviral drug tests for patients infected by SARS-CoV-2. And the vaccine may be available by next fall. Therefore, the more we slow down the spread, the better we can deal with this virus.

In this long historical process, vaccination can be said to be the only more effective preventive measure. During the influenza epidemic season, comprehensive measures such as influenza vaccination are taken to prevent it, which has shown being very effective in controlling the influenza epidemic.

In response to SARS-CoV-2, more and more vaccine candidates are currently undergoing evaluation and clinical trials worldwide and this number will increase substantially in the future.  What distinguishes humans from other creatures is that humans have sufficient wisdom, courage, and perseverance. It’s believed that with global efforts, a safe and effective vaccine can be available as soon as possible.

 

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The early success rate of establishing the PDX model is extremely low, which is largely due to the rejection of the transplanted material by the host immune system. Although methods such as X-ray irradiation and thymectomy have been used to suppress the host's immune system, the development of the PDX model has largely benefited from the production of immunodeficient host mice.

The first breakthrough was the development of nude mice. In 1962, Scottish doctor Issacso and others first discovered individual hairless mice in non-inbred albino mice, but did not conduct an in-depth analysis of them. In 1966, Flanagan from the Institute of Animal Genetics in Edinburgh confirmed that this hairless mouse was caused by allelic mutations on the chromosomes. He believed that it was a new spontaneous mutant and named it "Nude" mouse. Flanagan also found that female nude mice had extremely low fertility and could not raise their offspring due to poor breast development, but unfortunately he did not find the most important feature of nude mice, athymic glands. In 1968, Pantelouris from the University of Strathclyde in Scotland discovered athymic glands in nude mice obtained from the Institute of Animal Genetics in Edinburgh, which aroused great interest among medical biologists around the world. Nude mice are congenital hairless, athymic, and completely lacking in T lymphocyte function, and do not produce rejection response to xenotransplantation. Therefore, they are especially suitable for xenotransplantation of xenogeneic animal tissues and human tumor xenotransplantation, and are expected to replace thymectomy mice.

In 1969, Danish scholar Rygaard successfully transplanted human colon cancer tumors into three nude mice for the first time. Six days after tumor transplantation, tumor tissue was found at the transplanted site. On the 40th day of tumor transplantation, the original 2×2×2mm tumor tissue had grown to 10×20×25mm in two of the nude mice, and 5×5×20mm in the other nude mouse. No signs of tumor growth were found in normal mice in the control group. As a result, scholars have established many PDX tumor models by transplanting various types of tumor tissues into nude mice.

In 1983, Bosma, GC and others discovered a severe combined immunodeficiency CB17-SCID mouse, which lacks mature T cells and B cells, but the innate immune system retained in the body such as normal NK cells is not good for humans. Long-term colonization of source cells.

In 1995, Jackson Lab used non-obese diabetic mice NOD/Lt to cross with SCID mice, and NOD-SCID mice were produced. This hybrid breed inhibits the activity of NK cells and is accompanied by defects in the innate immune system. Therefore, it is an animal model with more severe immunodeficiency, easier xenotransplantation success and stable application. However, NOD-SCID develops thymoma in an average of 8.5 months, so long-term models are not suitable.

In 2002, the Japan Institute of Experimental Animals (CIEA) bred NOG mice. They successfully established a severely insufficiency immune system by crossing NOD/scid mice and γ-chain IL-2 receptor knockout (IL2rγKO) mice. Compared with NOD-scid mice, the survival rate of human cell and tissue transplantation in NOG mice is significantly improved. At the same time, it can implant a higher proportion of normal or cancerous human cells and tissues.

In 2005, Jackson Lab in the United States developed NOD-scid gamma (NSG) mice. Due to genetic mutations, these mice have defects in T and B lymphocytes and the gamma chain gene of IL2 receptor, which has important immunoregulatory functions, was knocked out. In addition, NK cells also lose their function and can be used to transplant human cells and tissues without causing traditional immune responses. They are currently the most ideal recipient mice for humanized tissue transplantation.

Subsequently, some scholars transplanted human hematopoietic stem cells (HSC) into these mice to form humanized hematopoietic and immune system reconstruction mice, which proved to be very effective human-derived mice. In addition, there are PBMC models in which human peripheral blood mononuclear cells are directly injected into severe immunodeficiency mice for immune reconstitution; human embryonic liver and thymus are co-transplanted under the kidney capsule of immunodeficiency mice, and at the same time BLT models are established by injecting hepatic hematopoietic stem cells from the same embryo into mice. These models are also widely used in the research and construction of PDX models and are effective tools for studying human diseases.

In recent decades, with the widespread use of immunodeficient mice, a large number of PDX tumor models have been successfully established. These models retain the histopathology, molecular characteristics and drug response of their parent tumors, can reproduce tumor heterogeneity that cell line systems have not captured, and show potential clinical trial response prediction capabilities at the population level. In view of this, the PDX model provides a powerful tool and has been widely used to study cancer biology and assist in the preclinical setting of personalized cancer treatment and drug screening.

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Antibody-drug conjugate (ADC) is a humanized or human monoclonal antibody that is conjugated to a highly cytotoxic small molecule (payload) through a chemical linker. It is a novel form of treatment and is used in cancer chemotherapy. This new antibody-based molecular platform can selectively deliver effective cytotoxic payloads to target cancer cells compared with traditional chemotherapy, thereby improving efficacy, reducing systemic toxicity, and having better pharmacokinetics (PK)/Pharmacodynamics (PD) and bio-distribution.

Driven by the success of Adcetris® and Kadcyla® approved by the FDA, this drug class is developing rapidly along with approximately 60 ADCs currently in clinical trials. In this article, we briefly reviewed the molecular aspects of each ADC component (antibody, payload, and linker), and then mainly discussed traditional and new technologies for successfully constructing clinically effective ADC coupling and linker chemistry. From the point of view of medicinal chemistry and pharmacology, current efforts in conjugation and connection chemistry will provide deeper insights into the molecular design and strategies of clinically effective ADCs. The development of site-specific conjugation methods for the construction of homogeneous ADCs, especially ways to improve ADC design will open the way for new cancer treatment methods.

An introduction

In the past half century, with the development of chemotherapy, the treatment of cancer has improved significantly. In addition to surgical resection, radiotherapy, targeted therapy using small molecules or monoclonal antibodies, and recent immunotherapy, chemotherapy using cytotoxic agents is the main treatment option. Chemotherapy is perfected by screening and developing small molecules that can selectively cause cancer cell death by inhibiting microtubule function, DNA synthesis or protein function. Although chemotherapy has achieved great success in the treatment of cancer, especially leukemia, there are still difficult problems, such as the development of drug resistance mechanisms. Serious adverse reactions caused by off-target cytotoxicity may worsen the patient’s quality of life, leading to drug withdrawal. This fact makes clinicians and medicinal chemists reluctant to pursue more potent cytotoxic drugs to treat cancer. In this case, the use of highly cytotoxic agents combined with cell targeting molecules has become a potential clinical strategy. In particular, antibody-drug conjugates (ADC), humanized or human monoclonal antibodies conjugated to cytotoxic small molecules via chemical linkers, may undergo fundamental changes in the design and administration of cancer chemotherapy. The platform can target cancer cells and selectively deliver highly cytotoxic drugs, thereby creating a broad therapeutic Indeed, successful clinical results using ADCs have inspired scientists in the biomedical research community to further advance this new platform to the next generation of cancer treatments. In this article, we reviewed the molecular aspects of ADCs, the latest developments in ADCs that have been successfully used in clinical applications, and the coupling and connection technologies used to successfully construct ADCs.

A brief history

In 1913, German physician and scientist Paul Ehrlich first proposed the concept of selectively delivering toxic drugs to target cells. Forty-five years later, his targeted therapy concept was proved for the first time in the form of ADC, where methotrexate was conjugated with a leukemia cell targeting antibody. In early research, polyclonal antibodies were the main targeting agent. In 1983, the first use of anti-carcinoembryonic antigen antibody-vinblastine conjugate for ADC human clinical trials, and reported promising results. Advances in antibody engineering technology, including the production of humanized antibodies, have promoted ADC research. The first-generation ADC consisting of chimeric or humanized antibodies was tested in the 1990s. Finally, further major efforts on practical therapies led to the FDA-approved ADC: Gimumab ozogamicin (Mylotarg®) for CD33-positive acute myeloid leukemia in 2000, and for CD30-positive relapsed or refractory Hodgkin’s lymphoma in 2011 brentuximab vedotin (Adcetris®). Anaplastic large cell lymphoma and trastuzumab Emtansine (Kadcyla®) were used in HER2-positive breast cancer in 2013. However, Mylotarg® was withdrawn from the market in 2010 due to lack of clinical benefit and lethal high toxicity compared to standard chemotherapy. Despite this setback, ADC technology is still developing rapidly, and about 60 ADCs are currently in clinical trials. In addition to immunotherapy using checkpoint inhibitors, this emerging chemotherapeutic molecular platform is expected to significantly increase its market share as one of its largest markets. The most effective anti-cancer therapy in the near future.

The structure and mechanism of ADC

Another important consideration is the limited number of payload molecules that can be effectively delivered to target cells. If it is assumed that the efficiency of each step in the ADC mechanism is 50% (biodistribution, binding to antigen, internalization, release of payload, intracellular stability of payload, and binding of payload to target), then only 1.56% administered drug molecules can enter the target cell. In fact, the estimated actual intake is much lower than this assumption (less than 0.01% per gram of tumor injection). Therefore, in order to maximize the therapeutic efficiency of using ADC, the cytotoxicity of the payload must be high enough to effectively eradicate target cells, and ideally should be in the picomolar range. It is very important to choose important toxic drugs as payloads, and ideal drugs have inherent selectivity to target cancer cells. Certain types of non-cancer cells may be able to internalize ADC through non-specific pinocytosis or crystallizable fragmentation (Fc) region receptor-mediated endocytosis. In addition, the payload can be released when it degrades into circulating blood. Therefore, the payload is mainly selected based on the above considerations; anti-mitotic agents are usually less toxic to non-cancer cells than to cancer cells, and are payloads that are mainly used in FDA-approved ADC and ADC clinical trials. In addition to calicheamicin (for Mylotarg®), auristatin (for Adcetris®) and maytansinoid (for Kadcyla®), a new type of highly effective anti-mitotic compound has been explored for ADC loading: Carmycin, pyrrolobenzodiazepine dimer (PBD) mannosides, mannosins, and tubulolysin analogs are such examples.

To be continued in Part II…

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CD Genomics, an innovative sequencing and genotyping company, recently announced the launch of its new website with brand new product and service ranges and easy-access custom design.

Gene Panel is an invaluable tool to analyze parallel gene expression for disease-associated mutations. Among them, next-generation sequencing (NGS) technologies are perhaps the most widely used approaches, which can analyze genetic mutations of large sample-size projects and facilitate research on human well-being. In the meantime, some non-NGS technologies may offer alternations for gene panel with a much more affordable price.

CD Genomics Disease Panel is a one-stop destination for kinds of sequencing and reagents needs. Their catalog features hundreds of products such as Ready-to-Use NGS Panel for oncology, genetic disorder, genotyping, pathogen infections, Custom Panel Product and Non-NGS Panel, which can promote researchers’ disease research, biomarker discovery, molecular diagnostic application and targeted drug development.

The newly launched website also features updated service descriptions and details, and a new Resource page. The services range from Predesigned NGS Panel for cancer, inherited disease, pathogens and pharmacogenomics testing, Tumor Mutational Burden Analysis, Custom NGS Panel, to Non-NGS Panel, for example, MassARRAY, Multiplex SNaPshot, Multiplex Ligation-Dependent Probe Amplification and ARMS-PCR for Sequence Variation Analysis. Moreover, customers searching for a new addition to the backyard can decide between the listed items and others.

“CD Genomics can quickly respond to customers’ primers design or probes determination requirements. The accumulation of each measurement ensures a shorter turnaround time. Their comprehensive quality control and verification ensure the accuracy of results. I’d like to recommend the appropriate technology for gene-targeted detection to other researchers or institutions.” Said by a client, Jonson Smith.

The company has an Illumina platform for variant detection. Multiple disease-related genes and regions of interest can be detected in a single assay. CD Genomics is a professional sequencing company with extensive experience in detecting genetic mutations such as SNPs, indels, copy number variations, and DNA methylation.

