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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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immunodiagnosis, an important component of in vitro diagnosis, is used for the diagnosis of various diseases and the determination of immune status. In medicine, it is an important method to determine the cause of disease and lesion site, or to determine whether the immune status of the body is normal. In addition, it is also used in forensic blood stain identification, biochemical serum composition identification and research of species evolution. In addition, it has also been used in forensic medicine for the identification of blood stains, the identification of biochemical serum components and the study of species evolution, etc., which can be carried out in vivo and in vitro. Immunodiagnostic reagents are the most widely used in diagnostic kits and are widely used in hospitals, blood stations and physical examination centers. They are mainly used for hepatitis detection, venereal disease detection, tumor detection and pregnancy detection. Among them, immunodiagnosis includes radioimmunoassay, enzyme-linked immunoassay, chemiluminescence, etc. Enzyme-linked immunity: ELISA has the advantages of low cost and large-scale operation. Meanwhile chemiluminescence immunoassay: CLIA has the advantages of sensitive, rapid, stable, selective, reproducible, easy to operate, flexible and diverse methods.

In medicine, it is an important method to determine the cause of disease and lesion site, or to determine whether the immune status of the body is normal. Western blot theory is a hybridization technique that combines high-resolution gel electrophoresis with immunodiagnostic methods. Western blotting is also called enzyme linked immunoelectrotransfer blot (EITB) It has the advantages of high capacity, high sensitivity and high specificity, and is the most commonly used method for the detection of protein characteristics, expression and distribution, such as qualitative and quantitative detection of tissue antigens, mass determination of polypeptide molecules and antibody or antigen detection of viruses, etc.

Ria (radioimmunoassay) is a method to study the occurrence, development and transformation of the body's reaction to antigens by competitive inhibition reaction between isotopically labeled and unlabeled antigens. Radioimmunoassay involves two techniques: the first is biological. It USES the response of specific antibodies to identify a given organic substance; The second is physics. It introduces radioactive atoms into organic matter and marks them. It can measure almost any substance in the organism, including various hormones secreted by the organism itself, various drugs taken orally or injected by the patient, some virus antigens, etc., and has been widely used in clinical routine tests.

Latex enhanced immunoturbidimetric assay is a method based on the immunological precipitation reaction principle. Its basic principle is to combine the substance to be tested (antigen) in the sample with the human IgG(antibody) coated on polystyrene particles in the reagent to form insoluble immune complex. Compared with the traditional turbidimetric method, the turbidity of the immune complex is further amplified due to the coated polystyrene particles, which overcomes the disadvantage that it is difficult to form turbidity when the amount of antigen-antibody complex is small, improves the sensitivity of detection, meets the requirements of rapid reaction and micro quantization, and is suitable for automatic biochemical instrument detection. Latex immunoassay is not only simple and rapid, but also can be automated, suitable for batch detection, and the reagent cost is relatively low, is one of the best choice of laboratories with automatic biochemical instrument to carry out PCT detection, has a wide range of application prospects.

Lateral flow test principle is in the 1990 s in monoclonal antibody technology, colloidal gold immune chromatography technology and new materials technology developed on the basis of a new type of in vitro diagnostic technology, is fast, simple, single copy detection and economic advantages, has been widely used in medical tests, food quality monitoring, environmental monitoring, agriculture and animal husbandry, entry-exit inspection and quarantine, forensic finalized and other fields. With the rapid development of immune detection technology, quantitative, high-sensitivity, multivariate detection and system integration have become a new research focus in the field of IVD. Compared with traditional ELISA and colloidal gold qualitative lateral chromatography, lateral flow test has many advantages, such as good stability, wide linear range and high sensitivity.

Immunological detection method is a series of experimental methods for the determination of antigens, antibodies and immune cells. With the interdisciplinary infiltration, the scope of immunology is expanding, and new immunological detection methods are emerging one after another. The application of IVD assay is also expanding, which not only becomes an important method for the diagnosis of a variety of clinical diseases, but also will continue to provide convenience for the research of many disciplines.

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There is a large demand for Chinese goods especially in the electronics sector and one can find many options when it comes to choosing the right dropship Chinese wholesaler for obtaining these goods. From digital devices like cameras, picture frames and watches to audio and video systems, solar gadgets, and appliances related to health and lifestyle, a dropship Chinese wholesaler can be the one stop shop to get everything at very reasonable prices.

A dropship Chinese wholesaler usually ensures shipping of the goods within 3 -5 days and the delivery is through UPS, DHL or EMS. These dealers like BigBoxStore and Ownta, are secure and certified dealers. Such dealers will have most of the personal information of the consumer and it is good to check if they are PayPal verified sites with SSL encryption.

The other advantage in buying goods from a dropship Chinese wholesaler is the customer service, which is usually available round the clock. The dealers also give warranty for their goods which in sometimes is coupled with the product company's extended warranty. The dealers for the sake of their own business ensure that the customer gets to know the best prices for various stock like cell phones, dvd players etc. Unlike most wholesale stores, one can get good prices even when they buy in small quantities from a dropship Chinese wholesaler.

The dropship Chinese wholesaler would package the goods at attractive prices and usually for the sake of reputation ensures that they are not damaged. Many sites like Chinavasion offer products with a 12 month warranty which is a decent time period to test the quality of a product. The sites are also advanced and give the customer a real-time peek into the supplies, so customers do not have to go through the disappointment of trying to book an article only to know later that the stock is not available.

Importers also do not have to worry about the paper work and clearance required for the items as it is usually taken care of by the courier companies most of the times. As a dropship Chinese wholesaler deals in constant shipping of goods, one can ensure the best prices in shipping which sometimes might not be charged on the customers at all. This comes as stark contrast to buying from individual stores where customers usually pay the shipping charges which could be substantial if you are from a different part of the globe altogether.

Import Express is a wholesale online site from China. Find cheap import products at wholesale price, including Clothing, Mobile Phone, Computers, Jewelry, Beauty, Sports, Home decor and so on, Shopping on Import-express For Small Businesses now.

Import Express is supposed to be the first priority for new comers. They have global reach servicing customers in more than over 200 countries.

Import Express, a B2B based wholesale website. Since 2005, Import express has played a critical role in helping more retailers grow faster around the world. As an experienced end-to-end wholesale solution company, Import-express provides professional wholesale and drop ship service. The website continue to provide high-quality wholesale business service that empowers retailers to do more business all over the world.

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  1. Introduction

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide; with an estimated 35,000 to 50,000 cases and 10,000 deaths annually. Approximately 25% of encephalitis patients die while about 50% of the survivors develop permanent neurologic and/or psychiatric sequelae, including memory loss, impaired cognition, behavioral disturbances, convulsions, motor weakness or paralysis, and abnormalities of tone and coordination. The virus is a member of the JE serogroup of the genus Flavivirus, family Flaviviridae, and is transmitted between vertebrate hosts by mosquitoes, principally by Culex tritaeniorhynchus. Flaviviruses are known to cause many diseases in humans. Japanese encephalitis virus (JEV), West Nile Virus (WNV)Dengue Virus (DENV)Yellow Fever Virus (YFV) and Zika Virus (ZIKV) are the most common flaviviral infections.

Japanese encephalitis was recognized in horses and humans as early as 1871. In 1924 a severe epidemic was reported from Japan. In 1934, Hyashi reproduced the disease in monkey by intra-cerebral inoculation. In 1935, JE virus was isolated from human brain in Tokyo, Japan, and its virological and serological prototype, Nakayama strain, was established. Five genotypes of JEV are known at present. Different genotypes of JEV (associated with different virulence patterns) thrive in a particular climatic condition: genotypes IV (the oldest) and V are isolated in the tropical endemic region of Indonesia–Malaysia, whereas genotypes III and I are found in the epidemic region.

  1. Signs and Symptoms

Most JEV infections are mild (fever and headache) or without apparent symptoms, but approximately 1 in 250 infections results in severe clinical illness. Severe disease is characterized by rapid onset of high fever, headache, neck stiffness, disorientation, coma, seizures, spastic paralysis and ultimately death (Figure 1). The case-fatality rate can be as high as 30% among those with disease symptoms. Of those who survive, 20%–30% suffer permanent intellectual, behavioural or neurological problems such as paralysis, recurrent seizures or the inability to speak.

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Figure 1. Common symptoms of Japanese encephalitis.

The incubation period of JEV is about 10-15 days. Most of the infected patients have no symptom or mild symptom, only a few with central nervous system symptoms, such as high fever, headache, cachexia, disturbance of consciousness and convulsions, etc. The manifestation can be divided into four levels according to the severity of the disease:

a. Mild symptoms. Patients remain conscious, but with cachexia in different degrees. Usually with no convulsions except several child patient caused by high fever. The temperature of the patient could between 38~39℃. Most mild symptom patient recovery within one week. The infection usually needs CSF or serological test to confirm.
b. Common symptoms. Patients with conscious disturbance such as lethargy or mild coma, abdominal reflex and cremasteric reflex disappearing, may have sort term convulsion. The temperature of the patient could be about 40℃ last for 10 day, no sequela.
c. Severe symptom. The temperature keep above 40℃, patients become unconscious and with repeated or continuous convulsion. Usually have localizing symptom and sign. Central respiratory failure could also appear. The progress could last for more than two weeks. Patients could have mental disorder and paralysis among recovery phase, some would have sequela after recovery.
d. Burst symptom. The temperature rising quickly, patient become high or over fever, and with repeated or continuous strong convulsion. Could be die due to the respiratory failure if not rescue in time. Survival usually suffer serious sequela.
  1. Viral Structure

JEV is an enveloped virus about 50 nm in diameter with a single stranded (ss), plus sense, RNA genome of ~11 kb in length. The genome is organized into a capsid formed by multiple copies of capsid C protein; which is covered by a host derived lipid bilayer. The genome has one open reading frame (ORF) encoding for a single polyprotein, which is cleaved into 3 structural proteins e capsid C, precursor to membrane (prM), envelope E and 7 non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, by viral proteases and host signalases, post translationally.

C protein has w120 amino acids and forms homodimers. It is involved in packaging of the viral genome and formation of the nucleocapsid. prM (~165 aa) and E (~495 aa) are glycoproteins having two transmembrane helices. They are released from the nascent polyprotein following co-translational cleavage by signal peptidases. In the immature virions, prM protein might serve as a chaperone for folding and assembly of the E protein. The prM gets cleaved by cellular furin-like protease to form M (~75 aa) and the protein peptide during the maturation of the flaviviruses in the Golgi complex. Ninety homodimers of E protein present in the host derived lipid bilayer form the major mature virion component. The E protein is the main target of neutralizing antibodies and contains a cellular receptor-binding site(s) and a fusion peptide.

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Figure 2. Viral structure of Japanese encephalitis virus. The viral particle (A), the E protein structure (B) and the cross-section profile of JEV (C).

  1. Diagnosis, Treatment and Prevention

Diagnosis:
Individuals who live in or have travelled to a JE-endemic area and experience encephalitis are considered a suspected JE case. To confirm JEV infection and to rule out other causes of encephalitis requires a laboratory testing of serum or, preferentially, cerebrospinal fluid. Surveillance of the disease is mostly syndromic for acute encephalitis. Laboratory diagnosis of JE is generally accomplished by testing of serum or cerebrospinal fluid (CSF) to detect virus-specific IgM antibodies. JE virus IgM antibodies are usually detectable 3 to 8 days after onset of illness and persist for 30 to 90 days, but longer persistence has been documented. Therefore, positive IgM antibodies occasionally may reflect a past infection or vaccination. Serum collected within 10 days of illness onset may not have detectable IgM, and the test should be repeated on a convalescent sample. For patients with JE virus IgM antibodies, confirmatory neutralizing antibody testing should be performed. In fatal cases, nucleic acid amplification, histopathology with immunohistochemistry, and virus culture of autopsy tissues can also be useful.

Treatment:
There is no antiviral treatment for patients with JE. Treatment is supportive to relieve symptoms and stabilize the patient.

Prevention:
Safe and effective JE vaccines are available to prevent disease. There are 4 main types of JE vaccines currently in use: inactivated mouse brain-derived vaccines, inactivated Vero cell-derived vaccines, live attenuated vaccines, and live recombinant vaccines. Over the past years, the live attenuated SA14-14-2 vaccine manufactured in China has become the most widely used vaccine in endemic countries, and it was prequalified by WHO in October 2013. All travelers to Japanese encephalitis-endemic areas should take precautions to avoid mosquito bites to reduce the risk for JE. Personal preventive measures include the use of repellents, long-sleeved clothes, coils and vaporizers. Travelers spending extensive time in JE endemic areas are recommended to get vaccinated.

Article source: https://www.creative-diagnostics.com/Japanese-Encephalitis-Virus.htm.

References:

1. Misra U K, Kalita J. Overview: japanese encephalitis[J]. Progress in neurobiology, 2010, 91(2): 108-120.
2. Johri; Sunit K. Singh S. Japanese encephalitis virus: from genome to infectome[J]. Microbes and Infection, 2011 (4).
3. Solomon T, Ni H, Beasley D W C, et al. Origin and evolution of Japanese encephalitis virus in southeast Asia[J]. Journal of virology, 2003, 77(5): 3091-3098.
4. Luca V C, AbiMansour J, Nelson C A, et al. Crystal structure of the Japanese encephalitis virus envelope protein[J]. Journal of virology, 2012, 86(4): 2337-2346.
5. Ghosh D, Basu A. Japanese encephalitis—a pathological and clinical perspective[J]. PLoS negl trop dis, 2009, 3(9): e437.
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Antibody-drug conjugates (ADCs) consist of recombinant monoclonal antibodies (mAbs) that are covalently bound to cytotoxic chemicals (called warheads) through synthetic linkers. ADCs not only have the anti-tumor efficacy of highly cytotoxic small molecule drugs, but also combine the high selectivity, stability and favorable pharmacokinetic characteristics of the mAb. In this article, the selection of antigen targets in ADC development, the warheads used in ADCs in clinical stages, the design and optimization of linkers, antibody selection and optimization, site-specific and alternative conjugate chemistry, and strategies to enhance potency are discussed. Non-oncology ADCs are also included.

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  1. Antigen target selection

A major problem in the development of cancer ADCs is the identification and validation of sufficient antigenic targets for the mAb. Several factors need to be considered in antigen selection. First, an ideal antigen of interest has a high level of expression in a tumor, but little or no expression in normal tissues, or at least expression is limited to a given tissue type to reduce ADC target toxicity and result in an acceptable therapeutic index. Second, the target antigen should be present on the cell surface so that circulating mAbs can enter. Third, the target antigen should be an intrinsic antigen, so that after binding, the ADC is transported into the cell, and the cytotoxic agent can exert its function.

However, non-internal ADCs may show significant toxicity in some cases and often cause a strong "bystander effect." Future designs should consider the relative role of cytotoxic drugs and antibodies in the antitumor activity and toxicity profile of the entire ADC.

  1. Warhead for clinical ADC

ADCs currently undergoing clinical trials, most targeting DNA or microtubules, have been efficiently optimized. Since the number of antigens on the surface of tumor cells is limited (about 5,000-106 antigens per cell) and the average drug-to-antibody ratio (DAR) of ADC is limited to 3.5-4, that is, the amount of ADC transferred to tumor cells is very low, and the cells that are combined with cell toxicity may cause clinical failure.

Auristatin and maytansin, which act by inhibiting tubulin assembly, are the most commonly used warheads for ADCs today. Other warheads used are based on pyrrolobenzoquinazoline (PBD), dihydroindolothiazepine, Tubulysins, calicheamicin, irinotecan derivatives, docamomycin, camptothecin analog, adrenaline and doxorubicin and so on. In fact, due to fierce competition, more and more ADCs in early clinical trial studies did not disclose the chemical structure of antigen targets and/or warheads and linkers.

  1. Linker design and optimization

Premature release of the drug in the circulation can lead to systemic toxicity and lower therapeutic indicators. An effective linker design must balance good stability over a few days in the cycle as well as efficient lysis when delivered to target cells. To enhance the solubility and DAR of ADCs and overcome the resistance induced by extracellular proteins (such as MDR1) delivered by chemotherapeutic agents, several strategies are being investigated: conditional release of drugs in the cytoplasm of target cells (removable and non-removable cleavage of the linker); the enhancement and limitation of the bypass effect is achieved by the ability to cross the biofilm-linker-drug metabolite; the polar linker increases solubility and reduces MDR.

  1. Antibody selection and optimization

Both antibody and ADC are forced to increase the homogeneity and developability of the antibody to reduce the rate of drug loss. Analytical techniques such as liquid chromatography, electrophoresis and mass spectrometry not only help to select clones of the best antibodies with suitable glycosylation characteristics, but also for the comprehensive structural identification of research fields and potential clinical candidates, as well as for identification "Hot spots" on antibodies that are detrimental to stability and pharmacokinetic and pharmacological properties. Mass spectrometry also helps to optimize the structure of next-generation mAbs from a pharmaceutical perspective, allowing the development of drug candidates (OptimAbs) and ADCs (OptimADCs) with reduced CMC loading and better drug properties. Selection of antibodies includes chimeric, humanization and selection of human antibodies as well as isotype selection.

  1. New coupling strategy

The second-generation of ADCs are controlled mixtures of different drug loading materials with a typical average DAR of 3.5 or 4. Species with DAR greater than 4 show lower tolerance, higher plasma clearance and reduced in vivo efficacy. Most ADCs today have a common structural feature, such as a thiosuccinimide linkage formed by a maleimide reaction of a thiol and an alkyl group. However, most ADCs cause measurable removal of maleimide during long-term circulation, which can be solved by site-specific coupling and alternative conjugate chemistry: for example, engineering cysteine, unnatural amino acid engineering, enzymes auxiliary ligation, sugar recombination and sugar binding, amino-terminal engineered serine, ligation to the Fab nucleotide binding site, re-bridging of native cysteine, avoiding retro-Michael disintegration and high load of ADC.

  1. Improve the performance of the ADC

Additional strategies to improve ADC performance can be designed to avoid potential resistance to warheads, by using smaller protein scaffolds to enhance tumor penetration, or by combining ADCs with recently approved mAb-based immune checkpoint inhibitors.

  1. ADC for non-oncological indications

There are few studies on ADCs for non-cytotoxic drugs. For example, an ADC directed against C-X-C chemokine receptor type 4 is an antigen that is selectively expressed on serum cells; antibody-antibiotic conjugates (AAC) against S. aureus in cells.

  1. Conclusions and future development

The development of ADCs has benefited from general improvements in the design of therapeutic mAbs, as well as specific improvements in conjugate synthesis methods that enhance homogeneity. The linker strategy and the diversity of warheads provide new opportunities to improve drug delivery in tumors, while also reducing the chances of drug exposure to normal tissues. In fact, it is important to better understand the toxicity determinants of ADC, whether it is used as a single drug or in combination with other therapies. To increase the therapeutic index, the ADC needs to reduce the minimum effective dose under the potency of the cytotoxic agent, or increase the maximum tolerated dose in terms of tumor selectivity. The next generation of ADCs relies on the synthesis and characterization of more homogenous and stable ADCs with macromolecular structures with medicinal chemical-like control. Recent ADC developments have revived interest in cytotoxic natural products, and in the future, the breakthrough in ADC performance may involve warheads with new combat mechanisms. In addition, alternative forms of new mAbs have emerged, but must be compared to full mAb in terms of therapeutic indicators such as toxicity, efficacy, and pharmacokinetics.

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7. The remaining kit materials should be properly preserved

After the standard product is packed, it can be stored according to the temperature required by the manual. This can avoid contamination. It can avoid repeated freezing and thawing of the standard products that require cryopreservation. The enzyme label plate is put back into the foil paper bag, desiccant is added, the sealing is sealed, and stored at 4 ° C. Do not completely balance the enzyme plate to room temperature, then put it back into the foil paper bag, because at this time due to the temperature difference, there will be water vapor on the microplate, which is not conducive to stable storage.

8. Add samples in a fast and accurate way, avoiding air bubbles

The loading time should be controlled within 15 minutes. Mix the sample before loading, especially the sample that is thawed after cryopreservation (the sample that is naturally melted will have stratification), and mix it upside down, but pay attention to the movement and avoid bubbles. If the frozen sample is precipitated, it can be centrifuged again. Care should be taken to avoid air bubbles throughout the loading process. Air bubbles affect the mutual binding of proteins and affect the reaction.

9. Ensure uniform temperature during incubation to prevent evaporation and drying

The sealing film is compressed during incubation. When multiple plates are tested together, lay the plates flat and do not stack them to avoid drift or edge effects due to uneven temperature.

10. Application of the plate washer

The use of a plate washer not only reduces labor intensity, but also helps to ensure the stability of the ELISA test. At present, the automatic plate washer not only has many practical washing actions, such as bottom flushing, two-point aspiration, vibration, and cross-priming; it can also adjust parameters such as flushing pressure, flushing distance, and flushing time as needed. By optimizing these parameters, a good washing effect can be ensured. The performance of the plate washer has a great influence on the accuracy of the results. A good plate washer should ensure that the residual liquid after washing is as small as possible, generally not exceeding 2 μL, and the artificial clapper paper is not wet after washing. The suction needle should be checked regularly for blockage.

11. Manual washing instruction

Add lotion quickly. The lotion should be freshly prepared to avoid contamination by other reagents or bacterial. Soak it for 30s-1min. The pouring of the seesaw should be quick and easy. After pouring, draw the board on the absorbent paper and use high-quality absorbent paper without dust or debris. It is necessary to force the plate so that there is no visible liquid hanging in the hole; but it should not be too heavy to allow the sample to dry for too long. Immediately do the subsequent dosing after the ELISA plate is patted.

12. Key points of color reaction

The ELISA assay is an enzymatic substrate color reaction, which increases with time and darkens, so choosing the optimal time to terminate the reaction is an important factor in obtaining an accurate standard curve and sample concentration. The termination process should be complete. When using the TMB substrate, the complete termination point is yellow, and there will be no blue or green color. If necessary, the 96-well plate can be oscillated if necessary during the termination process, or mixed with a pipette tip (the stop solution contains strong acid to avoid corrosion).

13. Data analysis

Perform curve fitting and analysis according to the data analysis method recommended in the manual. The standard curve in the specification is the result obtained by a very experienced operator in the manufacturer's laboratory. The customer's standard curve and the standard curve in the specification may differ, but it’s not means bad. Generally, the criteria for judging the standard curve are: background OD value <0.2; highest point OD value>1.0; correlation coefficient R2 is close to or equal to 1. As long as the standard curve that meets these conditions is available.

Summary

When you understand and master these unique tips for ELISA, you are very close to make an excellent ELISA experiment.

Creative Diagnostics provides a wide range of enzyme-linked immunosorbent assay (ELISA) kits for the detection of hundreds of different proteins and molecules, including cytokines, growth factors, markers for infectious diseases, diabetes and tumor, drugs and small molecules, etc. You can search for the ELISA kit you need by target name, species, etc. on their website. Should you need any assistance, the company’s experienced scientists are more than happy to provide optimized protocols and helpful technical support to accelerate your research.

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An overview of several commonly used antibodies for neurological and neuroendocrine tumors

1. Neuron-specific enolase (NSE)

It has been found that there are three kinds of the same enzymes in the brain tissue, namely dd.dr and rr. NSE belongs to the rr shape. It exists not only in nerve cells and their axons, but also in various endocrine cells and their associated tumor cells. Previous experiments detected 69 cases of APUD system tumors by NSE, and all of the results were positive; while only 2 of the 16 cases of secretory glands such as pancreatic cancer were positive. It can be seen that NSE is a very useful antibody for detecting tumors in the APUD system. It is suitable for a variety of tissue sections, with monoclonal and polyclonal antibodies, and better results can be obtained with antigen retrieval.

2. Chromogranin A

Chromogranin A antibody detects neuronal and neuroendocrine cells and its related tumors, such as pheochromocytoma, medullary thyroid carcinoma, islet cell tumor, pituitary tumor, carcinoid and so on. Monoclonal antibodies and polyclonal antibodies are available, which are suitable for various types of slices and antigen retrieval is required.

3. Glial fibrilary acidic protein (GFAP), clone 6F2

GFAP is one of the intermediate filament proteins and is an effective marker for astrocytes and ependymal cells and their derived tumors. According to the immunohistochemistry of 60 astrocytomas cases, the results were all positive. Therefore, it is the prior antibody choice for astrocytoma and ependymoma. Polyclonal antibodies have been used in the early stage, and monoclonal antibodies have been recently used, which do not require any treatment and are suitable for various sections.

4.S-100

S-100 is a protein isolated from bovine brain. Three sub-shapes of S-100 have been discovered through extensive research, i.e. S-100ao, S-100a and S-100b. It can be detected in human Schwann cells, ependymal cells, astrocytes, nerve fibers, nerve sheaths, skin melanocytes, and the like. Therefore, it can be used to detect tumors of the above tissues and their sources.

The antibody is suitable for various sections and does not require any treatment. The dilution should be 1:200 and the background should be controlled as much as possible.

Other antibodies for cancers

1. Thyroglobulin (TG), clone 2H11

TG is a glycoprotein synthesized by thyroid follicular cells. It is an antibody with high specificity among many antibodies. It is only used to detect thyroid tissue and tumors derived from it, without any treatment and good results can be obtained. It adapts to a variety of slices, but background control should be noted.