About CD Genomics

CD Genomics enjoys a high reputation for sequencing, microarray analysis, library construction and genotyping, providing reliable services to pharmaceutical and biotechnology companies as well as academia and government agencies. Based on rich experience in targeted sequencing, CD Genomics has developed a specialized platform for targeted sequencing of disease-related genes to accelerate research on disease pathogenesis, disease identification, biomarker discovery, targeted drug development, etc. CD Genomics offers predesigend NGS panels, which include a designed library of targeted sequencing, as well as a custom panel that allows customers to select genes of interest to design their own sequencing panels.

 

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In 1991, Lynne Burek published a brief commentary in the 75th anniversary issue of the American Journal of Epidemiology entitled, “The Interaction of Basic Science and Population-Based Research: Autoimmune Thyroiditis as a Case History” (1). He traced the pathways that led us from fundamental investigations in the laboratory to population studies designed to determine the factors that predict the risk of autoimmune thyroid disease before the onset of diagnostic clinical signs. In the present article, He will especially comment on the changes in the field that mark the transition into the 21st century, which reflect a combination of broadly based systematic research.

The autoimmune diseases, which collectively affect more than 20 million Americans, are clinically diverse but share a fundamental etiology: a self-reactive adaptive immune response. By definition, these diseases are distinguished by the presence of B cells and T cells that recognize antigens present in the body of the host, in the form of self-reactive antibodies or T cells. In recent years, a related group of diseases has been described as sharing many of the same inflammatory mediators but lacking self-reactive lymphocytes, which are often to referred as “auto-inflammatory responses,” and these disorders usually have a clear genetic component and evidence of activation of innate immune system. According to epidemiologic studies, the incidence rates of autoimmune disorders is increasing in industrialized countries, which probably reflects some environmental changes. In addition, greater attention has been directed to these diseases because of improved diagnostic procedures and therapeutic interventions. However, for any of them, there is essentially no definitive cure. A common feature of most autoimmune diseases, is that they develop gradually, so serious tissue damage might occur before the clinical diagnosis diseases. This observation strengthens the efforts to diagnose autoimmune diseases earlier in their courses before irreversible damage occurs. These studies are interdisciplinary in nature and require the combined efforts of immunologists, epidemiologists, environmental scientists, geneticists, and clinicians. In the past decades, such collaborative teams have conducted many researches on autoimmune diseases.

The primary early risk factors for induction of an autoimmune response have been identified in the genes of the major histocompatibility complex (MHC) (2). Heightened susceptibility depends on potent activation of self-antigen–specific T lymphocytes by antigen-presenting cells bearing the same MHC. In sub-sequent studies of thyroiditis in humans, an autoimmune response to particular epitopes of the thyroglobulin molecule predicted progression of disease. Other genes, inside and outside of the MHC, modify the initial autoimmune response. These immune-regulatory genes are often expressed through particular cytokines and other inflammatory media- tors and enhance or limit the disease of genetically susceptible animals selected on the basis of their MHC haplotype (3). Beyond these genetic risk factors, many internal variables can shift autoimmune responses to clinical disease that are less likely to occur. These variables include such factors as gender, age, pregnancy, and even neurologic and emotional signals. Combined with genetic factors, characteristic autoimmune antibodies are the best predictors of impending autoimmune diseases. On the basis of the earlier decades of fundamental research, the practical application of this knowledge in human susceptibility predictors is described. Because of the high genetic susceptibility of thyroiditis in their family members, children with high genetic predisposition to thyroiditis were studied, and the onset of autoantibody formation and occurrence of clinical signs in normal children were tracked. Through years of follow-up research, children with a high risk of autoimmune thyroiditis can be identified. For example, among the 19 siblings who share the MHC haplotypes with a brother or sister who has been clinically diagnosed with autoimmune thyroiditis, 17 (89%) have thyroid antibodies, and 6 of these 17 (35%) showed biochemical or physical evidence of thyroid dysfunction during the first decade of observation. Since the beginning of this study, researchers have revisited these children in disease- susceptible families on several occasions and have proposed a gradual “natural history” of autoimmune thyroiditis. The step 1 in the "natural history" begins with a combination of genetic traits, and then the step 2 is environmental exposure, such as excessive dietary iodine (5). Step 3 is marked by the production of characteristic autoantibodies to specific determinants of thyroglobulin, as well as the production of antibodies against additional thyroid-specific antigens, such as thyroid peroxidase. In step 4, subclinical thyroiditis is evidenced by a decrease in thyroxine levels. Then in step 5, thyroid-stimulating hormone is compensatoryly increased, and thyroxine is rised to normal level. Finally, step 6 is characterized by overt hypothyroidism in which the thyroxine level cannot rise, the level of thyroid-stimulating hormone is increased, and there is pathologic and clinical evidence of thyroid goiter or an atrophic thyroid gland. Because this series of steps can take many months or years, there is an opportunity to identify individuals at highest risk of developing autoimmune disorders by using genetic and functional markers and to intervene with preventive measures.

The Autoimmune disease model

  1. Childhood Asthma

Asthma is a chronic airway inflammatory disease. Due to the persistence of this chronic inflammatory reaction, the airway is highly reactive, and the symptoms recur when exposed to the cause. The study of the pathogenesis of asthma has evolved from the theory of sputum to the theory of chronic inflammation of the airways, and has now developed into a parallel theory of smooth muscle dysfunction and airway inflammation. Clinical treatment can also be done by repeated treatment, with emphasis on anti-inflammatory and relief of smooth muscle spasm. In the 1950s, non-selective adrenaline was used as an antispasmodic agent to treat asthma. In 1956, a selective strong short-acting β2 agonist (Short Acting Beta 2 Agonist, SABA) was introduced. In 1971, a β2 agonist (Long Acting Beta2 Agonist, LABA) came out. Oral glucocorticoids were used in the 1960s to antagonize airway inflammation, which is effective but has many side effects. In 1972, beclomethasone dipropionate (BDP) was successfully developed. In the 1980s, budesonide (BUD) and fluticasone propionate (FP) were developed. By analogy, these inhaled corticosteroids have a stronger anti-inflammatory effect on the airway, and their side effects are significantly reduced.

When children are stimulated by allergens, cold air or other incentives, they often first manifest as symptoms of upper respiratory tract allergy, such as itchy eyes, itchy nose, sneezing, etc. Because infants and young children are difficult to express itching, their symptoms often only express as blinking, blowing nose, etc, and further manifest as itching, dry cough and expectoration. These symptoms usually last for hours or days before the onset of asthma. Sudden onset of wheezing is a major feature of childhood asthma. The wheezing symptoms of asthma in children vary greatly depending on the severity of asthma. Children may have high-pitched wheezing that can be heard without a stethoscope or at a distance. Due to frequent breathing or difficulty breathing, asthma symptoms in infants and young children can be expressed as mouth breathing and nose flapping, and many children may be accompanied by a cough. Under normal circumstances, dry cough will appear at the beginning of the disease. When the seizure subsides, a white mucus-like phlegm will be coughed out. In severe cases, the symptoms of asthma can be expressed as irritability, purpura, pale, and cold sweat. Physical examination revealed an increase in heart rate and wheezing in both lungs. Symptoms of heart failure may be further aggravated with jugular vein engorgement, edema, middle lung, small blisters, and enlarged liver. Signs of emphysema can be seen in children with chronic asthma, such as barrel chest and chest percussion. In the remission period, children with asthma may have no symptoms or signs, have no effect on activities, or only manifest as symptoms of allergic rhinitis and pharyngitis. A small number of children may have chest discomfort, with or without wheezing in the lungs.

To be continued in Part II…

 

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To develop drugs that target specific cell surface proteins, it is helpful to understand other proteins in its vicinity. The pathology of many diseases can be understood by elucidating local biomolecular networks or microenvironments. To this end, the enzymatic proximity labeling platform is widely used to map a wider range of spatial relationships in subcellular structures. However, there has long been a search for techniques that can map the microenvironment more precisely.

 

Geri et al describe a microenvironmental profiling platform that uses photocatalytic carbon ene generation to selectively identify protein interactions on cell membranes, a method called MicroMap (μMap). A light-triggered labeling technique that improves the spatial resolution of this type of mapping. Specifically, they relied on a photocatalyst with a very short range of energy transfer to activate a carbine-based tag that could only diffuse a small distance in water before the reaction.

 

Using photocatalytic-antibody conjugates to spatially localize carbene generation, they demonstrated selective labeling of antibody-bound targets and their microenvironment protein neighbors.

 

They used this technique to identify the constituent proteins of the programmed death ligand 1 (PD-L1) microenvironment in living lymphocytes and selectively label them in immune synaptic junctions. Therefore, it its a valuable system in cancer immunotherapy.

 

About author

 

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.

 

The company has developed all-in-one, gene-to-structure pipelines for the structure determination of macromolecules. 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. The company also provides a comprehensive list of products and other related services to facilitate 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, the company provides gene-to-structure services on the purification, crystallization, structure determination and analysis of various membrane proteins.

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Abstract: Since SARS-CoV-2 is similar to SARS-CoV, the research of 2003 that ACE2 is an essential receptor for SARS-CoV infection can be further explored for unveiling the secret of SARS-CoV-2.

An ongoing outbreak of a novel coronavirus (SARS-CoV-2) has raised global concerns. It is identified as the cause of pneumonia with unknown etiology. Since the early outbreak in Wuhan, China, it has subsequently spread to all provinces of China and many other countries. In fact, coronavirus is a relatively common human virus, which causes 10% of the common colds. However, some coronaviruses are more harmful, such as SARS-CoV and MERS- CoV and the novel coronavirus, named SARS-CoV-2.

How does SARS-CoV-2 infect patients?

Spike protein (including S1 and S2 subunits) is the most important pathogenic protein of coronavirus, which helps the virus to bind to the transmembrane receptor protein on the human cell membrane, thereby helping itself to enter the cell.

The latest research shows that the Spike proteins of SARS-CoV-2 and SARS-CoV are more conserved, and in vitro experiments have proved that as long as the cell expresses ACE2, SARS-CoV-2 can infect cells; on the contrary, if there is no ACE2 on the cell protein, it will not be infected. Therefore, it is believed that SARS-CoV-2 is mediated into the interior of cells through the binding of Spike proteins to ACE2 proteins, which may become a breakthrough in the study of SARS-CoV-2.

What is ACE2 protein?

The ACE2 gene is located on the X chromosome, which encodes a type I transmembrane glycoprotein with a single extracellular catalytic domain. ACE2 has a well-known homologous gene, ACE, which is also an angiotensin-converting enzyme. ACE and ACE2 both have two domains: the amino-terminal catalytic domain and the carboxy-terminal domain).

Despite similarities, ACE2 and ACE function differently. ACE's role is to convert angiotensin I (AngI) to active angiotensin II (AngII), thereby increasing hypertension. The role of ACE2 is to convert AngII to heptapeptide angiotensin 1-7 (Ang1-7), and then antagonize the blood pressure-increasing effect of AngII, which has a negative regulatory effect on the RAS system. Therefore, ACE2 functions completely differently from ACE and the two work together to balance blood pressure.

Lessons from SARS

Due to the function of ACE2, previous basic clinical studies have linked it with hypertension and cardiovascular disease, but in 2003, after ACE2 was identified as an essential receptor for SARS coronavirus infection, its research in this area has been carried out. And because the pathogenic mechanism of SARS-CoV-2 is highly similar to SARS of 2003, the current research of SARS is extremely informative.

In 2003, researchers identified ACE2 as a functional SARS-CoV receptor by co-immunoprecipitation technology. Subsequently, ACE2 was identified as an essential receptor for SARS infection in vivo in the ACE2-knockout mouse model. However, previous studies have found that ACE2-knockout mice with lung injury have a more severe acute injury and higher mortality than the wild-type mice, suggesting that ACE2 deficiency may worsen the symptoms of acute lung injury. Therefore, ACE2-mediated degradation of AngII is important for lung protection against the pathogenesis of pneumonia causing by SARS-CoV infection.

To unveil the secret of SARS-CoV-2, more research is needed, and at the same time, scientists worldwide are dedicated to finding methods of more accurate diagnosis and more efficient treatment.

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Despite the widespread use of radiological techniques in diagnosis and treatment, there is still no material that can protect people from radiation.

Recently, researchers at the Nanoparticle Research Center at the Korea Institute of Basic Science, in collaboration with colleagues at Seoul National University, the College of Dentistry, and the Institute of Dentistry, reported an efficient and safe nanocrystal that is effective against dangerous dose radiation.