2. Bovine Dapiuomavirus (BPV)

The antibody is mainly used for detecting infectious diseases of the reproductive system such as condyloma acuminata. The virus is present in the hollowed cells of the epithelium. According to experiments, not all the hollow cells contain virus after color development detecting, which may be due to loss of cells on slices in the process of production. In the markers of 50 cases, viruses of varying contents were found. The antibody is a polyclonal antibody, and the dilution can be 1:400 when vacuum negative pressure detection is used. The background control should be noted, and good results can be obtained without any treatment, and are suitable for various slices.

3. Proliferation cell nuclear antigen (PCNA)

PCNA is an acidic protein with a molecular weight of 36 kD, which is an aid to DNA polymerase when DNA is replicated. This protein appears when the cell enters the proliferative cycle. It is a marker of the cell proliferation cycle, especially the "S" phase. Suitable for all kinds of slices.

4. Cow calf alkaline phosphatase (CAP)

The antibody detects normal osteoblasts, small intestinal cells, prostate epithelial cells, and cells containing CAP throughout the body. However, it is commonly used in clinical tests to detect the content of CAP in bone tumors and prostate tumors. The antibody does not require any treatment and is suitable for various sections.

5.Estrogen receptor (ER)

ER is an acidic protein with a specific function. It transmits hormone information. The study found that this substance is present in the nucleus of cancer cells, such as breast cancer, endometrial adenocarcinoma, cervical cancer and other cancer. At present, in the treatment of these cancer patients, it is first necessary to detect whether the ER is positive. If positive, anti-hormone therapy will be taken, if negative, hormone therapy will be taken. In conclusion, the positive or negative result of ER test is directly related to the choice of treatment plan, and is a hard indicator to judge the prognosis of patients. At present, when detecting ER, it is necessary to perform thorough and complete antigen retrieval. According to experiments, in order to obtain satisfactory results, it is necessary to perform long-time bath-type antigen retrieval, that is, immersing the slices in the container of the antigen repair solution, and then put it together in the pot and boil for forty minutes. If the antigen retrieval time is not enough, the slice can also be negative. In addition, high pressure retrieval, vacuum negative pressure and microwave retrieval can also be used.

6. Progesterone receptor (PR)

PR, like ER, is listed as a mandatory test in the treatment of breast cancer, cervical cancer, endometrial adenocarcinoma, etc. In order to develop a medical plan and judge the prognosis hard indicator, the general test is performed simultaneously with ER and PR.

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Abstract: In recent years, the development of immunology research has brought many exciting breakthroughs in the innovation of cancer diagnosis and treatment strategies. Immunotherapy has become the fourth tumor treatment mode after surgery, radiotherapy and chemotherapy, and has been successfully used in the treatment of various tumors. Among them, adoptive immunotherapy has received great attention in the past few decades. In vitro cultured tumor infiltrating lymphocytes or lymphocyte-activated killer cells can affect tumor regression in a specific cancer. However, adoptive immunotherapy is not a first-line treatment in clinical practice as adjunctive therapy due to certain limitations. With the development of genetic engineering, immunotherapy using genetically modified T cells is more attractive in the treatment of human tumors. Chimeric antigen receptor is one of the strategies for genetically modified T cells. CAR-T cell therapy is a method of combining the high specificity of antibodies to antigens and the cytotoxic activity of T cells to target cells. T cells expressing a chimeric antigen receptor are obtained by genetic recombination, purified, expanded and activated in vitro, and infused into a patient to perform the function of killing tumor cells. In the past 25 years, CAR-T has shown good targeting, killing activity and persistence in both in vivo and clinical trials, in the treatment of malignant tumors such as leukemia, lymphoma, glioma and lung cancer. A delightful effect has been achieved. Through artificial genetic modification, T cells express single-chain antibodies that recognize tumor-specific antigens and signals that activate T cells, thereby achieving targeted killing of tumor cells. At present, clinical studies have confirmed that CAR-expressing T cells can be effectively used to treat refractory or relapsed acute B-lymphocytic leukemia, and thus promote the use of CAR-T for the treatment of other hematological malignancies and solid tumors. This article mainly introduces the research progress and broad prospects of chimeric antigen receptors.

Keywords: chimeric antigen receptor, T cell, tumor, immunotherapy

Car-T cell structure and preparation

Car is located on the surface of T cell membrane and is a Tran’s membrane structure composed of extracellular binding domain, hinge region, Tran’s membrane region and intracellular signal region. Although the adverse effects of CAR-T treatment have not been fully resolved, the success of anti-CD19CAR-T has pushed the treatment of CAR to industrialization. Currently, most CAR-T production processes are cumbersome and expensive. Production procedures include isolation, activation, transduction, proliferation, and reinfusion of T cells: 1 Acquisition of peripheral blood T cells from patients using apheresis: 2 Activated T cells: Activated T cells typically use anti-CD3 and anti-CD8 immunomagnetic beads, Cytokines such as interleukins can also be added to enhance the activation effect; 3 CARs synthesized by genetic engineering are transduced into T cells, and the transduction methods can be divided into viral transductionand non-viral Transduction (electroporation, transposon-based or gene editing system transduction methods) 4CAR-T will be placed in medium with IL-2 and other cytokines for 2 weeks; 5CAR-T is washed and concentrated, And quality inspection, only after the quality inspection, CAR-T can be returned to the patient.

How does CAR-T work?

T cells are specific anti-tumor effector cells in humans, but their function is MHC-restricted. The construction of a specific chimeric antigen receptor was first proposed in 1989. This process first combines the T cell receptor (TCR) subunit with both single-chain variable regions of tumor antigen monoclonal antibodies, and then transfects by retroviral, lent viral vector or transposon gene transduction methods. T cells are stained, and the finally modified T cells are expanded and purified in vitro and then returned to the body. The constructed CAR-T cells can specifically recognize and bind tumor antigens in vivo, and have the ability of T cells to self-renew and kill. T cells are genetically modified to obtain targeted killing activity, which is not inhibited by the local immune microenvironment of the tumor, thereby breaking the host's immune tolerance state, and allowing T cells to specifically recognize specific antigens on the surface of tumor cells in a non-MHC-restricted manner. Enhance its ability to recognize and kill tumor cells.

Application of Car-T cells in tumor therapy

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Traditional radiotherapy, chemotherapy, surgery and other cancer treatment methods still have great limitations in the treatment of refractory and recurrent tumors. Cellular immunotherapy has pointed out a new direction for cancer treatment. Among them, the therapeutic effect of Car-T cells is particularly prominent. Since the birth of Car-T cells, with the continuous improvement of its functions, it has gradually moved from scientific research to clinical practice. It has been widely used in hematological tumors, lymphomas, melanomas, ovarian cancers, neuroblastomas, kidney cancers, etc. In the treatment of malignant tumors, CD19 is the initial and most studied target of Car-T cells, and has outstanding clinical efficacy in the treatment of leukemia and lymphoma. CD19 is only expressed on the surface of B lymphocytes, so CD19Car-T cells input into the body do not damage normal cells of the body. It has been reported that CD19Car-T cells in patients with refractory relapsed chronic lymphocytic leukemia, acute lymphocytic leukemia, and non-Hodgkin's lymphoma have achieved satisfactory response to complete remission and no recurrence.

Several security issues that cannot be ignored

Off-target effect

What is the off-target effect? Most CAR-targeted target antigens are not tumor-specific and have varying degrees of expression in normal tissues, so CAR-modified immune cells can also damage normal tissues while attacking tumor tissue. Therefore, the chimeric antigen receptors expressed by CAR-T cells can be two or more, and generally normal tissue cells expressing only one antigen can be prevented from being damaged by CAR-T, or can be selected only for tumor cells. The target antigen on the surface is specifically bound to tumor cells without binding to normal tissue cells.

Cytokine release syndrome (CRS)

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CRS is a syndrome in which CAR-T cells are proliferated in a large amountafter exposure to cells with tumor antigens, and a large number of tumor cells are killed. In this process, a series of cytokines are formed, which form a cytokine storm and cause serial damage to the patient's body. It is the most common and most adverse reaction in the treatment of CAR-T cells. In response to this problem, not only the design of safe CAR-T cells, but also the number of cells injected into the patient each time is strictly limited, thereby reducing the occurrence of such adverse reactions.

Prospects of chimeric antigen receptors

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In the field of tumor immune cell therapy, chimeric antigen receptor T cell therapy (CAR-T) represents the most advanced technology of today. With the development of modern biological science, CAR-T has received more and more attention. In terms of tumor immunotherapy, CAR-T clinical trial results show that it has unparalleled advantages, but in the technical improvement and large-scale clinical application, CAR-T still has many challenges. How to improve the ability of CAR-T cells to specifically kill tumors and the safety and effectiveness of clinical treatment is an urgent problem to be solved in CAR-T treatment. With the deepening of research and the increasing technology, it is believed that CAR-T therapy will play an increasingly important role in the immunotherapy of tumors for the benefit of medical development and human health.

References

[1]Mo Z, Du P, Wang G, et al. The Multi-Purpose Toll of Tumor Immunotherapy: Gene-Engineered T cells [J]. Journal of Cancer, 2017, 8(9): 1688-1705.

[2] Olbrich H, Slabik C, Stripecke R. Reconstructing the immune system with lentiviral vectors [J]. Virus Genes, 2017, 53(5):722-728

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What are the glyconegineering and glycosylation?

Glycoengineering, as well as protein engineering and gene engineering, belongs to the category of biotechnology, and its basic research is also called glycobiology. The research of glycoengineering is still in the stage of basic and applied basic research. glycoengineering, which was born in 1990s, is a discipline to study the structure, function, metabolic regulation and application of glycoconjugates.With the research and elucidation of the biological functions of sugar chains, a variety of glycan antibodies are being developed, some of which have gone into clinical practice.

Among many post-translational modifications of proteins, glycosylation modification is one of the most important and complex modifications, and also one of the key quality attributes for evaluating antibodies. The function of monoclonal antibodies is closely related to glycosylation modification. Glycosylation modification can affect the properties of proteins, such as conformation, stability, solubility, pharmacokinetics, activity and immunogenicity.

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Figure1.Types of glycosylation

Different glycosylation modifications have different effects on the stability, half-life, safety and biological activity of the antagonists, which are described below.

Stability and half-life

The most obvious effect of glycosylation modification is that it can increase the stability and solubility of protein. Studies have shown that glycosylation protects proteins by hiding binding sites between proteins and proteases. Rudd et al found that the steric protection of N-glycosylation on adjacent polypeptides was due to the formation of hydrogen bonds between glycosylation and hydrophilic amino acids. Glycosylation modification can also hinder the binding of protease and antibody, thereby increasing the stability of antibody.

Sialic acid glycosylation can prolong the existence time of glycoprotein in serum. When the glycoprotein chains are terminated by galactose modification or desalicitated, they can be recognized by the sialic glycoprotein receptor (ASGPR), resulting in a significant reduction in half-life. The ASGPR located on the surface of hepatocytes can not recognize the glycoprotein of total sialic acid, but it will be desalibrated by the non-specific sialidase in the blood as the blood circulates. The galactose glycosyl group of bare can be recognized by ASGPR, and the glycoprotein is degraded. Other studies suggest that high levels of mannose reduce the half-life of antibodies in serum.

In general, the more stable the drug is in the body and the longer its half-life, the more effective it will be. As for antineoplastic drugs, if the stability of drugs is high and the half-life is long, the dosage can be reduced appropriately and the interval between doses can be designed longer, and the effect is the same. However, with the increase of drug stability and half-life in human body, drug safety will become one of the factors to be considered because of potential miss-target.

Safety

The key quality attributes of antibodies (CQAs) are the criteria to judge whether they are qualified or not. CQAs must be within an appropriate range to ensure the effectiveness of drugs and, more importantly, the safety of drugs. By influencing the effect function of Fc terminal, glycosylation modification of Fc terminal can affect the safety of antibodies in the form of immunogenicity, PK/PD.

The development of antibodies has experienced the development of murine primordial antibody, chimeric antibody, humanized antibody, and finally the whole human antibody. The immunogenicity of antibodies has been continuously decreasing.Antibodies have developed into human antibodies, and the immunogenicity caused by different glycosylation modifications should not be considered. However, in order to increase the efficacy of antibodies or prolong their half-life, some amino acid mutations and glycosylation modifications are often introduced artificially, and their potential immunogenicity should be considered when making such modifications. Moreover, immunogenicity is not only caused by internal factors, but also by external factors such as materials, formation of polymers and introduction of surfactants.

The main consideration of PK/PD is the minimum dose and time of drug action in vivo. It is particularly important to minimize the immunogenicity of antibodies that can not completely eliminate the immunogenicity. Glycosylation affects PK mainly by affecting the stability and half-life of antibodies. The effect of glycosylation on the activity of antibodies acts on PD, which will be discussed in the following sections. The clearance of antibodies that bind strongly to Fc receptor is low, and glycosylation can affect the binding strength of antibodies to Fc receptor or C1q complement. If the clearance rate of antibody is low, antibodies bound to Fc receptor or C1q complement may cause the immune system to attack normal cells, resulting in serious miss-target effect.

Biological activity

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are two important pathways for the efficacy of antibodies in cancer therapy. The glycosylation modification sites of Fc end of antibody are usually the binding sites of Fc receptor (ADCC mechanism) and C1q (CDC mechanism). By adjusting glycosylation, the binding of Fc receptor and C1q with antibody can be increased, thus the activity of antibody ADCC and CDC can be increased.It has been reported in many studies that glycosylation of sialic acid can affect the binding of antibody to FcγRIIa, thus reducing the activity of ADCC.

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Figure2.Mechanisms of action of ADCC and CDC

Conformation determines performance. The effects of glycosylation modification on the stability, half-life, safety and biological activity of antibiotics are cross-cutting. It is the best choice to control the glycosylation modification of antibodies within an appropriate range so as to balance the three.

Expectation

Drug glycosylation is a unique post-translational modification. Their synthesis is not a mode-driven process, so it is a difficult task to obtain a consistent glycosylation. Sugar forms are known to regulate a range of complex functions, and it is pertinent to invent strategies that are easy to monitor sugars in real time. In addition to significant progress in process analysis, glycosylation level control remains an unrealized goal. Biopharmaceutical industry and academic researchers have made great efforts to bridge this gap, and have made great progress in understanding the complexity of post-translational modification. They have also understood the effects of process variables on sugar types and the effects of polysaccharide types on the efficacy of biopharmaceuticals. This also clearly implies that in the development of bio-analogue drugs, similar drug manufacturers can not obtain information about the original research and development of drugs.

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Tumors are high-risk and high-mortality diseases that pose a serious threat to human health. A large number of studies and prevention data confirm that early diagnosis and early treatment are the most effective ways to prevent and treat cancer and reduce mortality. Therefore, the search for cancer markers that can be used for early diagnosis has become the focus of attention.

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However, in addition to poor differentiation, easy metastasis and high-speed proliferation, malignant tumor cells are completely equivalent to normal cells in cytological behavior. No specific expression products or shedding components have been found so far, or various malignant cancer markers have not yet been discovered. This brings problems to tumor diagnosis and immunotherapy.The ideal specific cancer markers must meet the following requirements: (1) unique to a certain tumor; (2) related to tumor size and stage; (3) therapeutic monitoring; (4) prognosis; (5) Does not intersect with healthy people and other diseases. Cancer markers that meet these criteria have not been discovered so far and may never be found. In recent years, all cancer markers used in clinical practice are mostly tumor-associated antigens, or some normal people also have trace amounts, but the amount is different. In any case, finding tumor-specific antigens is still a hot spot for cancer research because of the refractory tumors and the need for biotherapy.

Classification of common cancer markers:

1. Cancer Antigen 19-9 (CA19-9)

The carbohydrate antigen CA19-9 is a monoclonal antibody numbered 1116NS19-9 obtained by Koprowski et al. in 1979 using a human colon cancer cell line to immunize BALB/c pure mice and hybridize with myeloma. A class of tumor-associated carbohydrate antigens, the antigen recognized by the monoclonal antibody is named the carbohydrate antigen CA19-9. CA19-9 is a tumor-associated antigen, which is related to Lewis blood group components and can be present on normal tissue cells. It is not unique to tumor cells. It is mainly found in epithelial cells such as the stomach, intestine and pancreas of the fetus. The biliary cells and the stomach, colon, endometrium and salivary gland epithelium can also be synthesized. In certain malignant tumors, CA19-9 in body fluids is often at a high level. Observing the changes of CA19-9 in vivo is conducive to early detection of the above-mentioned malignant tumors of organs, and has certain differential significance for the benign and malignant tumors.

CA19-9 is detectable in serum, tumors, non-neoplastic diseases, and normal humans, but there are significant differences in CA19-9 levels in benign and malignant diseases. In benign diseases, normal people, serum CA19-9 elevation is usually low or transient, mostly below 100U/ml.In malignant tumors, the detection rate of different organs is different. At present, the increase of serum CA19-9 is more common in digestive tract tumors, especially pancreatic tumors, followed by biliary tract, colon, liver and stomach. In lung cancer, breast cancer and ovarian cancer. There is also a certain detection rate. The concentration of CA19-9 and the positive detection rate in the same organ tumor are related to the pathological type. For example, the CA19-9 concentration of colorectal cancer is higher in adenocarcinoma, mucinous carcinoma and signet ring cell carcinoma, but in papillary gland. Cancer and phosphorus cancer are lower. There are many clinical reports on the sensitivity and specificity of serum CA19-9 in various tumors, which are different from the disease and the number of cases in the selected cases, but the general trend is consistent. In the pancreatic cancer, the detection rate of serum CA19-9 is the highest. The sensitivity of each family is about 79%, even as high as 90.2%, and the specificity is between 70% and 90%. It has been used as a test for pancreatic cancer. Preferred serological indicators. The sensitivity in cholangiocarcinoma, gastric cancer, colon cancer, and liver cancer was 67% to 86%, 31.5% to 68%, 50%, and 49% to 60.9%, respectively. The sensitivity in lung cancer and breast cancer is low, about 10%. Many studies have shown that the concentration of CA19-9 is related to tumor size, especially in pancreatic cancer and colon cancer. A large number of studies have shown that preoperative detection of CA19-9 levels can help to determine the prognosis of patients with cancer, especially for patients with pancreatic cancer, colon cancer, hepatobiliary tumors, and gastric cancer. Preoperative high CA19-9 is a prognostic factor. The chances of recurrence and metastasis are high and the survival time is often short.

The tumor marker CA19-9 is a tumor-associated antigen. This index has important significance in the diagnosis, curative effect, prognosis and postoperative monitoring of malignant tumors such as pancreatic cancer, cholangiocarcinoma, liver cancer, gastric cancer and colon cancer. However, its specificity is still low, and the detection rate varies from place to place. How to improve the clinical value of this marker remains to be further studied. The detection rate of CA19-9 varies from one report to another, and it is related to the selected method and the sensitivity of the selected kit, in addition to the difference in the condition and number of cases in the selected case. With the deepening of research, the emergence of more sensitive, more reliable, more reproducible, more convenient, more environmentally friendly and economical assays, the development of highly specific CA19-9 antibody will increase the value in tumor diagnosis.

2.Cancer Antigen 125 (CA 125)

CA125 is a heterogeneous high molecular weight mucin-like glycoprotein containing mainly galactose, N-acetylglucosamine and N-acetylaminogalactose chains. The protein is partially rich in serine and can be recognized as an antigen by the monoclonal antibody OC125, hence the name CA125. Although the CA125 antigen is a glycoprotein, it has both membrane-bound and episomal states. CA125 in plasma and body fluids binds to glycoproteins of different relative molecular masses, respectively, and has the smallest subunit of CA125 immunoreactivity.

CA125 is present in the body cavity epithelial cells of embryonic development and disappears several hours after birth, but reappears in ovarian cancer cells. Immunohistochemistry revealed that CA125 was found in fetal digestive tract epithelial cells, amnion, adult pleura, peritoneal mesothelial cells, fallopian tube endothelium, uterus and cervix, but no CA125 was found in adult and fetal ovarian epithelial cells. Under normal circumstances, CA125 does not enter the blood circulation, so the quality of CA125 is very low in healthy people and most benign diseases. CA125 can enter the blood circulation and various body fluids and exhibit high expression during the growth and metastasis of malignant tumors and when some non-tumor diseases progress. Elevated serum CA125 marker is a direct contact between the shed tumor cells and the systemic circulation when the "natural barrier" in the human body is destroyed, resulting in elevated serum CA125 levels. If the tumor is confined, CA125 has not yet reached the systemic circulation, and the level of CA125 in the blood is not high.

Serum CA125 has been widely used in ovarian cancer screening, diagnosis, disease monitoring, prognosis and treatment. Serum CA125 combined with a variety of tumor markers can greatly improve the diagnosis and differential diagnosis of ovarian cancer. The half-life of CA125 used during chemotherapy can better predict the patient's response to treatment and monitor disease. The quality of serum CA125 can predict the trend of the subject to develop ovarian cancer. Serum CA125 detection combined with CT for the follow-up examination of patients with ovarian epithelial cancer is more valuable for detecting tumor recurrence. In addition, the current experimental and clinical studies on the treatment of ovarian cancer with CA125 monoclonal antibody have made great progress, and may bring new hope for the treatment of ovarian cancer. Examining the test results of elevated serum CA125 to patients can greatly improve the rate of return visits and improve the enthusiasm of women for regular gynaecological examinations.

3. Cancer Antigen 27.29

Breast carcinoma-associated antigen CA27.29 is a glycoprotein antigen produced by the MUC-1 gene. The product of the MUC-1 gene is MUC-1, which is a transmembrane glycoprotein expressed by glandular epithelia. The function of MUC-1 is associated with anti-pathogens and cell signaling. While overexpressed MUC-1, glycosylation changes, and abnormally intracellular localization have been related to cancers. Besides, MUC-1 was found in many types of malignant cells such as breast, ovarian, pancreatic, lung and prostate carcinomas. It can be shed into the circulation of the patients who have breast cancer. Hence CA27-29 is a part of MUC-1 and it can be detected by specific antibodies.

Alterations in glycosylation of MUC-1 have great influence on its physiological and pathophysiological activity by affecting the interaction between the MUC-1 and other target proteins. It has been reported that the abnormal glycosylation of MUC-1 induces novel protein-protein interaction and increases the expression of phospho-IκB kinase (IKK)-β and phospho-inhibitor of nuclear factor kappa-B (IκB)-α which are the membranes of NF-κB family. By stimulation with inflammatory factors such as TNF-α, the cancer form MUC-1 induces activation of NF-κB members and interact with phopsho-p65, then moves to the nucleus to regulate the production of pro-inflammatory factors such as IL-6 and TNF-α. Then, the inflammatory cells like myeloid cells and neutrophils are recruited to the tumor site by the inflammatory cytokines. Moreover, the inflammatory cells are capable of enhancing tumor growth and progression by producing pro-inflammatory and pro-tumorigenic cytokines.

For patients with breast cancer and ovarian cancer metastasis, especially those who have no clinical symptoms after treatment, when the serum CA27.29 is checked regularly, it is found that patients with CA27.29 measured value greater than 40 U/mL are less than the measured value. Patients with 40 U/m L or 40 U/m L have a much lower survival rate. Serum C A27.29 levels can also be used as a predictor of cancer recurrence in other adenocarcinomas, such as metastatic colorectal cancer and pancreatic cancer patients, and tumor metastasis can be found significantly earlier than other imaging techniques. This method is of little significance for the diagnosis and diagnosis of breast cancer patients during the construction period. In addition, in some diseases or physiological conditions, such as benign tumors, serum CA 27.29 values may also increase in the first 3 months of pregnancy, so attention should be paid to differential diagnosis.

4. Cyclin D3 (CCND 3)

Encoded by CCND3 gene, CCND3 protein belongs to the highly conserved cyclin family. All the cyclin members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins act as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns. Different expressions contribute to the temporal coordination of each mitotic event. CCND3 forms a complex with CDK4 or CDK6, and functions as a regulatory subunit of those two proteins. A number of reports have revealed that CCND3 are involved in the phosphorylation of tumor suppressor protein Rb. The CDK4 activity associated with CCND3 was reported to be necessary for cell cycle progression through G2 phase into mitosis after UV radiation. Several transcript variants of CCND3 have been found so far.

CDKs promote cell cycle transitions in mammalian cells by phosphorylating key substrates cyclins. Abnormal expression of cyclins was reported to be involved in many cancers progression. Highly expression of Cyclin D3 was reported in a number of tumor cells. Cyclin D3 gene is amplified in bladder carcinoma in situ. Genomic changes disrupting the expression of Cyclin D3 are involved in the aberrant growth of several human B-lymphoid malignancies. Targeting Cyclin D3 by miR-138 induces cell cycle arrest in hepatocellular carcinoma. Furthermore, Cyclin D3 is selectively required for proliferative expansion of germinal center B cells. For breast cancer, CCND3 is overexpressed in human breast cancer cell lines and primary invasive breast cancers. In addition, E1AF promotes breast cancer cell cycle progression via upregulation of CCND3 transcription. All the studies reveal that CCND3 is the marker of many tumor cells.

Research and development of highly specific CCND3 antibody widely used in disease screening and therapeutic monitoring, providing key products to the research market and providing the highest value for the diagnosis and prognosis of many tumor cells

Reference

[1] Ni X G, Bai X F, Mao Y L, et al. The clinical value of serum CEA, CA19-9, and CA242 in the diagnosis and prognosis of pancreatic cancer [J]. Eur J Surg Oncol, 2005, 31(2):164-169.