 

By growing manganese oxide (Mn3O4) nanocrystals on cerium oxide (CeO2) nanocrystals, the group improved the catalytic activity of CeO2/Mn3O4 nanocrystals, thus avoiding the side effects of lethal radiation.

HYEON Taeghwan, director of the IBS Nanoparticle Research Center (Seoul National University), said: "Reactive oxygen species (ROS) are found in many major diseases, including sepsis, cancer, cardiovascular disease and Parkinson's disease. When our body is exposed to high levels of radiation, large amounts of ROS are generated within milliseconds due to the breakdown of water molecules. These ROS severely destroy cells and eventually lead to their death."

The research team used CeO2 and Mn3O4 nanoparticles for their excellent ROS scavenging ability. The challenge is how to use these antioxidant nanomaterials in a safe and economical way: although effective, CeO2 and Mn3O4 nanoparticles can only act to remove ROS at high doses, which is not only difficult to apply but also very wasteful.

In recent studies, researchers have borrowed from methods commonly employed in the field of catalysis: stacking nanoparticles with different lattice parameters results in surface strain and increases oxygen vacancies on the surface of nanocrystals.

"The synergistic effect of the strains produced on Mn3O4 and the increase in oxygen vacancies on the CeO2 surface improve the surface-binding affinity of ROS, thereby enhancing the catalytic activity of nanocrystals," said Han Shangen, lead author of this study. "Strain engineering of nanocrystals, which are mainly studied in the catalyst field, has now been extended to the medical field to protect cells from ROS-related diseases," said CHO Min Gee, co-first author of the study.

The team examined the safety and efficacy of this novel antioxidant nanocrystal. Molecular dynamics were analyzed using an acute radiation model of human intestinal organoids. "Genes expressed by organics pretreated with CeO2/Mn3O4 nanocrystals were associated with the proliferation and maintenance of intestinal stem cells compared with the group without pretreatment, while fewer genes were associated with cell death," explained Sang-woo Lee.

In a mouse study, with only a very small dose (1/360 of the amifostine injection dose), CeO2/Mn3O4 nanocrystals significantly increased the survival rate of animals to 67% and reduced oxidative stress to internal organs, circulatory system and bone marrow cells without obvious signs of toxicity.

"To ensure the safe and widespread use of radioprotectants in clinical practice, it is critical to maintain high catalytic efficacy at low doses." PARK Kyungpyo, a professor in the Department of Dentistry at Seoul University, said: "This CeO2/Mn3O4 nanocrystal demonstrates its strong antioxidant effect and effectively protects our entire body even in small doses."

 

About the Author

Collected by Matexcel. At Matexcel, many surface modification techniques have been developed so that our expertise in surface chemistry made post-synthesis functionalization possible, conveniently incorporating polymers, proteins, DNA, and antibodies to synthetic nanoparticles and other nanostructured surfaces.

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Carbomer is a homopolymer of acrylic acid, which is cross-linked or bonded to any of several polyol allyl ethers. The chemical formula of Carbomer is C3H4O2, and the compound is usually a white powder. And it can be dissolved in water, ethanol and glycerin. Carbomer's molecules contain 56% to 58% carboxyl groups, which are weakly acidic, so its aqueous solution should be used after neutralization with alkali to reduce its irritation to the skin and mucous membranes. When carbomer is dispersed in water, due to the repulsion between the negative charges generated by the ionization of the carboxyl group, the curled polymer stretches out and expands in volume. The 1% carbomer aqueous dispersion can be neutralized with an alkaline substance to form a gel. The concentration of the commonly used carbomer aqueous dispersion is 0.1% to 3.0%.

 

Carbomer is well known for its use in the cosmetics industry and personal care industry, and it is often used as a thickener and emulsion stabilizer, mainly to help control the viscosity and flow of cosmetics. It helps to disperse and suspend insoluble solids in the liquid and prevents the oil and liquid parts of the solution from separating. Carbomer has the ability to absorb and retain moisture, and when swelled in water it can swell to 1000 times its original volume.

 

Carbomer series products (Carbomer 934, Carbomer 940, Carbomer 941, Carbomer 980) are all similar in chemical properties, but their molecular weight and viscosity are different from each other. The codes of the Carbomer series products (ie 910, 934, 940, 941 and 934P) indicate the molecular weight and specific components of the polymer. Powder carbomer is commonly used in skin care products and cosmetics, while liquid carbomer is mainly used in cleaning products. At present, powder carbomer is generally added to disinfection and sterilization products on the market.

 

In the past few years, Carbomer's global market has developed rapidly, with an average growth rate of 11.98% from 2013 to 2017. In 2018, the global carbomer market was valued at US$722.9 million and is expected to reach US$17.951 billion by 2028, with a compound annual growth rate of 9.3%. Carbomer's series products include Carbomer 940, Carbomer 980, Carbomer 934, etc. In 2018, Carbomer 940's market share is about 37%. Carbomer is widely used in the pharmaceutical, personal care and cosmetic industries. Carbomer is most used in the personal care and cosmetics industries, with a proportion of about 54%.

 

Carbomer is commonly used as a thickener, dispersant, suspending agent and emulsifier in cosmetics and personal care products. By adding carbomer to shampoo, conditioner, cream and lotion, these makeup and cleansing products will become smoother. It can also be used in styling gel, sunscreens, eye creams, and scrubs. Clinical studies have shown that carbomer polymers are less likely to cause skin irritation and sensitization at concentrations as high as 100%. Furthermore, carbomer polymers show low phototoxicity and photocontact sensitization potential.

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Updates:

BMS recently published the result of the critical phase III True North study (NCT02435992) that assessed Zeposia (ozanimod) as an induction and maintenance therapy for adult patients with moderate to severe ulcerative colitis (UC). It is worth mentioning that Zeposia is the first in history an oral sphingosine-1-phosphate (S1P) receptor modulator to show clinical benefits.

 

Facts

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) manifesting bloody stools, severe diarrhea, and frequent abdominal pain, usually over time rather than suddenly, which is characterized by abnormal immune response, long duration, long-term inflammation and ulcer in the colonic mucosa. To the therapies currently available, many patients do not respond adequately or at all, seriously influencing their physical function, social and emotional health and work ability. It is estimated that 12.6 million people worldwide are suffering from IBD.

 

Pathopoiesis Mechanism

1. Sulfide in Diet

The toxic effect of sulfide on colon cells may be a major cause. The proportion of protein in diet increases as the adjustment of people's dietary habits, resulting in the sharply climbing intake of sulfur-containing amino acids (including methionine, cysteine, cystine and taurine). Through the degradation and fermentation of such amino acids by intestinal bacteria, a variety of sulfur-containing compounds are produced. The accumulation of hydrogen sulfide in the intestine may have a certain direct toxic effect on colon cells, and may also indirectly change the protein function and antigenicity. Research shows that the intake of meat (rich in protein), especially red meat and processed meat, increases the risk of UC recurrence. In addition, due to the non organic sulfate (including sulfur dioxide, hydrogen sulfide, sulfite) is widely used as preservative in the storage and storage of food and beverage, such as hamburger, concentrated beverage, sausage, beer and red wine, etc. Therefore, these foods and drinks also increase the risk of UC.

 

2. Dietary Fat

Excessive intake of fat or unsaturated fatty acid will damage the colonic mucosa, result in colitis, and may also affect the absorption and secretion of cholesterol. The hypercoagulable state formed by hypercholesterolemia can cause vasospasm, increase the tension of blood vessels, and affect the blood supply of mucosa, which therefore may cause colon mucosa damage. Excessive intake of monounsaturated fatty acids and polyunsaturated fatty acids may increase the incidence of UC.

 

3. Carbohydrate

High sugar intake may be related to UC. According to an epidemiological survey, people who often eat foods with high sugar content, such as cola drinks and chocolate have a positive correlation with UC, compared with those who jointly eat vegetables and fruits. The pathogenesis of UC caused by high sugar diet is not clear so far.

 

Diagnosis

Conventional approaches of UC diagnosis like clinical evaluation (symptoms), radiology and endoscopy, in conjunction with histological examinations and imaging techniques are costly and time-consuming. Non-invasive diagnostic tests on serological biomarkers (p-ANCA, ASCA, CBir1, anti-I2, etc.) and stool markers (infectious pathogens like calprotectin and lactoferrin) in easily obtained biological samples with IVD immunoassays are receiving popularity recent years, which have been well developed into various platforms, including ELISA, LFIA, CLIA, IHC, etc.

 

In March 2020, Zeposia was approved by the U.S. FDA for the treatment of adult multiple sclerosis (clinically isolated syndrome, recurrent remission disease and active secondary progressive disease), which in May got approved by the European Commission to treat adult patients with relapsing remitting multiple sclerosis (RRMS) with active disease manifesting clinical or imaging characteristics.

 

UC is an unpredictable and potentially debilitating disease. The results of the True North study are very encouraging for patients with moderate to severe UC due to the consistent efficacy at key clinical and endoscopic endpoints, suggesting that Zeposia may address the need for new oral therapies.

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The Parallel Artificial Membrane Permeation Assay (PAMPA) is a non-cell based assay designed to predict passive, trans- cellular permeability of drugs in early drug discovery. In this assay, we will discuss the major concerns related to PAMPA.

 

PAMPA analysis measures the permeability of artificial membranes that provides an in vitro model of passive diffusion. Passive diffusion is an important factor that determines transport through the gastrointestinal tract, penetration of the blood-brain barrier, and transport across cell membranes. Permeability may also be affected by several other mechanisms, including paracellular transport and active uptake or efflux not evaluated in PAMPA. Therefore, PAMPA can provide a simplified permeability method by measuring a single mechanism which avoids the complexity of active transport/outflow and enables compounds to be ordered by a single permeability.

 

Generally, PAMPA is used as the main permeability barrier, in which a simple measurement of passive diffusion is required. However, separation using PAMPA may misunderstand the true understanding of permeability in vivo. Cell-based analysis (e.g. Caco-2) evaluates permeability through passive diffusion across cells and active and paracellular transport. Therefore, Caco-2 permeability screen can provide more detailed mechanical information.

 

What is the relationship between Caco-2 and PAMPA?

PAMPA measures permeability only by passive diffusion, while the Caco-2 permeability assay also evaluates active uptake/efflux and paracellular transport. Therefore, if the compound only passes through the membrane by passive diffusion, a good correlation is observed between the Caco-2 permeability measurement and PAMPA. If the compound is an active substrate, PAMPA will overestimate the permeability, if the compound undergoes active uptake or paracellular absorption, PAMPA will underestimate the permeability. The relationship between Caco-2 permeability and PAMPA permeability can be used to diagnose the penetration mechanism.

 

The penetration of these compounds is dominated by passive diffusion, unrelated compounds are divided into two subsets. A subset has a higher PAMPA permeability than the cell monolayer permeability and is composed of compounds affected by secretion mechanisms: efflux of alkali or reduced passive diffusion under Caco-2 when operating under a pH gradient. The cell monolayer permeability of the other subset is higher than that of PAMPA, and consists of compounds with an absorption mechanism: when running under a pH gradient, the absorption of acid under the Caco-2 is increased by the paracellular uptake, active transport or passive diffusion. In view of the characteristics of these two methods, these studies show how to synergistically apply PAMPA and Caco-2 to the rapid and effective study of the penetration mechanism in drug discovery.

 

In the early discovery process, PAMPA can be used to quickly screen all compounds at low and neutral pH to evaluate passive diffusion permeability to indicate the potential of gastrointestinal tract and cells to measure penetration. In the middle of the discovery process, selected compounds can also be analyzed by Caco-2, which goes to the outside of the basal. This result, combined with PAMPA data, shows sensitivity to other penetration mechanisms (secretion and absorption). During the mid- to late-stage discovery, selected candidates can be examined in detail through multiple targeted Caco-2 experiments and transporter inhibitors to fully characterize the penetration mechanism. All compounds can be quickly screened using PAMPA at low and neutral pH to assess passive diffusion permeability to indicate the potential of the gastrointestinal tract and cells to measure penetration.