[2] Gorp T V, Cadron I, Despierre E, et al. HE4 and CA125 as a diagnostic test in ovarian cancer: prospective validation of the Risk of Ovarian Malignancy Algorithm[J]. 2011.

[3] Cen P, Duvic M P, Kurzrock R. Increased cancer antigen 27.29 (CA27.29) level in patients with mycosis fungoides[J]. Journal of the American Academy of Dermatology, 2008, 58(3):382-386.

[4] Büschges R, Weber R G, Actor B, et al. Amplification and expression of cyclin D genes (CCND1, CCND2 and CCND3) in human malignant gliomas.[J]. Brain Pathology, 2010, 9(3):435-442.

[5] Zheng G, Patolsky F, Yi C, et al. Multiplexed electrical detection of cancer markers with nanowire sensor arrays[J]. Nature Biotechnology, 2005, 23(10):1294.

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Simple Introduction

First of all, let’s start with its definition: Peptide mapping is the main method for the analysis and identification of protein products and preparations. In addition, there are some other core techniques such as peptide mass mapping and peptide mapping ms.

 

The following are functions of Peptide mapping:

To confirm that a protein primary structure (amino acid sequence) including the N and C terminals.

  • To provide protein loci and the proportion of modified groups, such as glycosylation, acetylation, sulphates a00nd phosphorylation.
  • To provide qualitative and quantitative information on protein degradation products
  • To get information on protein oxidation and deamination directly.

According to these useful features above, peptide mapping has already gained in popularity in biological field. After getting numerous data from peptide mapping, the next step is deeper analysis.

 

A sample graph of peptide mapping

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Peptide Mapping Analysis

Based on graphs of peptide mapping and also peptide mass mapping, scientists can get a lot of useful information. According to the size of molecular weight of proteins, peptides and amino acid composition characteristics, the use of strong specificity of proteolytic enzyme [is commonly endopeptidase] on the special peptide chain site will peptide fragment into smaller fragments, through the separation of a certain form characteristic fingerprint detection methods. eolytic enzyme [is commonly endopeptidase] on the special peptide chain site will peptide fragment into smaller fragments, through th

The picture below shows the steps of peptide mapping method:

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Step 1:

Protein digestion: Immobilized trypsin provides rapid and simple protein digestion with high reproducibility, high sensitivity and excellent data quality in a format that is compatible with automated operations.

Step 2:

Peptide separation: this technique provides a complete set of chromatographic tools for all development applications requiring peptide separation and analysis

Step 3:

Biological mass spectrometer: It provides a fast and easy to solve multicomponent analysis method, Used for sequence determination, structural analysis, molecular weight determination and component content determination of polypeptides. What’s more, it has the characteristics of high sensitivity, strong selectivity and good accuracy.

Step 4:

Peptide analysis software: Integrated software can save time and identify more materials. Simple software workflows can guide biotherapy, characterization pathways, and provide comprehensive coverage, including peptide sequence validation and identification of all variants and modifications.

 

So peptide mapping analysis is quite effective and important in some specific aspect of research. Let’s take an example here:

Study on quality control of impurities and harmful substances in Genetic Recombinant Drug is a crucial project., in particular, when the recombinants are used in the production of genetically engineered drugs undergo mutations. Probably, they would bring some mutations into drugs. Besides strengthen control of original material and process of production, peptide mapping analysis is necessary method to ensure safety and consistency.

 

As a result, peptide mapping analysis has given rise to a number of new things. For instance, HPCE, A new electrical technology for separation which emerged in 1980s. Because of its relatively high resolution, HPCE has started a wider road for structural analysis and quality control of protein drugs.

 

At present, in addition to the routine analysis of amino acid sequence of some small peptides, the peptide graph analysis is one of the important conventional indicators to control the consistency of most gene engineering products. All in all, technologies on peptide mapping have already penetrated into every corner of our everyday life.

 

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What is an autoantibody?

Autoantibodies are antibodies that target tissues, organs, cells, and cellular components. The growth, development and survival of the human body have the maintenance of a complete autoimmune tolerance mechanism. The normal immune response has a protective defense effect, that is, it does not react to its own tissues and components. Once the integrity of self-tolerance is destroyed, the body regards its own tissues and components as "foreign substances", and an autoimmune reaction occurs to produce autoantibodies. Normal human blood may have low titers of autoantibodies, but no disease occurs. However, if the titer of autoantibodies exceeds a certain level, it may cause damage to the body and induce disease.

There are many kinds of autoimmune diseases antibodies , the most important of which are antinuclear antibodies. In addition, anticardiolipin antibodies, neutrophil cytoplasmic antibodies, anti-mitochondrial antibodies, anti-erythrocyte antibodies, anti-platelet antibodies, anti-endothelial cells antibodies, anti-neurovirus antibodies, rheumatoid factor, anti-thyroglobulin antibodies, anti-insulin bodies Antibodies and the like are also autoantibodies.

Reasons for autoantibody production:

Antibodies are generally produced by the immune system by foreign proteins or other substances (especially pathogenic bacteria) that enter the body and are used in immune reactions to eliminate harmful foreign substances. Usually, the immune system can recognize and ignore the body's own cells, and does not produce antibodies to it; at the same time, the immune system does not overreact to substances (such as food) that are not threatened in the environment. However, under certain circumstances, the immune system recognizes the body's own substances and treats them as foreign invaders, thereby producing antibodies (ie, autoantibodies) against these substances, triggering autoimmunity. These autoantibodies attack the cells, tissues, and organs of the body, causing an inflammatory reaction and causing damage to the body.

The production of autoantibodies may be due to the presence of some of the same molecular structures between pathogenic antigens (bacteria, viruses, etc.) and their own components: an immune response that cross-reacts with autoantigens; or some infectious agents that cause autoantigens Denatured, the immune system produces autoantibodies to these exposed new antigens.

The pathogenic effect of autoantibodies is still unclear. Whether it is the "cause" or "consequence" of autoimmune diseases has different opinions. For patients with high titers of autoantibodies, those without clinical symptoms may not need treatment, but should go to the hospital regularly. Review and review.

 

In tumors, inflammation, autoimmune diseases (such as lupus erythematosus, genital warts, Crohn's disease, multiple sclerosis), neurodegenerative diseases, infectious diseases, etc., a large number of autoantibodies are produced and accumulated in patients. Some autoantibodies have emerged in the early stages of a specific disease, even before the onset of symptoms of the disease, providing a reliable disease biomarker for the early diagnosis; some autoantibodies are the body's own protection against disease. Sexual antibodies, which provide new ideas for the treatment of the disease, as data from the world-renowned pharmaceutical giants show that 60% of the profits of large pharmaceutical companies have come from drugs that belong to antibodies. So how do you discover these potential autoantibodies?

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Methods for screening for autoantibodies:

At present, the most suitable method for screening autoantibodies is the protein chip method. A protein chip often has thousands of protein spots, which can screen one autoantibody that can interact with these proteins at one time, and then pass fluorescent These autoantibodies can be found by incubation of the marker against the anti-Human IgG secondary antibody and fluorescence detection.

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The principle is simple, but it is very difficult to do, why? This has to say about the binding process of antibodies to antigens. In short, the corresponding antibody recognizes a specific epitope on the antigen and then binds it. The epitopes are divided into two types, linear epitopes and non-continuous epitopes. A linear epitope consists of a contiguous sequence of amino acids. An antibody that recognizes a linear epitope recognizes this amino acid sequence and produces an antigen-antibody binding reaction; a non-contiguous epitope is composed of a discontinuous amino acid, which is correct by the antigenic protein. After folding, they are close together and recognized by the corresponding antibody, producing an antigen-antibody binding reaction.

 

In the body, most autoantibodies are non-continuous epitopes that recognize antigens. Correct identification and screening of these autoantibodies requires that the proteins on the protein chip be full-length, correctly folded, and biologically functional. However, the traditional protein chip can only guarantee that the protein synthesized on the chip is full-length (some can not be guaranteed), can not guarantee the correct folding of the protein, and can not guarantee the normal biological function of the corresponding protein. Other problems affecting the screening of autoantibodies by traditional protein chips include high CV values (>30%), poor reproducibility, low resolution, high background signal, and inability to distinguish autoantibodies with low expression levels. Screening for autoantibodies with such protein chips is like fishing with a large network full of loopholes. Screening of autoantibodies with such protein chips has brought great resistance to researchers and companies in the research of antibody screen. Sengenics' ImmunomeTM Protein Chip Research Platform, invented by Professor Jonathan Blackburn at the University of Cambridge in 1996, is a collaboration between Oxford and Cambridge. It is the only one in the world that is fully-length, correctly folded and functionally validated. Protein chip platform. Autoantibodies that recognize non-contiguous epitopes can be screened for advantages that cannot be replaced by other protein chip products.

The ImmunomeTM Protein Microarray Research Platform contains 1631 full-length, correctly folded and functionally validated human proteins, covering cancer antigens, transcription factors, kinases, signaling pathway molecules, etc., to meet the research needs of users in different directions. Daban's low autoantibodies provide strong technical support as biomarkers. Compared to traditional protein chip products in the screening of autoantibody applications, Sengenics ImmunomeTM protein chip products can be described as "Skynet is restored, not leaking", full-length, correct folding, functional verification, wide coverage, low coverage CV low background signal, high-resolution full-generation protein chip platform technology helps researchers and business users to "capture big fish" in the field of autoantibody research, and return home.

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Researchers from the University of North Carolina Lineberger Comprehensive Cancer Center have discovered how different mutations in a specific gene help drive glioblastoma, the most lethal form of brain cancer.

In the preclinical study, researchers investigated whether the location of where the mutation occurred within the sequence of the PIK3CA gene affected the mutation's ability to help drive cancerous growth. They also tested whether mutations within certain sequences of the gene were linked to better responses to particular drugs. They found mutational status was not linked to a response to a single targeted drug, but it was to a combination of treatments.

UNC Lineberger's C. Ryan Miller, MD, an associate professor in the UNC School of Medicine Departments of Pathology and Laboratory Medicine, Neurology, and Pharmacology, said the findings, published in the journal PLOS One, call for a more refined approach to precision medicine for glioblastoma, requiring more information about mutations that occur in a particular tumor.

"One approach to personalized medicine has been to sequence the tumor to find any type of mutation in genes for which there are drugs that target them, and then treat all patients the same," Miller said. "We think it's going to need to be more nuanced than that. We will need to take into consideration not only whether there are mutations in genes like PIK3CA, but also: where the mutation is in the gene, and what is its biochemical mechanism? Not only that, but what are the other concurrent mutations in the tumor -- what other genes are mutated, and are they druggable too?"

Glioblastoma is the most common primary malignant brain tumor in adults. Current treatments, which can include surgery, radiation and chemotherapy, have had limited effectiveness. A newly diagnosed patient has median survival of 12-15 months, and the five-year survival rate is less than 5 percent. Clinical trial results have been disappointing for drugs targeting certain molecular pathways driven by mutations commonly found in the disease.

Studies have found that the PIK3CA gene is mutated in about 10 percent of glioblastoma cases. Unlike some cancers in which mutations frequently occur in a specific location, or "hotspot," of a gene, mutations in PIK3CA can occur in multiple different parts of the gene in glioblastoma.

"For most of the successful examples of personalized medicine in solid tumors, the targets are hotspot mutations that activate a gene, which activates its signaling pathway, which drives the disease," Miller said. "Those tumors are addicted to that signaling. What interested us about this target is it didn't really fit that profile. The mutations were in multiple protein domains, and based on the sequence you could predict that they would have different biochemical mechanisms of action."

In studies in glioblastoma cells, researchers found that specific mutations in the PIK3CA gene help to drive the cancer. And while the presence of mutations was not linked to a greater response to treatment with a single drug targeting the PIK3CA pathway, they did see improve response with two different therapies: buparlisib and selumetinib.

"When we did the drug studies, we found that the mutation status of the cells really didn't predict response to the drug, unless we included a second drug in combination that targeted a parallel pathway," Miller said. The next step for the researchers will be to evaluate drugs targeting the particular downstream effects of the different pathways. Miller said researchers hope future studies could help identify additional potential therapeutic targets in glioblastoma, while helping to guide clinical trials using existing drugs.

Individual authors were supported by the University Cancer Research Fund, the NationalCenter for Advancing Translational Sciences, the National Cancer Institute, the Robert H. Wagner Scholar, Bill Sykes Scholar in Pathobiology and Translational Science award, the UNC Graduate Training Program in Translational Medicine, and the National Institute of Environmental Health Sciences.