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Oncolytic virus (OV) is a kind of natural or genetically modified virus that can specifically infect and kill tumor cells without causing too many harmful effects on normal cells. Initially, the world authoritative medical journal TheLancet reported that influenza viruses will cause tumors in patients to regress, resulting in the oncolytic virus, and then researchers around the world have carried out a series of studies on this, and now there are more than 160 different oncolytic viruses preclinical research and clinical trials are underway. However, due to the strong immunogenicity of oncolytic viruses, when injected into the body intravenously, it will trigger the body to produce different degrees of immune responses, which will quickly clear the virus by the body. In addition, the tumor targeting of oncolytic viruses is limited, resulting in their inability to specifically reach the tumor site through the blood circulation, which seriously affects the tumor suppressive effect of oncolytic viruses, and produces different degrees of adverse reactions. In order to solve the above drawbacks, researchers construct different types of vectors to load oncolytic viruses to block their immunogenicity, extend their blood circulation time, and further combine oncolytic viruses with chemotherapy, radiotherapy, immunotherapy, photodynamic therapy, photothermal therapy and other combination of treatment means to improve its tumor treatment effect.

Construction of oncolytic virus vectors based on biological materials

Biomaterials can be used to load oncolytic viruses to achieve the purpose of blocking viral immunogenicity and achieving targeted delivery of tumors. The biomaterials currently used for oncolytic virus loading mainly include liposomes, cells or extracellular vesicles.

  1. Liposome expressing oncolytic virus

Cationic liposomes constructed with (2,3-dioleoyl-propyl)-trimethylamine (2,3-dioleoy-loxy-propy)-trimethylammonium (DOTAP) encapsulate oncolytic adenovirus, which can block the surface of adenovirus, reduces its immunogenicity, and allows the oncolytic adenovirus to enter the Coxsackie virus-adenovirus receptor (CAR) low-expressing tumor cells to replicate through endocytosis. However, after entering the body, cationic liposomes will adsorb some negatively charged serum proteins, causing the liposome nanoparticles to agglomerate, which seriously affects their access to the tumor site through the enhanced permeability and retention effect (EPR effect). Anionic liposomes are endogenous components of eukaryotic cell membranes. Compared with cationic liposomes, they have the advantages of low toxicity and low immunogenicity. They have been widely used in recent years. At the same time, there are studies that claim that anionic liposomes can undergo a phase change under the action of calcium ions, which is conducive to the liposomes encapsulating oncolytic viruses through electrostatic adsorption. PEG (polyethylene glycol) anionic liposome based on matrix metalloproteinase and substrate peptide/cholesterol, successfully encapsulated oncolytic adenovirus, its research team confirmed the high gene expression efficiency of the material in vitro, while the safety of the material was confirmed by C57BL/6N mice.

  1. Cell or extracellular vesicles expressing oncolytic virus

Commonly used cell carriers such as stem cells, lymphocytes, and tumor-related cells have good tumor chemotaxis. Coupling the oncolytic virus on the surface of the cell or loading it into the cell can not only effectively block the immunogenicity of the virus, but also improve its targeted enrichment effect on tumors. Studies showed that stem cells have a tendency to affect the tumor microenvironment and have a low immunogenicity. On this basis, JOSIAH and others used adipose-derived stem cell (ADSC) from healthy mice for the first time to load myxoma oncolytic virus, then it was injected into tumor-bearing mice to achieve the targeted delivery of the virus to glioma. In addition, some researchers used mesenchymal stem cell (MSC) expressing Newcastle discase virus (NDV). The results found that in vitro MSC can not only improve the targeting of NDV to glioma cells, but also the secretion of TNF-related apoptosis-inducing ligand (TRAIL), which significantly improve the oncolytic effect of the virus on glioma stem cells. In addition, there are studies used immature dendritic cells (iDC) and lymphokine-activated killer cells (LAK) to coordinately transport reovirus, which killed ovarian cancer cells in vitro by resisting neutralizing antibodies in patients' ascites. LANKOV et al. reported for the first time that using tumor-associated macrophages as a carrier of oncolytic measles virus to delivery mice ovarian cancer, this strategy allows the virus to evade the neutralization of antibodies and complement, and then the virus can be transferred to tumor cells through in situ cell fusion.

Extracellular vesicles are a subcellular structure with membrane components secreted by almost all cells, including exosomes, microvesicles, and membrane particles, which also play an important role in drug delivery. LV et al. designed an engineered cell membrane nanovesicle containing targeting ligands for delivery of oncolytic adenoviruses (OA@BCMNs). This system showed strong resistance in a variety of tumor-bearing mouse models. The tumor effect can significantly prolong the survival time of mice without major adverse reactions.

Tumor extracellular vesicles with a similar membrane structure to tumor cells can also be used as an ideal carrier for oncolytic viruses. Due to their homologous targeting, this system can also achieve tumor-targeted delivery of oncolytic viruses. A study used tumor cell-derived microparticles (T-MP) to deliver oncolytic adenovirus, and the constructed nanoparticles (oncolytic adenovirus-microparticle (OA-MP) can not only make the virus escape the body's resistance viral effect, and can make the oncolytic virus enter into the nucleus of tumor cells or tumor stem cells to self-replicate without relying on the receptor-mediated infection. Therefore, this system is also more effective for tumor cells with low CAR receptor expression, such as human chronic myeloid leukemia cells (K562 cells).

Construction of oncolytic virus vectors based on non-biological materials

The non-biological carrier material mainly encapsulates the virus through two reaction modes: covalent action and non-covalent action. Covalent modification is to use amino acid residues on the envelope or capsid protein of the virus surface to couple with other molecules through covalent action, and non-covalent modification is to use electrostatic adsorption, hydrogen bonding, van der Waals force, or antigen-antibody action. The modification is coupled to the virus.

  1. Oncolytic viruses with high molecular polymers

Due to the ease of preparation and good biocompatibility of high molecular polymers, they have the advantages of being metabolizable and degradable in the body, which has attracted extensive attention in the medical field, such as drug loading, delivery and release. Since most viral proteins are negatively charged under physiological conditions, many cationic polymer complexes can be adsorbed on the virus surface by electrostatic action. TESFAY et al. encapsulated FSL-PEG2000 on the surface of vesicular stomatitis virus (VSV) through electrostatic adsorption; therefore, the virus surface protein was effectively blocked, thereby effectively reducing the antibody neutralization effect in mouse serum and the effect of mice liver and spleen on virus storage. However, because virus infection depends on the specific recognition of its surface proteins and host cell surface receptors, and the coating of FSL-PEG2000 affects the recognition and infection of viruses and tumor cells, the follow-up research aims to find some controllable polymer, which allows the oncolytic virus to be released in a targeted manner when it reaches the host cell without affecting its ability to infect. Environmental stimuli-responsive polymer can respond to small changes in the outside world, so this feature can be used to achieve on-site release of oncolytic viruses. CHOI et al. used physical adsorption method to wrap the pH-sensitive polymer (mPEG-b-pHis) on the surface of adenovirus to prepare a stable oncolytic adenovirus complex, which not only reduces the immune response of adenovirus, but also release the oncolytic virus under the slight acid environment of the tumor, and then restore the virus to the tumor cells. Based on the same principle as above, LEE et al. utilized biodegradable macromolecular polymer cholic acid coupled polyvinylamide [bileacid-conjugated Poly(ethylencimine), DA3] encapsulates adenovirus, and the constructed viral nanocomposite particles (Ad/DA3) can also effectively increase the effect of virus in vitro to a variety of tumor cells.

To be continued in Part Two…

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1.3. Blood system diseases

HSC was first used for blood diseases, and it is the most mature. Both autogenous HSC and foreign HSC are used. And they are mainly used for some hematological malignancies, such as acute myeloid leukemia, acute lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, aplastic anemia, etc. All have achieved good results and improved the 10-year survival rate of patients, but it is ineffective for some patients, such as low diploid leukemia. Hematopoietic stem cell transplantation is generally used after chemotherapy. It can differentiate into myeloid progenitor cells, lymphoid progenitor cells, etc. in the human body, and then produce blood cells of various lineages. The treatment of blood system diseases is mainly through cell replacement. Eventually The effect is to rebuild the functional hematopoietic system and immune system. It is now found that multiple myeloma and sickle cell disease can also benefit from HSCs transplantation. For patients with poor conventional treatment or relapse, stem cell transplantation is still effective and can improve the cure rate. However, before transplantation, the patient's physical tolerance, blood system status and human leukocyte antigen typing must be fully considered to avoid the occurrence of GVHD.

1.4. Bone and joint system diseases

The human osteoblasts have the ability to proliferate and differentiate. When the damage is not serious, the bone itself can undergo a certain degree of self-repair. For more severe bone and cartilage injuries, especially when there are a large number of bone defects, local blood supply, and oral and maxillofacial fractures that do not heal, stem cell transplantation still plays a very important role. From the source of bone and joint development, mesenchymal stem cells derived from mesoderm are the most widely used in bone and joint diseases. Mesenchymal stem cells can migrate to the site of injury and differentiate into osteoblasts and chondrocytes to repair bone damage. In osteoarthritis and sports injuries, stem cell therapy has been found to reduce pain, repair cartilage degradation, and promote motor function recovery. The mechanism of stem cell treatment of bone and joint diseases is multi-faceted, including paracrine function, improving microenvironment, promoting angiogenesis, replacement repair, etc. It is currently believed that the main role is to improve the local microenvironment of bone injury.

1.5. Eye diseases

The application of stem cell transplantation in ophthalmology is mainly to promote the repair of cornea and retina. There are a certain number of stem cells inside the eye tissue, mainly limbal stem cells (LSC), which is also a class of stem cells that are widely used in ophthalmology. It has a certain therapeutic effect on corneal injury and limbal stem cell deficiency, but it faces a limited source and immune rejection during allogeneic transplantation, which limits a large range of clinical applications. Fat or bone marrow-derived MSCs are another ideal cell type, and have certain therapeutic effects on glaucoma, retinal pigment degeneration, and macular atrophic damage combined with retinal yellow spots (Stargardt disease). Animal studies have found that MSCs can differentiate into photoreceptors and retinal pigment epithelial cells, secrete neurotrophic factors, and inhibit the apoptosis of optic nerve cells, and transplantation under the retina is more effective than intravitreal. Clinical studies have found that MSCs can migrate into the macular area of the retina and improve the patient's vision, but the effect almost disappears after 1 year. Considering that the cell type is not suitable for growth in the eye, it is only a short-term effect that occurs through the secretion of trophic factors. In addition: ESCs or i PSC-derived retinal pigment epithelial cells and oral mucosal epithelial cells can also be selected, and it has a therapeutic effect on patients with age-related macular degeneration and retinitis pigmentosa.

Because MSCs and HSCs have a wide range of sources, the preparation difficulty is relatively low, the safety is good, the paracrine and immunomodulatory effects are strong, so they are currently widely used. In addition to the diseases mentioned above, there are many diseases that may benefit from stem cell transplantation. Type 2 diabetes is essentially an autoimmune disease mediated by T cells. Therefore, the use of MSCs or HSCs can regulate immune function, inhibit T cell activation and inflammatory factor production, so as to relieve the symptoms of diabetes and reduce the amount of insulin. In addition, other diseases such as hepatitis, cirrhosis, burns, skin damage, systemic lupus erythematosus, rheumatoid arthritis, intestinal Crohn's disease, acute respiratory distress syndrome and other diseases have also been studied using mesenchymal stem cell therapy.

To ensure the success of stem cell therapy, we must first solve the problem of stem cell survival and migration after transplantation. Many studies have found that the stem cells transplanted into the body have a short survival time and cannot differentiate into target cells and function. Therefore, ensuring the homing, survival, differentiation and normal migration of transplanted stem cells is the key to effective treatment. There have been attempts to combine drugs (sodium ferulate, lithium valproate, erythropoietin), or related physical therapy (shock wave), or pre-treatment of stem cells (endothelial nitric oxide synthase) during the culture stage Enhancer, and embedding stem cells into biological materials (such as hydrogels, fabricated scaffolds) to increase the success rate of stem cell transplantation. For the problem of low cell differentiation efficiency in vivo, direct transplantation of totipotent stem cells should be avoided as far as possible, and high-purity tissue-specific precursor cells can be differentiated in vitro for transplantation, which also reduces the risk of tumorigenesis. Secondly, stem cells exist in many tissues and organs of the human body. Extracting and applying these stem cells is a good choice, but the problem is to obtain sufficient and effective stem cells. During the in vitro expansion process, the characteristics of stem cells may change to some extent, making them no longer suitable for repairing damaged tissues. Moreover, after the occurrence of the disease, the microenvironment of the lesion has undergone tremendous changes, which will also adversely affect the survival and migration of stem cells after transplantation. Finally, the standardization of stem cell preparation and transplantation, including the number and purity of stem cells, cell viability, route of administration, frequency, transplantation site, and the optimal time for treatment, all require careful evaluation and selection. It is important to establish a set of stem cell preparation and expansion standardized procedures for augmentation, storage and transplantation to achieve a stable treatment effect.

References

[1] Ferraro F,Celso CL,Scadden D. Adult stem cels and their niches[J].Adv Exp Med Biol,2010,695:155-168.

[2]  Ito Y,Nakamura S,Sugimoto N,et al. Turbulence activates platelet biogenesis to enable clinical scale ex vivo production[J]. Cell,2018,174(3):636-648.

[3] Regueiro A,Cuadrado-Godia E,Bueno-Beti C,et al. Mobilization of endothelial progenitor cells in acute cardiovascular events in the PROCELL study:time-course after acute myocardial infarction and stroke[J]. J Mol Cell Cardiol,2015,80:146-155.

[4] Bochon B,Kozubska M,Suryga?a G,et al. Mesenchymal stem cells-potential applications in kidney diseases[J]. Int J Mol Sci,2019,20(10):2462. 

[5]  Ming GL,Song H. Adult neurogenesis in the mammalian brain:significant answers and significant questions[J]. Neuron,2011,70(4):687-702.

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The principle of SNP genotyping technology is PCR amplification of genomic fragments containing SNP. The main features are high accuracy, strong flexibility and high throughput. The main method is the TaqMan probe method.

Technical principles

SNP genotyping

First, the SNP-containing genomic fragments are amplified by PCR, and then single-base extension is achieved by sequence-specific primers. Then, the sample analyte and the chip matrix are co-crystallized and then excited by an intense nanosecond (10-9s) intense laser in a vacuum tube. Nucleic acid molecules are desorbed into single-charged ions. Because the flight time of ions in the electric field is inversely proportional to the ion mass, the precise molecular weight of the sample analyte is obtained by detecting the flight time of the nucleic acid molecules in the vacuum tube, thereby detecting SNP site information.

Main features

The accuracy of SNP detection achieved by time-of-flight mass spectrometry (MALDI-TOF) can reach 99.9%. In addition to the advantages of high accuracy, flexibility, high throughput, and short detection cycle, the most attractive should be its cost performance. The time-of-flight mass spectrometry platform (MALDI-TOF) is an internationally-used research platform for genetic single nucleotide polymorphism (SNP). This method has become a new standard in this field with its scientificity and accuracy.

Main methods

  1. TaqMan probe method

Design PCR primers and TaqMan probes for different SNP sites on chromosomes respectively, and perform real-time fluorescent PCR amplification. The 5'-end and 3'-end of the probe are labeled with a reporter fluorescent group and a quenching fluorescent group, respectively. When the PCR product is present in the solution, the probe is annealed to the template, which produces a substrate suitable for exonuclease activity, thereby cleaving the fluorescent molecule attached to the 5'-end of the probe from the probe, destroying the two PRET between fluorescent molecules emits fluorescence. Usually used for small SNP loci analysis.

  1. SNaPshot method  

This technology was developed by American Applied Biology Company (ABI), and it is a typing technology based on the principle of single base extension of fluorescent labeling, also known as small sequencing, which is mainly aimed at medium-throughput SNP typing projects. In a reaction system containing sequencing enzymes, four fluorescently labeled ddNTPs, different length extension primers and PCR product templates immediately adjacent to the 5'-end of the polymorphic site, the primer extension is terminated by one base, and after detection by the ABI sequencer, The SNP site corresponding to the extension product is determined according to the shifting position of the peak, and the type of the incorporated base can be known according to the color of the peak, thereby determining the genotype of the sample. The PCR product template can be obtained by multiple PCR reaction system. Usually used for 10-30 SNP loci analysis.

  1. The HRM method

high-resolution melting curve analysis (HRM) is a SNP research tool that has emerged in recent years. It detects the presence of SNPs by monitoring the combination of double-stranded DNA fluorescent dyes and PCR amplification products during real-time heating. Moreover, different SNP sites and whether they are heterozygotes will affect the peak shape of the melting curve, so HRM analysis can effectively distinguish between different SNP sites and different genotypes. This detection method is not limited by the location and type of the mutated base. No sequence-specific probes are required. After the PCR is completed, high-resolution melting is run directly to complete the analysis of the sample genotype. The method does not need to design a probe, and the operation is simple, fast, low in cost, accurate in result, and realizes the real closed tube operation.

  1. Mass Array method  MassARRAY molecular weight array technology is the world's leading gene analysis tool launched by Sequenom. It combines primer extension or cleavage reaction with sensitive and reliable MALDI-TOF-MS technology to achieve genotyping detection. The iPLEX GOLD technology based on the MassARRAY platform can design up to 40-fold PCR reaction and genotype detection, with flexible experimental design and high accuracy of typing results. According to the needs of the application, when testing hundreds to thousands of samples from dozens to hundreds of SNP sites, MassARRAY has the best cost performance, especially suitable for verifying the results of genome-wide research findings, or a limited number of the situation where the research site has been determined.
  2. Illumina BeadXpress method uses Illumina's BeadXpress system for batch SNP site detection, which can detect 1-384 SNP sites at the same time. It is often used to confirm the results of genomic chips and is suitable for high-throughput detection. The microbead chip has the characteristics of high density, high repeatability, high sensitivity, low sample load, flexible customization, etc., and extremely high integration density, thus obtaining extremely high detection screening speed, and can significantly reduce costs during high-throughput screening.

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We work hard to offer you the same dependable services to pharmaceutical and biotech companies, as well as academia and government agencies for satisfying all your sequencing or array needs. Through nearly ten year's hard working and depend on our professional work team, we are proud of satisfying the needs of our clients both at home and abroad, which across more than 50 countries and districts. We always devote ourselves to providing you with the best and professional service. Here are some: pacific biosciencesTotal RNA Sequencinghuman genomegbs sequencing, etc.

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When it comes to R&D of new drug, people would think about high investment, long term, and high risk. Although drug design and screening technology has been improving, the disease is complex. Even if the drug was successfully marketed, it will still face many unknown factors such as adverse reactions, market sales, and national policies. In fact, it is not impossible to solve these problems, which is to design a few good enough drug candidates.

Does it easy to find preferable candidates?

With the rapid development of X-ray diffraction, NMR, electron microscopy and other technologies, the structural parameters of the target protein are gradually clear. On the other hand, the significant increase in computing performance and the reduction in hardware prices have made computer-aided drug design tools an indispensable helper for scientists. Various computer algorithms can now perform virtual screening or directly give new molecular structures, and help scientists optimize the medicinal properties and physical and chemical properties of seedling compounds. So, can a computer now automatically design a drug molecule from scratch by giving it a chemical structure like artificial intelligence?

In the field of drug design, computer programs have assisted researchers in developing some drugs in the last century. The most common method is to use a virtual screening technique such as molecular docking to target a protein with a known structure in a massive chemical structure to find the hit compound. In addition, there are some drug design programs based on the chemical structure of small molecules, such as de novo design programs. Recently, Dr. Gisbert Schneider of the Federal Institute of Technology in Zurich (ETH Zürich) and others summarized the literature of automatic de novo drug design in recent years and detailed 10 drug target cases so that readers can get an overview of the overall situation or understand the frontier direction. Related papers were published in Angew. Chem. Int. Ed.

  1. β secretase inhibitor

BACE-1 is one of the targets of neurodegenerative diseases, but its patentability is quite challenging. Fishwick et al. reported a de novo drug design program SPROUT based on protein structure, and designed a non-polypeptide structure β-secretase BACE-1 inhibitor (J. Med. Chem., 2013, 56, 1843). Through the X-ray structure of the complex of BACE-1 and peptide-like ligands, SPROUT resolved key active sites and designed the original compound 1. After synthesizing Compound 1 and testing its biological activity, it was found that its activity is weak (IC50 = 323 μM). After structural optimization, compound 2 was obtained with moderate activity (IC50 = 27 μM). This case confirmed the feasibility of synthesizable compounds acting on targets based on automatic structural design.

  1. Aurora A kinase A inhibitor

VX-680 is a pan-Aurora kinase inhibitor with high anti-Aurora activity (Ki = 0.6 nM) and was terminated in clinical trials due to heart problems with prolonged QT intervals. Rodrigues et al. hoped to design a brand new chemical framework (Chem. Sci., 2013, 4, 1229) with the ligand-based de novo drug design program DOGS (Design Of Genuine Structures), which has similar activities as VX-680, but avoid heart problems while breaking through the intellectual property restrictions surrounding this structure. The DOGS program is based on 88 skeletons and produces 172 chemical structures. Since the aromatic sulfonamide structural fragments are often scored high in the scoring system, and the chemical synthesis is highly feasible, the corresponding compounds 3 and 4 were selected for synthesis. Another advantage of DOGS is that it can design synthetic routes, and design algorithms are derived from chemical transformation rules. In vitro experiments found that 100 μM 3 only inhibited 5% of Aurora A. The activity of 4 is IC50 = 10 μM, which is suitable for optimization from seedling compounds to lead compounds. The huge difference in activity between 3 and 4 is likely to come from imidazole structural fragments. Under this guidance, they obtained compound 5 by optimization (IC50 = 2.7 μM).

From this case, the following three points can be seen: (1) The structure of compound 5 is completely different from the original VX-680, and it is a completely new structure. (2) The program only needs one inhibitor structure to start the program, not even the protein structure, so this method has a wide range of applications. (3) Most of the designed compounds can be synthesized and used to test the activity.

Starting from the crystal structure of the complex of Aurora A and inhibitors, Park et al. used a structure-based virtual screening to find a key fragment structure MPPA in the fragment database. Their secret is to automatically dock and modify the scoring function (J. Chem. Inf. Model., 2018, 58, 700). Using the hinge-binding site as the starting point, the team used LigBuilder to focus on the fragment structure database of Aurora A. Designed new compounds using de novo drug design methods, followed the 5 rules of drug-like drugs in drug design, and finally selected 35 compounds and synthesized 17, the most active compound is 6 (IC50 = 0.012 nM).

  1. COX-2 and LTA4H dual inhibitor

Multiple pharmacology has also received attention in recent years, that is, a compound achieves a therapeutic effect by acting on more than one target. Shang et al. used a de novo drug design program LigBuilder to design compounds that act on cyclooxygenase-2 (COX-2)/5-lipid oxidase (5-LOX)/leukotriene A4 hydrolase (LTA4H) in order to achieve more Good anti-inflammatory effect (J. Chem. Inf. Model., 2014, 54, 1235). The authors extracted the fragment structure from the known inhibitors of these three targets and obtained 21 compounds, and then tested the activity at a high concentration (1 mM) to obtain 9 seedling compounds. The LigBuilder program performs a variety of binding mode calculations on these structural fragments, designed 1.1 million compounds, selected the first 1,000 of them to continue research, and finally synthesized 6 compounds, of which compound 7 has the best activity, and COX at a concentration of 100 μM The inhibition rate of -1 and COX-2 was 90%, and the inhibition rate of LTA4H was 43%, thus entering the second round of de novo drug design to obtain compound 8, and the third round to obtain compound 9. Compared with COX1, compound 9 has a very good selectivity for COX2 (about 11 times).

  1. Helicobacter pylori HtrA inhibitor

Helicobacter pylori can cause stomach/duodenal ulcers and even gastric cancer. The serine protease pathogenic factor HtrA (high temperature reguirement A) is the target of Helicobacter pylori and other pathogenic bacteria. The compound currently available is rhodanine derivative 10, but it has weak activity, poor solubility, and the rhodanine skeleton problem.

Perna et al. used DOGS software to start at 10 (Angew. Chem. Int. Ed., 2015, 54, 10244), designed 1707 compounds, screened by solubility combined with three-dimensional structure, and selected 65 of them for synthesis , Compound 11 best meets the following 4 requirements: solubility and fit rank top; no Michael addition acceptor structure; very different from the structure of Compound 10; a completely new type of chemical structure.

11 can be synthesized through a two-step reaction. Its affinity is similar to compound 10 (KD = 37 μM), but the ligand efficiency LE (ligand efficiency) is better. In the configuration relationship study, its analog 12 had the best activity (KD = 37 μM, LE = 0.3), and all had anti-Helicobacter pylori activity in vitro.