In addition to Miller, the study's other authors are Robert S. McNeill, Emily E. Stroobant,Erin Smithberger, Demitra A. Canoutas, Madison K. Butler, Abigail K. Shelton, Shrey D. Patel, Juanita C. Limas, Kasey R. Skinner, Ryan E. Bash, and Ralf S. Schmid.

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Positional mutation is a protein engineering technique that substitutes, inserts or deletes specific nucleotides in known DNA sequences based on the known structure and function of proteins to produce mutant protein (enzyme) molecules with novel traits. The technology is widely used in the biological and medical fields. Position mutation technology has the characteristics of high mutation rate, simple and easy to perform, and good repeatability. As a research method, localization mutation technology is also widely used to study the relationship between protein structure and function, so as to elucidate the regulation mechanism of genes, the etiology and mechanism of diseases.

 

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Introduction

The "small change" of protein molecules based on natural protein structure refers to the modification, substitution or deletion of a few residues of proteins of known structure. This is the most widely used method in protein engineering, and can be mainly divided into proteins. Two types of modification and gene location mutation. Gene-localized mutation refers to the transformation of protein molecules at the genetic level, that is, the method of site-directed mutagenesis, the insertion, deletion, substitution and reorganization of nucleotide codons of genes encoding proteins, and then the mutated genes are carried out. The protein expresses and analyzes the functional activity of the expressed protein, and the result provides a new design for protein molecular engineering.

 

Design goals and solutions for location mutation

The common design goals of localization mutations are to improve the heat and acid stability of proteins, increase activity, reduce side effects, improve specificity, and conduct structural-functional studies through protein engineering. Hartley is equal to 1986 to complete a design goal and solution that we want, and still has important reference value. The stability of protein is an important prerequisite for the normal biological activity of proteins. Therefore, improving the stability of proteins has become one of the important goals of protein design and transformation.

 

Type of mutation

There are many ways to change the nucleotide sequence of a gene, such as chemical synthesis of genes, direct modification of genes, and cassette mutation technology. Depending on the manner in which the gene is mutated, it can also be classified into three categories: insertion of one or more amino acid residues; deletion of one or more amino acid residues; replacement or substitution of one or more amino acid residues. In order to achieve the purpose of gene location mutation, in vitro recombinant DNA technology or PCR method is often used.

 

Site-directed mutation

The amino acids in a protein are determined by the triplet codon in the gene. By changing one or two bases, the amino acid species can be changed to produce a new protein. It is usually the amino acid that changes a position in the functional region to study the structure, stability or catalytic properties of the protein. The work of point mutation is the main body of current protein engineering research. So far, many kinds of proteins such as subtilisin, T4 lysozyme, dihydrofolate reductase, trypsin and ribonuclease have been modified. For example, replacing Asn117 of a tissue-type plasminogen activator (t-PA) with Glu117, thereby removing an original glycosylation site; since the original sugar chain can promote t -PA is cleared from plasma, so point mutations can reduce plasma clearance of t-PA and prolong plasma half-life.

 

Box mutation

In 1985, Wells proposed a genetic modification technique for a box-type mutation that can produce 20 different amino acid mutants at one site, and can perform "saturation" analysis of important amino acids in protein molecules. Using the localization mutation, two original vectors and endonuclease cleavage points not present on the gene are added on both sides of the amino acid code to be modified, and the endonuclease is used to digest the gene, and then the synthesized double-stranded DNA fragment with different changes is substituted for digestion. part. A variety of mutant genes can be obtained in such a single treatment.

 

Procedure for locating mutations

The protein molecular design program for gene localization mutation follows the procedure in the design principle, but the gene location mutation has its own particularity, and its specific procedure is as follows.

  1. Establish a structural model of the protein under study

Establishing a three-dimensional structural model of a protein is critical to establishing a mutation site or region and predicting the structure and function of the mutated protein. The structure can be determined by X-ray crystallography, two-dimensional nuclear magnetic resonance, or the like, or a structural model can be established based on the structure of the analog or other structural prediction methods.

  1. Identify locations that have a significant impact on the required properties
  2. Predict the structure of the mutant
  3. Construct mutants. Mutant protein
  4. Examination of mutant proteins

 

About us

We provide custom protein services in the biological sciences, enabling access to the latest tools, techniques, and expertise with competitive pricing and rapid turnaround time. We serve a broad spectrum of industrial and academic clients with a commitment to delivering high-quality data and customer services. Here are some our products: SPR, Co-Immunoprecipitation, Pull-Downs, CLIP-seq, etc.

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Positional mutation is a protein engineering technique that substitutes, inserts or deletes specific nucleotides in known DNA sequences based on the known structure and function of proteins to produce mutant protein (enzyme) molecules with novel traits. The technology is widely used in the biological and medical fields. Position mutation technology has the characteristics of high mutation rate, simple and easy to perform, and good repeatability. As a research method, localization mutation technology is also widely used to study the relationship between protein structure and function, so as to elucidate the regulation mechanism of genes, the etiology and mechanism of diseases.

 

%E5%9B%BE%E7%89%872-300x123.png

 

Introduction

The "small change" of protein molecules based on natural protein structure refers to the modification, substitution or deletion of a few residues of proteins of known structure. This is the most widely used method in protein engineering, and can be mainly divided into proteins. Two types of modification and gene location mutation. Gene-localized mutation refers to the transformation of protein molecules at the genetic level, that is, the method of site-directed mutagenesis, the insertion, deletion, substitution and reorganization of nucleotide codons of genes encoding proteins, and then the mutated genes are carried out. The protein expresses and analyzes the functional activity of the expressed protein, and the result provides a new design for protein molecular engineering.

 

Design goals and solutions for location mutation

The common design goals of localization mutations are to improve the heat and acid stability of proteins, increase activity, reduce side effects, improve specificity, and conduct structural-functional studies through protein engineering. Hartley is equal to 1986 to complete a design goal and solution that we want, and still has important reference value. The stability of protein is an important prerequisite for the normal biological activity of proteins. Therefore, improving the stability of proteins has become one of the important goals of protein design and transformation.

 

Type of mutation

There are many ways to change the nucleotide sequence of a gene, such as chemical synthesis of genes, direct modification of genes, and cassette mutation technology. Depending on the manner in which the gene is mutated, it can also be classified into three categories: insertion of one or more amino acid residues; deletion of one or more amino acid residues; replacement or substitution of one or more amino acid residues. In order to achieve the purpose of gene location mutation, in vitro recombinant DNA technology or PCR method is often used.

 

Site-directed mutation

The amino acids in a protein are determined by the triplet codon in the gene. By changing one or two bases, the amino acid species can be changed to produce a new protein. It is usually the amino acid that changes a position in the functional region to study the structure, stability or catalytic properties of the protein. The work of point mutation is the main body of current protein engineering research. So far, many kinds of proteins such as subtilisin, T4 lysozyme, dihydrofolate reductase, trypsin and ribonuclease have been modified. For example, replacing Asn117 of a tissue-type plasminogen activator (t-PA) with Glu117, thereby removing an original glycosylation site; since the original sugar chain can promote t -PA is cleared from plasma, so point mutations can reduce plasma clearance of t-PA and prolong plasma half-life.

 

Box mutation

In 1985, Wells proposed a genetic modification technique for a box-type mutation that can produce 20 different amino acid mutants at one site, and can perform "saturation" analysis of important amino acids in protein molecules. Using the localization mutation, two original vectors and endonuclease cleavage points not present on the gene are added on both sides of the amino acid code to be modified, and the endonuclease is used to digest the gene, and then the synthesized double-stranded DNA fragment with different changes is substituted for digestion. part. A variety of mutant genes can be obtained in such a single treatment.

 

Procedure for locating mutations

The protein molecular design program for gene localization mutation follows the procedure in the design principle, but the gene location mutation has its own particularity, and its specific procedure is as follows.

  1. Establish a structural model of the protein under study

Establishing a three-dimensional structural model of a protein is critical to establishing a mutation site or region and predicting the structure and function of the mutated protein. The structure can be determined by X-ray crystallography, two-dimensional nuclear magnetic resonance, or the like, or a structural model can be established based on the structure of the analog or other structural prediction methods.

  1. Identify locations that have a significant impact on the required properties
  2. Predict the structure of the mutant
  3. Construct mutants. Mutant protein
  4. Examination of mutant proteins

 

About us

We provide custom protein services in the biological sciences, enabling access to the latest tools, techniques, and expertise with competitive pricing and rapid turnaround time. We serve a broad spectrum of industrial and academic clients with a commitment to delivering high-quality data and customer services. Here are some our products: SPR, Co-Immunoprecipitation, Pull-Downs, CLIP-seq, etc.

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The genetic engineering was established in the early 1970s and it has played an important role in the field of biology after decades of continuous progress and development.

Plant genetic engineering technology uses recombinant DNA technology to systematically transform and recombine biological genes by artificial "shearing" and "splicing" in vitro, and then insert and integrate into the recipient plant genome to reorganize. The gene is expressed in the recipient cell, so that the recipient plant obtains a new shape and breeds a new variety with high yield, multi-resistance and high quality.

The research and application of plant genetic engineering is flourishing around the world and is considered to be the hope of agriculture in the 21st century. It plays an important role in the new agricultural revolution.

1. Plant genetic engineering and its technologies

Based on human's goals and design, plant genetic engineering was conducted through split, integrate, splice, etc. in vitro to make the genetic material is recombined, and those features are then transferred to the plant through a specific vector (such as plasmid, phage, virus, etc.) intracellularly, and the expression of the required genes are expressed in cells. With this, a new plant type is created. Genetic engineering has a superior development space for improving plant traits, quality, increasing yield, and improving plant resistance to diseases, insects, and stress resistance. Therefore, its future development is extremely broad.

Plant engineering genetic technology has greatly expanded the gene pool available to plants, and it has become a reality to initiate directional mutation according to people's preset plans, which has brought huge changes in plant breeding (mainly in the following aspects: break reproductive isolation, making it creates conditions for broadening the available gene banks of plants, and providing new techniques for manipulating mutations; most of the genes used for genetic engineering breeding have been studied more clearly. The purpose of improving plants is clear, and the selection means is effective, so that it is possible to produce directional variation and directional selection; by improving some key traits of plants, the original promotion varieties will be improved to a large extent, not only can shorten the breeding period, but also make a comprehensive breakthrough in different ecological regions; with the deepening of understanding of genetic engineering, the cloning of new genes and the improvement of transgenic technology, and the targeted operation of multiple genes will also be possible, which is difficult to imagine in conventional breeding, and may lead to new "green" Revolution").

2. The possible risk and mechanism of genetically engineered plants

People have gradually realized that because the current level of science and technology can not accurately predict all the manifestations of plant genetic engineering, the safety of plant genetic engineering has attracted people's attention.

2.1. Possible risk of genetic engineering plants

(1) The threat of genetically engineered plants to environmental safety:

The effect of insect-resistant genetically engineered plants on other organisms. The insecticidal effect of insect-resistant genetically engineered plants is non-selective. It can also kill beneficial insects or other organisms in the environment while killing pests. Even when their residues are degraded in the soil, they will also affect insects or microorganisms in the soil.

Antiviral genetically engineered plants pose a risk of new viruses. At present, in genetic engineering of antiviral plants, most of the genes introduced into plants are sequences derived from the viral genome, and the most widely used are CP gene, MP gene and replicase gene.

The potential threat of genetically engineered plants to the Earth's ecosystem. After the release of genetically engineered plants, gene drift is inevitable, which causes the transferred genes to be passed on to other crops. Metabolites of genetically engineered plants will spread to the external environment, causing a chain reaction.

(2) The harm of genetically engineered plants to human health:

Genetically engineered plants may contain known or unknown toxins that are toxic to humans.

Genetically engineered plants may contain known or unknown allergens, causing allergic reactions in the human body and even death.

Genetically engineered plants produce certain nutrients or nutritional qualities that cause certain symptoms in the body.

After the genetically engineered plant is eaten by humans, the food will pass the drug resistance gene to the pathogenic bacteria in the human body, which will make the body resistant.

2.2 The mechanism of genetic engineering plant to cause risk

(1). Genetic engineering causes mutations in DNA in plants. Genetic engineering introduces foreign genes into plant gene tissues, causing DNA mutations in plants. Different locations of foreign gene introduction may lead to changes in plant gene expression, enzyme expression, or in unknown growth and metabolism in plants, which may produce or polymerize certain harmful substances, thereby threatening human health.

(2). Genetic engineering causes plants to contain new proteins that directly or indirectly endanger human health. Plant genetic engineering introduces genetic information into plants, and this genetic information may come from any organism on the earth that allows plants to produce new proteins. This new protein may directly harm human health or this new protein affects plant cell metabolism and changes the nutrient composition of the plant, thereby affecting human health.