To be continued in Part Two…

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With the continuous maturation of single-cell transcriptomics research technology, it is estimated that in the next few years, single-cell gene expression and regulation research will become a popular topic, and the scientific research community will soon obtain enough quantitative transcript research data. This information can help us answer many important scientific questions and can also lay the foundation for quantitative research on cell types and heterogeneity in the future. Based on this information, the transcriptome of almost all types of cells in a complex multicellular organ can also be determined. Moreover, single-cell transcriptomics information can also help us improve the ability to manually manipulate gene expression regulatory networks, because a large amount of single-cell data truly reflects the biological disturbances faced by cells, and this information can help us deepen our understanding of regulatory networks.

  1. Prospects of single cell sequencing technology

Usually we will regard a cell with the same phenotype as a whole with a specific function, and call it a tissue or an organ. However, deep DNA and RNA sequencing of a single cell will reveal a variety of cell states that constitute a complex ecosystem, and then such a complex system forms the overall function of tissues and organs. The continuous development of high-information, real-time, multi-mode single-cell detection technology will help us truly understand the function of a single cell in a microenvironment system.

Although early morphological studies have clearly identified a variety of cell morphologies, recent studies have unexpectedly discovered many new and different cell states. A standard human cell contains approximately 6 billion DNA base pairs, and 600 million bases of mRNA (an mRNA of this size is already sufficient to provide super coding capacity). Deep sequencing of DNA and RNA of single cells can more fully grasp the functions of cells with unprecedented higher resolution. Scientists' ability to specifically recognize the state of cells helps us better understand the normal functions and abnormalities of cells.

Single-cell sequencing can detect differences between cells at a higher resolution, which also raises a series of new problems. The most fundamental problem may be that it is not necessarily meaningful to find and measure such cell-to-cell differences, that is, we do not know which cell state is the truly functional cell state. Since in a typical human cell, there are only dozens of copies of each mRNA on average. How do individual cells interact with each other to achieve functions at the organizational level? This essential study of cellular ecology is a brand-new field that is worth digging into. In addition, if we think that the phenotype of a cell is the function of a local ecosystem formed by multiple cells, then, in a tissue composed of multiple cells, how do so many local ecosystems coexist together? Is there a mutual exchange effect?

Although single-cell sequencing technology has brought us many surprises, and we are optimistic for the technology, but the technology is not currently a routine detection technology in the laboratory. Because the continuous advancement of basic technology and data analysis and interpretation technology is the key to improving the accuracy of single cell sequencing technology, and to understand the role of single cells at the system level, it is necessary to conduct single cell sequencing research on a large number of cells. We will comment on these issues next, will also focus on the future development of single-cell sequencing technology, as well as the newly emerging single-cell sequencing supplementary technology, and will introduce the specific functions of single cells in the overall ecological environment.

5.1 Important questions about single cell research

There are several important issues that affect the quality of data obtained by the Single Cell Sequencing Institute. Among them, the inevitable problem that needs special attention is that the transcriptome will change according to various stimuli, and this change is more prominent at the single cell level. With this in mind, we should treat single-cell transcriptome data with caution (at least to some extent) as the result of a perturbation experiment unless a less destructive RNA isolation technique can be developed.

5.1.1 Cell separation

Single-cell separation technology is almost the technology that needs to be developed most and needs to establish a standardized system. The use of patch pipettes or nanotubes to obtain the cytoplasmic content of individual cells is currently the conventional method of isolating cellular RNA, but this operation is prone to omission of organelle components. Using a microfluidic device can separate cells in individual reaction chambers, but the cells need to be separated from other substrates, and these substrates may interfere with the transcription status of the cells. During the process of cell dissociation, classification and enrichment, whether the transcription state of the cell changes is a question that needs special attention. Scattered cells are very easy to separate, but experiments with such cells require very good experimental design to avoid problems in interpreting the experimental results due to the lack of microenvironment. The most ideal situation is to separate the contents of single cells in a tissue or natural microenvironment. Only in this way, single cell mRNA detection can reflect the most real state of the cell under overall conditions, and only in this way can the effect of human operations on the cell be reduced as much as possible.

To be continued in Part X…

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In recent years, the incidence of cancer patients has been increasing yearly. Tumor markers have become mandatory items in many human examination projects. Among so many tumor indicators, how to identify their clinical significance? Let’s learn about the clinical significance of tumor marker detection in this article.

Tumor markers are a class of substances that are synthesized or released by tumor cells themselves or generated or elevated by the body's response to tumor cells. Tumor markers are present in blood, cells, tissues or body fluids, reflecting the presence and growth of tumors. Tumor markers are determined by methods such as chemistry, immunology, and genomics, for tumor diagnosis, curative effect and recurrence monitoring, and prognosis judgment Has a certain value. Tumor markers mainly include protein, sugar, enzyme and hormone tumor markers.

  1. Protein tumor markers

1.1 Alpha-fetoprotein (AFP) determination

Alpha-fetoprotein is a serum glycoprotein synthesized early in the fetus. After birth, AFP synthesis is inhibited. When malignant lesions occur in liver cells or gonad embryo tissues, cells capable of synthesizing AFP restart to synthesize, resulting in a marked increase in blood AFP content. Therefore, the detection of AFP concentration has important clinical value for the diagnosis of hepatocellular carcinoma and trophoblastic malignant tumors.

Reference value: <25μg/L

Clinical significance:

  • Primary liver cancer;
  • Gonad embryo tumor;
  • Viral hepatitis and cirrhosis (usually <300μg/L).

1.2 Carcinoembryonic Antigen (CEA) determination

The CEA content in the body after birth is extremely low and it is a broad-spectrum tumor marker that can be expressed in a variety of tumors. It is mainly used to assist the diagnosis, prognosis, curative effect monitoring and tumor recurrence of malignant tumors.

Reference value: <5μg/L

Clinical significance:

  • Increased CEA is mainly seen in patients with pancreatic cancer, intestinal cancer, gastric cancer, lung cancer, etc;
  • The concentration decreases when the dynamic observation improves, and vice versa;
  • CEA increased slightly in smoking patients.

1.3 Tissue peptide antigen (TPA) determination

The level of TPA in the blood is closely related to the degree of cell division and proliferation. The higher the level of TPA in the serum, the more commonly used clinically for the auxiliary diagnosis of rapidly proliferating malignant tumors, especially for monitoring the efficacy of known tumors.

Reference value: 130U/L

Clinical significance:

  • The serum TPA level of patients with malignant tumors can be significantly increased;
  • TPA level decreases after improvement, if it rises again, it indicates relapse;
  • The simultaneous detection of TPA and CEA is conducive to the differential diagnosis of malignant and non-malignant breast tumors.

1.4 Prostate specific antigen (PSA) determination

In prostate cancer, serum PSA levels are elevated. 80% of total serum PSA (t-PSA) exists in bound form, called composite PSA (c-PSA); 20% exists in free form, called free PSA (f-PSA). t-PSA and f-PSA increased, while the ratio of f-PSA/t-PSA decreased, suggesting prostate cancer.

Reference:

t-PSA<4.0μg/L

f-PSA<0.8μg/L

f-PSA/t-PSA>0.25

Clinical significance:

  • Prostate cancer;
  • If the concentration of t-PSA does not decrease or rises again after prostate cancer resection, it indicates tumor metastasis or recurrence;
  • PSA will increase after anal finger examination, prostate massage, and cystoscopy.

1.5 Squamous cell carcinoma antigen (SCC) determination

SCC is a subtype of tumor-associated antigen TA-4, a glycoprotein

Reference value: <1.5μg/L

Clinical significance:

  • Lung squamous cell carcinoma, cervical cancer, esophageal cancer;
  • Some benign diseases such as psoriasis and other skin diseases, renal insufficiency, upper respiratory tract infections, etc. can also cause increased SCC concentration;
  • Contamination of sweat, saliva and other body fluids can cause false positives.

1.6 Cytokeratin 19 fragment (CYFRA 21-1) determination

Mainly distributed in tissues or organs rich in epithelial cells, such as lung, breast, bladder, intestine, etc. When these tissues become malignant, the level of CYFRA 21-1 in the blood can be seen to increase. At present, it is mainly used for differential diagnosis and prognosis evaluation of non-small cell lung cancer.

Reference value <2μg/L

Clinical significance:

  • It is the preferred tumor marker for non-small cell lung cancer;
  • In addition to lung cancer, YFRA 21-1 increased in breast cancer, bladder cancer, colorectal cancer, etc.;
  • Other benign diseases such as pneumonia, tuberculosis, gastrointestinal diseases, etc., but their levels are slightly elevated (generally <10μg/L).
  1. Glycolipid tumor markers

2.1 Cancer Antigen 50 (CA50)

It is a tumor carbohydrate antigen and has no organ specificity for tumor diagnosis.

Reference value <20,000 U/L

Clinical significance:

  • Increased pancreatic cancer, gallbladder (tract) cancer, primary liver cancer, etc.;
  • Dynamic observation of its level is of great value for the prognosis of cancer tumors and judgment of recurrence monitoring;
  • It is valuable to distinguish benign and malignant pleural and abdominal effusions;
  • In chronic liver disease, pancreatitis, and bile duct disease, CA50 also increases.

2.2 Cancer Antigen 724 (CA724)

CA724 is a tumor-associated glycoprotein, which is a marker of gastrointestinal and ovarian cancer.

Reference value<6.7μg/L

Clinical significance:

  • The increase is seen in ovarian cancer, colorectal cancer, gastric cancer, pancreatic cancer;
  • Joint detection with CA125 can improve the detection rate of ovarian cancer;
  • Joint detection with CEA can improve the sensitivity and specificity of gastric cancer.

2.3 Sugar chain antigen 199 (CA199)

CA199 is a glycoprotein. Normal human salivary glands, prostate, pancreas, breast and other epithelial cells have a small amount of CA199.

Reference value <37,000 U/L

Clinical significance:

  • The preferred tumor marker for pancreatic cancer;
  • Can be found in acute pancreatitis, acute hepatitis, gallbladder cancer, cholangiocarcinoma, gastric cancer, colon cancer, etc.;
  • Continuous detection is of great value to the progression of the disease, surgical efficacy, prognosis estimation and diagnosis of recurrence;
  • Combined with CEA detection to improve the diagnosis rate of gastric cancer.

To be continued in Part II…

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Abstract: Science has made great progress in the fight against HIV and AIDS over the last decade. And a new platform called ConvertibleCAR has shown great potential.

AIDS has always been the focus of research in medical circles in various countries, because it is a disease that is relatively difficult to cure at the current medical level, and it also damages the human immune system greatly, and thus is easy to cause complications and eventually lead to death.

A study by the Gladstone Institute shows that a new technology based on CAR-T cell immunotherapy shows great hope in multiple therapeutic areas, especially in the fight against HIV. This new technology is called ConvertibleCAR®, and related research results were published in Cell on October 24, 2019.

The potential pool of HIV is the main obstacle to cure AIDS, and Dr. Warner C. Greene, who is the director of the Center for HIV Cure Research in Gladstone Institute as well as the corresponding author of the paper, has been hoping to target it and clear the virus.

Conventional CAR-T technology is required to transform an immune cell form, that is, cytotoxic T cells, to express antibodies on the surface. The antibody part allows cytotoxic T cells to reside in target cells (such as leukemia cells) and thus to attack and destroy them. Dr. Eytan Herzig of Gladstone Institute, the first author of the paper, said: "The disadvantage of traditional CAR-T is that they are designed to target a single molecule on cancer cells and once injected, they cannot be controlled.

In contrast, ConvertibleCAR technology makes it possible to combine cytotoxic "killer" T cells with any number of antibodies. This is essential for resistance to pathogens such as HIV, because there are hundreds of different variants of HIV. In this paper, scientists have overcome shortcomings by separating targeted antibodies from cytotoxic killer cells.

The modified NKG2D receptor can turn T cells into an effective killer only if it binds to its partner, which is a protein called MIC-A. The researchers trimmed and modified MIC-A so that it can specifically bind to the NKG2D receptor. Then, they fused it to the base of the targeted antibody and created the so-called MicAbody®. In this way, targeting MicAbody can be tightly combined with ConvertibleCAR-T cells.

To eliminate the potential pool of HIV, researchers have been testing a wide range of Broadly Neutralizing HIV-1 Antibodies (bNAb). Herzig explained: “They are called broadly neutralizing antibodies because they can neutralize a large number of strains of virus.”

But bNAb alone is not enough to kill HIV-infected cells. They need the help of killer T cells, and in HIV-infected patients, the problem is that the killer T cells have been exhausted, or the potential reservoir contains viruses that are resistant to these cells. Herzig and Greene believe that by combining bNAb and ConvertibleCAR-T cells, they may obtain the required lethality.