(3). The harm may be caused by the unpredictability of genetic engineering. Although DNA can be spliced very accurately under laboratory conditions, the effect of a foreign gene into a plant on the entire plant genome cannot be fully predicted and controlled. The plant is an extremely complex living system, and our existing scientific knowledge and technical means cannot fully control the inheritance of plants.

3. Suggestions on improving the safety management of plant genetic engineering

(1). Have a full understand on the strategic significance of genetic engineering safety management, and improve the safety awareness of genetic engineering of all people, especially leading cadres and related scientific research and staff, and popularize relevant laws and regulations on genetic engineering safety management.

(2). Formulate a comprehensive "Genetic Engineering Law" to ensure that all laws and regulations on the safety management of genetic engineering are available.

(3). Establish an effective and timely genetic engineering supervision and reporting system, and conduct long-term follow-up monitoring of projects with higher risk levels to ensure that genetic engineering safety is within the controllable range.

(4). Establish a scientific genetic engineering risk assessment mechanism and explore the establishment of an authoritative genetic engineering risk assessment social organization. Strengthen the approval of research, development, promotion, import and export, and extend the review time of high-risk projects.

With the rapid development of plant genetic engineering technology, the emergence of new gene introduction technology (such as CRISPR/CAS9, TALEN-Mediated DNA Insertion, Virus-induced Gene Silencing etc.)and the technology of crop tissue culture are more convenient and effective. In the near future, it is expected to produce high-yield, high-quality, high-efficiency, disease-resistant, insect-resistant and anti-adversity crops. A new crop variety with excellent traits will benefit human beings and become an important weapon for human beings to understand nature and transform nature. At the same time, when seeing the achievements of genetic engineering plant, we must also see the risk of genetically modified plants.

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Protein is mainly composed of chemical elements such as carbon, hydrogen, oxygen and nitrogen. It is an important biological macromolecule. All proteins are multimers formed by the connection of 20 different amino acids. After Forming proteins, these amino acids are also called as a residue.

 

The boundaries between proteins and peptides are not very clear. Some people believe that the number of residues required for a functionally acting domain is called a polypeptide or peptide if the number of residues is less than 40. To function biologically, proteins need to be properly folded into a specific configuration, mainly through a large number of non-covalent interactions (such as hydrogen bonds, ionic bonds, van der Waals forces and hydrophobic interactions); in addition, in some proteins (especially in the case of secreted proteins), disulfide bonds also play a key role. In order to understand the mechanism of action of proteins at the molecular level, it is often necessary to determine the three-dimensional structure of a protein. Structural biology has been developed by studying protein structure, using techniques including X-ray crystallography, nuclear magnetic resonance, etc. to resolve protein structures.

 

A certain number of residues are necessary to exert a certain biochemical function; 40-50 residues are usually the lower limit of the size of a functional domain. Protein size can range from such a lower limit up to thousands of residues. The current estimated average length of proteins differs between different species, typically about 200-380 residues, while eukaryotes have an average protein length of about 55% longer than prokaryotes. Larger protein aggregates can be formed by many protein subunits; for example, by the polymerization of thousands of actin molecules to form protein fibers.

 

Discovery history

In 1959, Perutz and Kendrew analyzed the structure of hemoglobin and myoglobin, solved the three-dimensional structure, and won the 1962 Nobel Prize in Chemistry.

Pauling discovered the basic structure of the protein. Based on the X-ray diffraction data, Crick and Watson proposed a model of the three-dimensional structure of DNA. Received the 1962 Nobel Prize in Physiology or Medicine. After the 1950s, Hauptmann and Karle established a purely mathematical theory for the direct determination of crystal structures using X-ray analysis, which has epoch-making significance in crystal research, especially in the study of macromolecular biological substances such as hormones, antibiotics, and proteins. And the molecular structure of new drugs played an important role. They were awarded the 1985 Nobel Prize in Chemistry.

 

Structure type

Protein molecules are covalent polypeptide chains formed by the condensation of amino acids end-to-end, but natural protein molecules are not loose random polypeptide chains. Each natural protein has its own unique spatial structure or three-dimensional structure, which is often referred to as the conformation of the protein, ie the structure of the protein.

The molecular structure of a protein can be divided into four levels to describe its different aspects:

Primary structure: A linear amino acid sequence that makes up a protein polypeptide chain.

Secondary structure: a stable structure formed by hydrogen bonds between C=O and N-H groups between different amino acids, mainly α-helix and β-sheet.

Tertiary structure: The three-dimensional structure of a protein molecule formed by the arrangement of multiple secondary structural elements in three dimensions.

Quaternary structure: used to describe a protein complex molecule that is functionally formed by interactions between different polypeptide chains (subunits).

In addition to these structural levels, proteins can be transformed in multiple similar structures to perform their biological functions. For functional structural changes, these tertiary or quaternary structures are usually described in a chemical conformation, and the corresponding structural transformation is referred to as a conformational change.

 

 

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Adjuvant Selection is the main method for finding molecular genetic markers, vaccines like bacterial vaccines, cancer vaccines, RNA vaccines, MAS breeding and so on.

Major methods for finding molecular genetic markers

DNA markers are divided into two types: type markers, mainly are some single genes, and used to compare the homologous loci varieties of relative distance and chain and linear correlation; type markers, mainly high polymorphism, information content rich DNA fragments, is one of the most commonly used microsatellite marker. Through Adjuvant Optimization, more and more kinds of molecular markers were introduced, including restricted fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and Microsatellites. At present, there are 2,505 markers in the world's pig research, including 1,391 microsatellite markers. type 873, type mark 1632.

The main methods to search for molecular genetic markers are candidate gene method and genome scanning method.

1. Candidate gene method as a candidate gene for a trait, it is usually some genes whose biological functions and nucleic acid sequences are known, and they are involved in the growth and development process of the trait. These genes may be structural genes, regulatory genes or genes that affect the expression of traits in biochemical metabolic pathways. Candidate gene method research should follow certain steps, such as candidate gene selection primer design, gene specific fragment amplification, polymorphic locus search and so on. Candidate gene search USES genes that are thought to have a direct physiological function for a trait to find QTLS. In addition, genes found in other species that control some traits can be studied as candidate genes for pigs. For example, the h-fabp gene affects the backfat thickness and intramuscular fat content of pigs (Gerbens et al., 1999). 2000; 2001); The melanocorticoid receptor 4 (MC4R) gene was significantly correlated with the intake, backfat thickness and growth rate of pigs (Kim et al., 1999; 2000).

2. Genome scanning: all genetic information is stored on the 19 pairs of chromosomes of the pig. Reference families were established, such as meishan European and American pig species, wild boar big white pig, and their hybrid offspring were used to find QTL through genetic markers. The most effective design is the genotype analysis of F2 generation isolation population. Figure 4-2 is a simple schematic diagram of single genetic marker and linkage QTL analysis. Alleles of genetic markers and their linkage QTL in F1 generation were heterozygous. In the F2 generation isolation population, the ratio of the three possible genotypes per seat should be 1:2:1, when the average performance of marker genotypes is compared, the existence of linkage QTL can be analyzed.

Anderson (1994), such as reported with a wild boar by the results of the large white building reference group, using the 105 DNA markers in the genetic map, the separation of F2 generation 200 pigs linkage analysis research found that on chromosome 4 seat back fat and control the growth rate, the average genetic effect 24 g/d and 5 mm, respectively, the equivalent of F2 DaiQun total phenotypic variation of 12% and 18%. Daily weight gain can differ by more than 50g between two extreme homozygous genotypes, resulting in a 10kg weight difference at market time in pigs.

(iii) MAS breeding

In pig breeding selection, it is difficult to determine the sex efficiency of low heritability (e.g., reproductive traits), high cost of measurement (e.g., disease resistance), phenotypic values (e.g., lean meat rate) or limited sexual performance (e.g., milk production) early in development. It is estimated that the selection of the marker before the determination of the offspring can increase the selectivity response by 10%~15%. The MAS of compatriots who choose to combine can be increased by about 40%. Combining multiple genetic markers and trait information, the selectivity response can be increased by 50%~200%. Using marker selection in cross breeding can predict and make full use of heterosis. Molecular genetic markers can also be applied to early selection and screening and detection of large populations to select populations with desired genotypes.

For example, there is little progress in the improvement of pig litter, a low genetic trait, by traditional methods. Rothschild et al. found in 1994 that the estrogen receptor (ESR) gene was one of the main genes responsible for the litter size of pigs, which could control the total litter size of 1.5 pigs and the live litter size of 1 pig in the meishan synthetic line of China. In the Chinese two-flower face hybrid population, the agricultural university of China not only confirmed the results of Rothschild et al., but also found another main gene locus controlling the number of piglets - FSH, which can control the total number of piglets and the number of live piglets by 2.0.

Although MAS can improve the effectiveness of selection and the annual amount of genetic improvement, its effectiveness is also affected by many factors. In addition to the heritability of traits, the intensity of selection, and the size of the selected population, the determinants are the linkage between genetic markers and QTLS. Zhang (1992) pointed out that each QTL could be specifically detected by using genetic markers closely linked to QTL, and the final selection of genetic markers would be equivalent to the selection of QTL itself. Therefore, genetic markers closely related to QTL must be obtained in order to improve MAS efficiency. Resources at present, through the establishment of the pig family, has some related to the growth, reproduction and carcass, meat quality of QTL mapping in some microsatellite nearby, such as on chromosome 3 microsatellite Sw2427 - Sw251 area and daily gain of pigs, on chromosome 4 S0101 - S0107 area and back fat belly fat, 7 chromosome S0064 S0066 regional composition and has a strong correlation between birth weight and body. It can be predicted that with the discovery of more genetic markers closely linked to QTL, MAS will be applied more effectively in practical breeding.

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3 Cancer vaccine adjuvant selection

The initial aim of formulating vaccines in adjuvants was to deliver the antigen in a poorly metabolizing and slowly degrading substance. The intention was to favor the slow and sustained release of the antigen to be captured by antigen-presenting cells (APCs) and be subsequently presented to T cells. Aluminum salts are widely used to favor T helper cell 2 (Th2)-mediated humoral immunity, but they are less efficient for promoting Th1-dependent immunity. To this aim, water-in-oil adjuvants have been developed to create a depot of the antigen at the site of the injection. The next generation of vaccine delivery agents includes nanoparticles such as silica or liposomes or synthetic polymers, which are ideal vehicles to be taken up by dendritic cells (DCs) patrolling within the subcutaneous tissues. However, the challenge with such supports is to selectively promote DC uptake while eluding the systemic reticuloendothelial network of macrophages, which routinely clear circulating particles. In addition to these substances designed to favor delivery of the antigen to APCs, today’s therapeutic vaccines also contain another class of adjuvants aimed to deliver danger signals to activate the immune system, as antigen alone may fail to prime effective T cell responses or even induce tolerance.

4 The choice of cancer vaccine delivery system

The choice of delivery systems and route of immunization depends on the end use of the vaccine. For practical reasons and minimal side effects, most prophylactic vaccines are administered via the skin, usually by subcutaneous injections in the epidermis or the dermis. These two locations are ideal, as they are enriched respectively in Langerhans DCs and dermal DCs, both cell populations being very efficient in capturing and processing antigens. The oral route is also very convenient and is used by vaccines against polio, typhoid fever, cholera, and rotavirus. The oral route is, however, more challenging in view of the extreme conditions in the gastrointestinal tract, including the low pH in the stomach and the presence of microbiota, which may degrade the antigen before it reaches the lymphoid organs. Moreover, the usually tolerogenic gut environment may not be ideal to generate a strong systemic immune response.

With regard to therapeutic vaccines used to treat chronic noncontagious diseases such as cancer, atopy, or diabetes, both immediate cellular effector responses and long-term immunity are desired to guarantee the continuous immune-surveillance of the disease. Although prophylactic vaccines for global immunization programs must be simple, inexpensive, and given via a noninvasive route, therapeutic cancer vaccines can benefit from more complicated technologies and use more invasive routes of delivery if beneficial for the patient. There is a very large array of cancer vaccines under development which use various delivery systems, and which are being tested in clinical trials. Other delivery routes tested in therapeutic cancer vaccines range from subcutaneous and intradermal to more invasive intraperitoneal and intranodal injections, to optimize antigen uptake by APCs and favor a local potent immune response. For instance, particulate therapeutic vaccines such as virosomes or nanoparticles can be injected in LNs using an ultrasound-guided imaging procedure. Although most of these strategies are still in the development stage, the potential to achieve strong and long-lasting antitumor responses is high, owing to new delivery systems and better understanding of T cell memory development.