Herzig and Greene tested whether the ConvertibleCAR-Mic-bNAb platform could attack the latent reservoir in the blood of HIV-infected people on antiretroviral therapy (ART). The results prove that the transformable CAR-T cells specifically bind to Mic-bNAbs to kill the infected CD4 T cells, but not the uninfected cells. It was also found that within 48 hours after exposure, more than half of the activated, HIV-expressing cells had been eliminated.

Dr. Eytan Herzig will explain more about this technology in the upcoming webinar held at 3:00 p.m. EDT on June 4th, 2020. The webinar entitled “ConvertibleCAR-T, a highly adaptable CAR-T platform to fight HIV” is sponsored by Creative Biolabs, a leading biotech manufacturer focusing on one-stop CAR-T/NK Cell therapy development service.

One thing interesting about CAR-T cells is that they first appeared in the late 1990s to fight against HIV, but failed. And then traditional CAR-T cells have achieved great success in relieving blood cancers such as lymphoma and childhood leukemia. Now, it comes back to HIV treatment with greater lethality and may even be the key method to overcome AIDS.

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Creative Biolabs has provided financial support for the young and talented college student in form of yearly scholarship for 4 years in a row and will keep it as a convention, with the aim to help them continue their study into the science-related fields.

 

MayNew York, USA Affected by the COVID-19, most American universities have switched their teaching mode from face-to-face to distance learning, hoping to proceed with the education plan of this semester to minimize the influence on graduation.

 

At Creative Biolabs there is a group of Ph.D. level scientists who have been exploring in the field of biology, biochemistry, chemistry and molecular biology. With the professional theoretical framework constructed in their specialties in university for years, plus practical experience gained in hundreds of projects, the experts deeply realize the longer they get involved in their fields, the more they love. Therefore, they firmly believe that encouraging the younger generation to discover in their interested scientific fields is of vital importance, which is also the original intention to set up the Creative Biolabs Scholarship Program.

 

The scholarship application procedure is just like those of American universities that students should submit an application form, transcripts, recommendation letter (from a lecturer, school counselor or community leader), an essay, or a PPT over certain topics.

 

Previously, reference letter was not a must but an additional material since the scholarship committee mainly evaluate the academic ability of candidates according to their academic records and propositional essays. This year, application materials from any one of the candidates without a reference letter with the designated format will be considered incomplete, and result in disqualification automatically. "We think the comprehensive ability of a candidate is also important, " said the head of the Scholarship Committee at Creative Biolabs, "the evaluation from others can be a good reference."

 

Another significant material other than the academic transcript is the essay, or the PowerPoint document that must be totally original and grammatically right, reflecting the real insight of students in their fields. The topics include:

l Overview on Clinical Applications of Oncolytic Viruses

l Novel Gene Delivery Systems for Gene Therapy

l Applications of Protac Technology in Disease Treatment

l Overview on Clinical Applications of Stem Cell Therapy

 

Considering the current situation that many students may have limited access to library resources or their referrers, Creative Biolabs modifies the submission deadline of all applications. "Not like last year, we extend the application to September 15," said the head of the Scholarship Committee, "to ensure the students have enough time to finish their essays, or find a referrer." 

 

As a leading custom service provider specialized in the field of antibody production and engineering, Creative Biolabs is always happy to support young and ambitious talents in the medical and science-related field. More details about the application procedures and requirements can be accessed at https://www.creative-biolabs.com/scholarship-program.html.

 

Company profile:

Creative Biolabs is a US based biotechnology company with specialties in antibody discovery, engineering and providing relative custom solutions.

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As the most extensive renewable energy (23,000 TW/year) in nature, how to realize its efficient and reasonable development and utilization has always been a research hotspot for scientific researchers. From the current development stage, the utilization of solar energy mainly focuses on three aspects: solar power system, solar thermal system and solar fuel system. However, the intermittent problem of regional light source caused by Earth rotation greatly limits the continuous conversion of solar energy to other energy sources, so that it cannot meet the continuous energy demand in daily production and life. In response to the problem, scientists put forward the corresponding energy reserve strategy. By combining the photoelectrochemical system with the secondary cell or liquid flow cell system, the conversion and storage of solar energy are realized, and the energy supply and demand caused by the intermittent light source are effectively solved. However, the multi-system connection has the disadvantages of complex system, high cost and serious energy transmission loss. Therefore, how to design the integrated solar energy storage system rationally has become the next research problem that researchers need to solve.

 

Recently, Shaojun Dongs team at Changchun Institute of Applied Chemistry, Chinese Academy of Sciences realized the continuous transformation and storage of solar energy under an integrated system by constructing a bio-photoelectrochemical model based on water/oxygen circulation, providing a new research idea for the efficient use of renewable energy.

 

The team designed the bio-photochemical system to use polypyrrole solid-state capacitive electrodes as energy storage modules to make the energy storage process safer and easier. In the process of solar energy storage, the water/oxygen molecules in the system can spontaneously form a cycle without the participation of additional sacrifice reagents and other redox pairs, which greatly improves the safety of the equipment and reduces the cost.

 

In addition, the modular and integrated structural design of this system enables it to be adjusted and optimized according to different circumstances and better meet the practical application needs of many aspects. Experimental data analysis showed that the conceptual model obtained maximum power density outputs of 0.34 ± 0.01 and 0.19 ± 0.02 mWcm-2 under light and dark field conditions, respectively, and showed stable solar accumulator cycle performance. By changing the capacitance of the energy storage module (polypyrrole capacitive electrode), the charge/discharge time can be effectively regulated. In this research work, the construction of bio-photoelectrochemical model of water/oxygen cycle promotes the development of cross-integration among photoelectrochemical system, bioelectrochemical system and capacitor system on the one hand, and also provides certain guiding significance for the future research and development of green rechargeable devices on the other hand. The results were recently published in the Journal of the American Chemical Society, (2019, 141, 16416-16421), and were supported by the National Natural Science Foundation of China and the Ministry of Science and Technology of China.

 

About Author

As a biotechnology company, Lifeasible is specialized in agricultural science, offering a wide variety of agro-related services and products for environmental and energy solutions.

 

Our plant breeding and culture services support increasingly stringent safety and quality standards in the agricultural industry. Relying on our revolutionary techniques, various molecular breeding services and molecular diagnostic methods are offered for a wider range of agriculture-related sectors.

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According to reports, the rapid spread of COVID-19 has led to a surge in the number of suspected infections and close contacts. The early detection of the virus can not only enable the infected person to be treated in time and reduce the risk of death, but also can effectively control the source of the infection and cut off the transmission route through isolation.

 

Generally, there are two detection strategies for infectious diseases: nucleic acid detection and immunological detection. Nucleic acid detection is the detection of viral RNA genome, including gene sequencing, quantitative real-time PCR, digital droplet PCR, gene chip and Loop-Mediated isothermal amplification. Immunological detection is the detection of specific antibodies produced by viral antigens or by human immune responses, including immunochromatographic strips, enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA). 

 

The following compounds provieded by BOC Sciences can be used in the production of COVID-19 test kits:

 

Guanidine thiocyanate

Guanidine thiocyanate is a colorless crystal or white powder, soluble in water. It is an anticoupling agent and a strong protein denaturation agent, which can dissolve proteins, and quickly separate the nucleic acid from nuclear proteins. The combination of guanidine thiocyanate and mercaptoethanol is able to inhibit the activity of RNase. The combined action of guanidine thiocyanate and sodium dodecyl sarcosine can denature proteins and release RNA.

 

Guanidine hydrochloride

Guanidine hydrochloride, white or yellowish in color, is used as a protein denaturator. As a strong denaturation agent for extracting total RNA from cells, guanidine hydrochloride solution can dissolve proteins, destroy the secondary structure of nucleoproteins dissociated from nucleic acids. In addition, guanidine hydrochloride and other reducing agents can inactivate RNA enzymes. Using dicyandiamide and ammonium salt (ammonium chloride) as raw materials, crude guanidine hydrochloride products can be obtained at 170-230.

 

Tris(hydroxymethyl)aminomethane

Tris(hydroxymethyl)aminomethane is a common buffer in molecular biology and cell culture. Tris buffer has a high buffering capacity, high solubility in water, and is inert to many enzyme reactions, which makes Tris a very satisfying buffer for many biochemical purposes. It is generally used to stabilize the pH of reaction system and has a strong buffering capacity between pH 7.5 and pH 9.0. Tris-HCl buffer system can also be used to stabilize the pH value in the gel. Tris buffer is widely used as a solvent for nucleic acids and proteins. Due to the low ionic strength, Tris buffer can also be used for the formation of intermediate fibers in nematodes. "TE buffer" prepared by adding EDTA into Tris hydrochloric acid buffer can be used for DNA stabilization and storage.

 

HEPES

HEPES buffer, mainly composed of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, is a non-ionic amphoteric buffer with good buffering capacity in the pH range of 7.2-7.4. Its greatest advantage is that it can maintain a relatively constant pH value in open culture or cell observation. Under these conditions, the cap of the cell culture bottle should be tightened to prevent the small amount of carbonate needed in the culture solution from being dispersed into the air. HEPES is difficult to dissolve in any other solvents, but it is readily dissolves in water, and does not precipitate with polyvalent metal ions. In addition, most natural factors such as temperature, concentration, medium salinity, and ion composition do not affect the dissociation of HEPES, so the stability of HEPES is excellent.

 

DTT

DTT, short for DL-dithiothreitol, is used to block the formation of intramolecular or intermolecular disulfide bonds between cysteines in proteins. The effect of DTT on proteins is that it can reduce disulfide bonds in proteins. One of the uses of DTT is as a reductant for sulfhydrylated DNA. Sulfur atoms at the ends of sulfhydrylation DNA tend to form dimers in solution, especially in the presence of oxygen. This dimerization greatly reduces the efficiency of some coupling experiments, such as DNA fixation in biosensors.

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3.2 The immunoregulatory effect of mesenchymal stem cells on B cells

B cells are pluripotent stem cells derived from bone marrow. With the stimulation of antigens and T cells, they proliferate in large quantities and further differentiate and develop into plasma cells capable of secreting antibodies. Mesenchymal stem cells have a certain inhibitory effect on the proliferation and differentiation of B cells. Mesenchymal stem cells can induce B cell proliferation and differentiation through cell contact. These conclusions may be related to the cell purification method, culture environment and detection time point, etc., and further research is needed. In addition, the functions of B cells secreting antibodies and expressing chemokine receptors CXCR4, CXCR5, and CCR7 are inhibited by mesenchymal stem cells, while the secretion of B cell costimulatory molecules and cytokines is not affected by mesenchymal stem cells.

The effect of mesenchymal stem cells on B cells is achieved through cell contact, and PD-1 / PDL-1 pathway is an important way for mesenchymal stem cells to inhibit B cells. Studies have shown that PD-1 can dephosphorylate some key signal transducers of BCR signaling by accumulating phosphatase, thereby hindering BCR signaling and inhibiting B cell activation. In addition, there are studies suggesting that T cells play an important role in the immune regulation of mesenchymal stem cells to B cells. Mesenchymal stem cells inhibit the proliferation and differentiation of B cells, but require the interaction between T cells and mesenchymal stem cells.

In addition to the key role of cell contact, mesenchymal stem cells regulate B cell function. Soluble cytokines also play a role that cannot be ignored. The study found that mesenchymal stem cells secrete CCL2, and achieve immune regulation of B cells by suppressing transcriptional activator protein 3 and inducing the production of paired box protein 5 (PAX5). In the mesenchymal stem cell co-culture system, the m RNA expression of Blimp-1 is significantly reduced, and the m RNA expression of PAX-5 is increased. Inhibiting the expression of Blimp-1 and promoting the expression of PAX-5 can inhibit the proliferation and differentiation of B cells.

3.3 The immunomodulatory effect of mesenchymal stem cells on NK cells

NK cells are short for natural killer cells. It is different from T cells and B cells; it does not require specific antibodies to participate in the killing of target cells or antigen-specific sensitization. The main role of NK cells in the innate immunity of the human body is to distinguish normal and abnormal cells by recognizing the ligands of corresponding activated receptors and inhibitory receptors on target cells. They also play a role in immune surveillance.

Mesenchymal stem cells can inhibit the proliferation and function of NK cells. NK cells are stimulated by activated cytokines, resulting in high expression of NK cell surface activated receptors NKp30, NKG2D, NKp44, CD69. The activated receptors on the surface of these cells are closely related to the function of NK cells. When NK cells and mesenchymal stem cells are co-cultured, the expressions of NK cell activation receptors NKp30 and NKG2D will be reduced, and NKp44 will not be expressed. Therefore, mesenchymal stem cells inhibit the cytotoxicity of NK cells by inhibiting the expression of NK cell activation receptors.

FN-γ plays a key role in the inhibition of mesenchymal stem cells on NK cells. The sensitivity of mesenchymal stem cells to NK cytotoxicity can be reduced by the presence of IFN-γ. The role of IFN-γ may be to induce mesenchymal stem cells to overexpress HLA molecules and interact with the inhibitory receptors of NK cells, thereby inhibiting the cytotoxicity of NK cells. Indoleamine 2,3-dioxygenase, prostaglandin E2, HLA-G play a major role in the inhibition of mesenchymal stem cells on NK cells. The HLA-G receptor is expressed on the surface of a leukocyte called LILRB, and can be expressed on the surface of T, B, and NK cells. The interaction of HLA-G / LILRB2 blocks the MEK / ERK signaling pathway and inhibits the cytotoxicity of NK cells. Adding indoleamine 2,3-dioxygenase or prostaglandin E2 to the mixed culture of mesenchymal stem cells and NK cells can restore the proliferation of NK cells to a certain extent. In the presence of IFN-γ and tumor necrosis factor alpha, mesenchymal stem cells interact with NK cells, mesenchymal stem cells secrete prostaglandin E2, and subsequently prostaglandin E2 can promote indoleamine 2,3-dioxygenase synthesis. In addition, the inhibitory effect of mesenchymal stem cells on NK cells is concentration-dependent. As the ratio of NK / mesenchymal stem cells increases, the inhibitory effect of mesenchymal stem cells on NK cells weakens.

3.4 The immunomodulatory effect of mesenchymal stem cells on dendritic cells

Dendritic cells are important antigen-presenting cells, derived from pluripotent hematopoietic stem cells. Immature dendritic cells have extremely strong antigen uptake, processing and processing capabilities. After ingesting the antigen or being stimulated, immature dendritic cells (CD14-, CD1a +) can differentiate into mature dendritic cells (CD80 +, CD83 +, CD86 +), and during this process, their antigen uptake and processing capacity will be significantly reduced, and their ability to present antigens and stimulate immune responses will be enhanced. Dendritic cells are presented to the corresponding CD8 + T cells and CD4 + T cells through the abundant antigen peptide-MHC class Ⅰ molecular complex and antigen peptide-MHC class Ⅱ molecular complex on their membrane surface, thereby activating T cell response and allowing T cells provide costimulatory molecules to fully activate T cells.

Mesenchymal stem cells can inhibit the differentiation of pluripotent hematopoietic stem cells into dendritic cells and the maturation of dendritic cells. Under the stimulation of granulocyte-macrophage stimulating factor / interleukin 4, the co-culture of mesenchymal stem cells and monocytes reduced the expression of CD1a and the high expression of CD14, indicating that the differentiation of dendritic cells was inhibited. In addition, under the stimulation of lipopolysaccharide, mesenchymal stem cells were co-cultured with dendritic cells, the expression of maturation marker CD83 of dendritic cells was decreased, the expression of costimulatory molecules CD80 and CD86 on the surface of dendritic cells was decreased, and the expression of costimulatory molecules CD80 and CD86 on the surface of DR was decreased, the expression of antigen presentation molecule HLA-DR was decreased, and the secretion of interleukin 2 was decreased, indicating that mesenchymal stem cells inhibited the maturation of dendritic cell. When the ratio of mesenchymal stem cells / monocytes is high (1:10), the inhibitory effect of mesenchymal stem cells on the differentiation of monocytes into dendritic cells can be completely achieved by soluble factors. When the ratio of mesenchymal stem cells / monocytes is low (1:20 or 1:40), mesenchymal stem cells exert their inhibitory effect mainly through cell contact. Mesenchymal stem cells can inhibit the differentiation of CD34 + -derived CD14 + precursor cells and monocytes into CD1a + dendritic cells, and inhibit the maturation and function of dendritic cells. The addition of macrophage colony stimulating factor and interleukin 6 antibody can reduce the expression of CD14, but cannot restore the expression of CD1a, suggesting that macrophage colony stimulating factor and interleukin 6 are involved in the suppression of mesenchymal stem cells.

The inhibitory effect of mesenchymal stem cells on the differentiation and maturation of dendritic cells indirectly leads to the inhibition of T cell activation, thereby suppressing the adaptive immune response. Mesenchymal stem cells have an inhibitory effect on dendritic cells through cell contact and soluble factors. This inhibitory effect may also involve macrophage colony stimulating factor, interleukin 6 and prostaglandin E2, and its specific mechanism needs further study.

At present, there are many clinical trials on the immune regulation of mesenchymal stem cells, and different mesenchymal stem cell isolation methods and different mesenchymal stem cell sources have different effects on the immune regulation of mesenchymal stem cells. Mesenchymal stem cells play an immunoregulatory role in the microenvironment, and different microenvironments also have different effects on their immune regulation.

References

[1] Che N, Li X, Zhang L, et al. Impaired B cell inhibition by lupus bone marrow mesenchymal stem cells is caused by reduced CCL2 expression. J Immunol.2014;193(10):5306-5314.

[2] Ribeiro A, Laranjeira P, Mendes S, et al. Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells. Stem Cell Res Ther.2013;4(5):125.

[3] Nekanti U, Dastidar S, Venugopal P, et al. Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia[J]. International Journal of Biological Sciences, 2010, 6 (5) :499-512.

[4] Boxall SA, Jones E. Markers for characterization of bone marrow multipotential stromal cells[J]. Stem Cells International, 2012, 2012, 975871.

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Potential therapies for SARS-CoV-2 can be divided into two categories: one directly targets the novel coronavirus, and the other is human-oriented. Viruses must enter the host cell to replicate in order to function. Therefore, antiviral drugs can be designed for a series of targets in the process of virus interaction with cell receptors, gene replication, transcription and translation when the virus invades host cells and completes its life cycle. In addition to the above drug research and development against the virus, we can also take the human body as a guide to find therapeutic drugs for the immune response caused by the virus. In the early stage of viral infection, the human body's own immunity can be appropriately improved/activated by the activation of interferon or innate immune receptors to fight the virus. For severely advanced/immune hyperactive patients, since patients are attacked by their own immune system, they can consider using immunosuppressive drugs or mechanisms to adjust. In addition, it can also be treated against the mechanism/target where the host and the pathogen interact with each other. Currently, the main treatment methods for the disease include small molecule drugs, biomacromolecule drugs, gene therapy, cell therapy, traditional Chinese medicine therapy, and medical devices. The potential applications of them in the treatment of SARS-CoV-2 are as follows.

 

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Chemical small-molecule drugs: Small-molecule drugs are currently one of the types of drugs that are expected to be used for the treatment of SARS-CoV-2. There are now multiple small-molecule drugs in clinical trials, and their effectiveness and safety against the coronavirus needs to be further verified.

 

Biomacromolecule drugs: These drugs have high specificity and strong continuity and compliance in the body, but their production cost is high, and because they only act on the cell membrane and outside the membrane and cannot enter the cell, so the target is limited. At present, no specific antibodies have been found to conduct clinical trials in the research of the coronavirus. At the same time, by extracting neutralizing antibodies from the plasma of some recovered covid-19 patients with sufficient concentration of effective antibodies, the potentially harmful components can be removed to treat critically ill patients, but it is unlikely to be promoted on a large scale.

 

Gene therapy: A disease treatment method developed in recent years. It usually uses transgenic methods to make up for missing functional genes or strengthen gene functions. A variety of methods have been developed, such as precise gene editing. However, there is no gene therapy for the coronavirus. This method is generally for chronic, long-term viral infections, such as gene editing of human cells, so that the pathogens lose the ability to infect the cells, or suppress the virus gene expression through RNA interference.

 

Cell therapy: Also a disease treatment method developed in recent years. One of the cell treatment methods is to supplement and replace the missing cells, such as the use of stem cells; one is to regulate the body's immunity by cytokines secreted by the transplanted cells, and this way is not to supplement or replace the cells, and then the transplanted cells will disappear; the other is that the transplanted cells can target and attack specific harmful cells, such as the specific recognition and removal of cancer cells by CAR-T cells. Cell therapy has been studied in the treatment of the SARS-CoV-2, such as the use of mesenchymal stem cells for immune regulation. In the future, we can also consider using specific stem cells for tissue repair and regeneration; organoids derived from stem cells can be used for disease modeling and drug screening.

 

Medical device treatment: In addition to drug treatment, respiratory support treatments that rely on medical devices, such as oxygen therapy, invasive mechanical  ventilation, extracorporeal membrane oxygenation (ECMO), and circulation support, also played an important role in the treatment of severe and critically ill patients with COVID-19.

 

Traditional Chinese medicine treatment: SARS-CoV-2 belongs to the category of "epidemic" diseases of traditional Chinese medicine. Its research idea is different from Western medicine. It does not aim at a specific target but cures the disease in a systematic way. Through in-depth observation and treatment of patients, on the basis of summarizing and analyzing the diagnosis and treatment schemes of traditional Chinese medicine across the country, combing and screening the treatment experience and effective prescriptions in various regions, it is necessary to judge its effectiveness and safety through rigorous clinical trials.

 

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RNA interference (RNAi) is a revolutionary discovery in genetic research. RNAi can specifically silence endogenous or exogenous target genes, which is a rapidly developing gene control technology with broad application prospects. Since Tuchl et al. found in 2001 that artificially synthesized double-stranded small interfering RNA (siRNA) can efficiently silence the expression of target genes in mammalian cells, the life science field has set off a wave of basic and applied research on siRNA. With the gene silencing mechanism, significant progress has been made in the development of siRNA drugs to treat diseases.

In August 2018, the world’s first siRNA drug, Onpattro (Patisiran) was approved by the FDA for the treatment of patients with polyneuropathy caused by hATTR.

Onpattro is the world's first drug developed based on the Nobel Prize achievement RNA interference technology. In 1998, Andrew Fire and Craig Mello revealed the RNAi phenomenon for the first time in C. elegans, and won the Nobel Prize in Physiology in 2006 for this discovery. The approval of the first siRNA drug in 2018 takes 20 years. Onpattro's approval marks a major breakthrough in the field of RNAi medicines, and small nucleic acid medicines are finally dawning.

According to statistics, among disease-related proteins in the human body, more than 80% of the proteins cannot be targeted by current conventional small molecule drugs and biological macromolecular preparations, and are non-drugable proteins. Gene therapy aimed at treating diseases through gene expression, silencing and other functions is considered by the industry as the third generation of therapeutic drugs after chemical small molecule drugs and biological macromolecular drugs. This kind of therapy achieves the treatment of diseases at the genetic level, not restricted by non-drugable protein. As the most mainstream type of RNA drugs in gene therapy, siRNA drugs are used to treat diseases from the level of mRNA. Compared with chemical small molecular drugs and biological macromolecular drugs, they have a higher efficiency at the protein level.

In 2004, Bevasiranib, an siRNA drug developed by Opko in the United States, launched a clinical trial for the treatment of wet age-related macular degeneration. This is the first clinical trial related to siRNA in the world. Subsequently, many global pharmaceutical giants, including Pfizer, Sanofi, Roche, and Merck, joined the development queue of siRNA drugs.

Unfortunately, the development of the Bevasiranib project has failed in phase III due to poor clinical results, and other latecomers have not been spared. The reason is that the poor targeting, off-target effects and stability of RNAi drugs are the most important constraints that affect their efficacy. Many factors have led to the efficacy of these drugs being far less than expected, but also accompanied by serious insurmountable medication side effect.

From the perspective of technical requirements, siRNA after intravenous injection is easily degraded by nucleases, has high renal clearance, poor cell uptake efficiency, and clinical application is limited. The success of siRNA drugs depends on the development of drug delivery system technology, especially the ability to delivery carrier technology that transports RNAi safely and efficiently to specific therapeutic targets in the body.

As the world's first approved siRNA drug, Patisiran adopts the LNP drug delivery system to encapsulate RNAi drugs in liposomes, which can be administered by intravenous injection. The liposome coating greatly improves the stability of the drug and the targeting of liver tissue can ensure that the siRNA will not be filtered by the kidney and will be gradually taken up by the target cells of the liver tissue during the blood circulation. This is the key to Patissiran to overcome the above major constraints and get approved for marketing.

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