Reference

[1] FUTURE II Study Group. Quadrivalent vaccine against HumanPapillomavirns to prevent high-grade cervical lessions. N Engl J Med, 2007, 356(19): 1915

[2] Olsson SE, Villa LL, Costa RLR, et a1. Induction of inmmne memory following administration of a prophylactic quadrivalent human papillomavirus (HPV) types 6/1 1/16/18 LI virus-like particle (VLP) vaccine. Vaccine, 2007 (25): 4931

[3] Harris JE, Ryan L, Hoover Jr HC, et a1. Adjuvant active specific immunotherapy for stage II and III colon cancer with an autologous tumor cell vaccine: Eastern Cooperative Oncology Group Study E5283. J Clin Orwol, 2000, 18(1): 148

[4] Berd D, Maguire Jr HC, Mastrangelo MJ, et a1. Treatment of human melanoma with a hapten — modified antologous vaccine. Ann NY Acad Sci, 1993, 690(8): 7

[5] Remann R, Goldschmidt AJ, Richter A. Adjuvant therapy of renal cell carcinoma patients with an autologous tumor cell lysate vaccine: a-year—follow—up analysis AnticancerRes, 2003, 23(2A): 969

[6] Mitchell MS, Kan — Mitchell J, Kempf RA, et a1. Active specific im-munotherapy for melanoma: phase I trial of allogeneic lysates and a novel adjuvant. Cancer Res, 1988, 48(20): 5883

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3 Benign Mucous Membrane Pemphigoid

3.1 Clinical manifestations

  Conjunctival and oral mucosal damage accounted for 60% to 90%, patients with nasal, pharyngeal, genital and anal mucosa accounted for about 25%, skin involvement is rare, skin damage is very similar to the lesions of bullous pemphigoid, but the time is short, and the stenosis or adhesion caused by scar healing and scar formation is characteristic.

3.1.1 Eye damage

The eye is the only affected part, and the damage usually occurs asymmetrically. After 1 to 2 years, the contralateral eye mucosa is involved. There are symptoms of catarrhal conjunctivitis. Transparent blisters can be found and quickly ruptured. Subsequently, scar atrophy, conjunctival and bulbar conjunctiva adhesions, eye movement involvement, tendon varus leading to secondary corneal changes, corneal opacity, scar formation of the tarsal plate with mucosa, atrophy of the gland and blockage of the lacrimal duct, thus corneal dryness, discoloration and blinding ulcers are formed.

3.1.2 Mucosal damage

Multiple blisters quickly form painful erosions and the scars heal. If the damage occurs in the tongue ligament, the contracted scar can limit the movement of the tongue. Damage that occurs in soft palate, tonsil, and buccal mucosa may limit food intake. The genital area of the vulva can form a head adhesion, a vaginal stenosis, and the like.

3.1.3 Skin damage

The incidence of skin damage is only about 25%, the damage is a tension blisters, the blister wall is not easy to rupture, occurs on erythematous skin, one or more parts. Healed with atrophic scars. If bullae occur in the head, scarring hair loss may occur. Generalized lesions are extremely rare, and even if there are secondary scar formation.

Benign mucosal pemphigoid can occur repeatedly for several years without significant effect on general health. The faster the disease progresses, the worse the prognosis. Due to eating difficulties, malnutrition and cachexia may occur, and the incidence of blindness is approximately 20% to 60%. Although extremely rare, there have been reports of cancerous changes in the scars of the oral mucosa.

3.2 Laboratory examination

3.2.1Histopathology: typical epidermis blister, no spine release. Infiltration of inflammatory cells composed of lymphocytes, plasma cells, and eosinophils can be seen in the upper dermis. Subsequently, there were a large number of fibroblasts with superficial dermal fibrosis and vascular hyperplasia and scar contracture.

3.2.2 Immunofluorescence: direct immunofluorescence of skin mucosa to detect IgG and C3 deposition in the basement membrane zone, and IgA and C3 deposition were also observed. The homogenous linear deposition was almost the same as that in bullous pemphigoid. The positive rate of indirect immunofluorescence detection of anti-basal membrane circulating antibodies was <10%, and the titer was low. If the DIF is negative, the fresh tissue should be removed from the erythema around the lesion for repeated testing.

4 Pathogenesis of Pemphigus

In the plasma of patients with pemphigus, there is anti-Dsg3 and/or anti-Dsg1 IgG, and no anti-Dsg2 antibody exists. This autoreactive antibody binds to its corresponding antigen, leading to a series of clinical pemphigus. The emergence of performance. In the active phase of pemphigus vulgaris, the serum is mainly pathogenic IgG4 subclass and IgG1, while in the serum of patients with long-term remission, there are low-valency IgG1 subclasses, IgG4 and Dsg1 and Dsg13 on keratinocytes. The combination causes the loss of adhesion between cells, leading to the release of intercellular cells and the formation of blisters in the epidermis.

The antigen Dsg produced in the MMP autoimmune disease belongs to the transmembrane component of desmos, belonging to the cadherin superfamily in the adhesion molecule, whose gene is located at 18q12.1. Dsg is divided into three categories: Dsg1, Dsg2, and Dsg3. Among them, Dsg2 is expressed in all tissues with desmosome, including monolayer epithelial cells and myocardial tissue. Dsg1 and Dsg3 are mainly restricted to stratified squamous cells. Current studies have shown that Dsg1 and Dsg3 are target antigens of pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. The relative molecular mass of Dsg3 is about 130 kD. which is mainly distributed on the surface of keratinocytes in the basal layer of the epidermis and the upper layer of the basal layer. The relative molecular mass of Dsg1 is about 160 kD, which is mainly distributed on the keratinocyte membrane in the upper layer of the epidermis, with the advantage of granular layer and subgranular layer expression. The Dsg3 and Dsg1 molecules, like other cadherin molecules, have five tandem repeats of approximately equal size extracellular domain (EC), each of which is approximately 100 amino acid residues in length, of which EC1 the region corresponds to the extracellular amino terminal residue and EC5 is at the carboxy terminus. The difference between Dsg1 and Dsg3 is mainly due to the homology of the 1 to 4 extracellular domains of Dsg1 and the homology of the 5 extracellular domains of Dsg3. Unlike classical cadherins, Dsg1 has a longer intracellular fragment, and the extracellular fragment of Dsg3 is slightly longer than Dsg1, but its intracellular domain is slightly shorter and has no glycine/serine-rich region.

In the five extracellular domains of the pemphigus antigen, EC1, EC2, and EC4 have relatively large homology and can be specifically recognized by the plasma of pemphigus patients. Therefore, these epitopes are called "an immune-dominant epitope, or a "pathogenic epitope," an antibody that specifically recognizes an epitope is called a "pathogenic antibody." The pemphigus antigen contains at least one "pathogenic epitope" in the EC1 -2 region, and antibodies against EC1 -2 are closely related to the pathogenesis of pemphigus. The recombinant protein expressed by the EC1 -2 and EC3 -4 gene sequences of Dsg3 only reacted with the plasma of pemphigus patients, and the response rates of the two were 57.9 % and 52.6%, respectively, and the bullous pemphigoid, system Lupus erythematosus and normal people do not respond. This indicates that EC1 -2 and EC3 -4 are antigen-specific and have a high affinity with pemphigus antibodies, thus providing a new approach for serological diagnosis and identification of pemphigus.

In Dsg1 and Dsg3, when either function is lost, the other can partially compensate for its function. This theory explains the tissue specificity of the loss of intercellular adhesion caused by autoantibodies in pemphigus patients. Because Dsg1 and Dsg3 have a certain distribution pattern, the difference in anti-Dsg antibodies also leads to differences in the clinical manifestations of pemphigus. By inserting the Dsg1 gene into Dsg3 knockout mice to form a transgenic mouse capable of expressing Dsg1, it was found that Dsg1 can compensate for partial Dsg3-mediated loss of intercellular adhesion function, which is confirmed genetically.

5 Treatment

Glucocorticoid is preferred for the treatment of pemphigus. Most scholars at home and abroad are empirical drugs. There are ethnic and regional differences in the choice of hormone dose. At present, the severity of the disease is not unified by using the scoring system. If the two are linked to the hormone dose and applied to the clinic, the treatment plan will be more reasonable. Determine the initial dose of hormone according to the ABSIS system: ABSIS skin severity score 10, give 30 ~ 40 mg / d, skin score of 10 ~ 50, 60 mg / d is appropriate, skin score > 50 points, give 80 mg /d. If the skin lesions are not controlled for 5 to 7 days, increase the dose by 50%. Reduced indications: Daily new blistering number <5, no new erythema; no obvious exudation of erosion surface; pemphigus antibody titer decreased earlier; ABSIS skin score decreased by more than 35%.

The long-term use of large doses of hormones can easily lead to many drug reactions. Therefore, the combination of immunosuppressive agents, including azathioprine, cyclophosphamide, mycophenolate mofetil, etc., is generally used to reduce the number of hormones and shorten the treatment time. At the same time, it can be combined with an immune-modulator (Amlalazine, etc.), plasma exchange method and in vitro. Photochemotherapy, etc. Randomized controlled observation of prednisone combined with mycophenolate mofetil, azathioprine and prednisone alone in the treatment of 42 cases of pemphigus, combined with the ABSIS system to assess the severity of the disease before and after treatment, the results show that the three treatment days The short-term efficacy and safety of acne are similar, but prednisone combined with mycophenolate mofetil reduces the dose of hormones most significantly. In recent years, some biological agents and treatments have become new choices for the treatment of pemphigus, such as rituximab, high-dose intravenous immunoglobulin (IVIG), immunosorbent assay, TNF-α antagonist and hematopoietic stem cell transplantation therapy.

 For the severe bullous disease such as pemphigus, detecting the Dsg ELISA index has certain guiding significance for judging its severity. However, if medical workers are to accurately grasp the changes in their condition, they also rely on an effective and widely recognized scoring system to provide a reliable clinical basis for the treatment and adjustment of pemphigus. At present, there are few clinical applications of ABSIS and PDAI. Both have their own advantages and disadvantages. If they can be combined, it is expected to improve the evaluation method. The correlation between the disease scoring system and antibody levels, and the number of hormones controlled by scoring will be important research topics. Many new drugs and treatments have emerged in recent years, and multi-center large-scale clinical trials are still needed to explore the best treatment options.

 

Reference

[1] Barnadas MA, Rubiales MV, Gich I, et al. Usefulness of specific anti-desmoglein 1 and 3 enzyme-linked immunoassay and indirect immunofluorescence in the evaluation of pemphigus activity [J]. Int J Dermatol, 2015, 54(11): 1261-1268.

[2] Daneshpazhooh M, Kamyab K, Kalantari MS, et al. Comparison of desmoglein 1 and 3 enzyme - linked immunosorbent assay and direct immunofluorescence for evaluation of immunological remission in pemphigus vulgaris [J]. Clinical & Experimental Dermatology, 2014, 39(1): 41-47.

[3] Nakahara T, Takagi A, Yamagami J, et al. High anti-desmoglein 3 antibody ELISA index and negative indirect immunofluorescence result in a patient with pemphigus vulgaris in remission: evaluation of the antibody profile by newly developed methods [J]. Jama Dermatology, 2014, 150(12): 1327-1330.

[4] Pfutze M, Niedermeier A, Eming R. Introducing a novel autoimmune bullous skin disorder intensity score ( ABSIS) in pemphigus [J]. Eur J Dermatol, 2007, 17(1): 4-11.

[5] Murrell DF, Dick S, Amagai M, et al. Consensus statement on definitions of disease, end points, and therapeutic response for pemphigus [J]. J Am Acad Dermatol, 2008, 58(6): 1043-1046.

[7] Chams-Davatchi C, Rahbar Z, Daneshpazhooh M, et al. Pemphigus vulgaris activity score and assessment of convergent validity [J]. Acta Med Iran, 2013, 51(51): 224-230.

[8] Sebaratnam DF, Frew JW, Davatchi F, et al. Quality -of-Life Measurement in Blistering Diseases [J]. Dermatol Clin, 2012, 30(2): 301-307.

[9] Mahajan VK, Sharma NL, Sharma RC, et al. Twelve-year clinicotherapeutic experience in pemphigus: a retrospective study of 54 cases [J]. Int J Dermatol, 2005, 44(10): 821-827.

[10] Agarwal M, Walia R, Kochhar AM, et al. Pemphigus Area and Activity Score (PAAS) --a novel clinical scoring method for monitoring of pemphigus vulgaris patients [J]. Int J Dermatol, 1998, 37(2): 158-160.

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What is 3D cell culture?

Three-dimensional cell culture is based on the common three-dimensional culture model of the scaffold, which can better simulate the natural environment in which the cells grow. Three-dimensional cell culture (TDCC) refers to the co-culture of vectors with different materials in three dimensions and various kinds of cells in vitro, so that cells can migrate and grow in the three-dimensional spatial structure of the carrier. Three-dimensional cell-carrier complex.

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Frontier knowledge about 3D cell culture research

  1. Three-dimensional cell tissue plus tensile culture model of the United States flexcell company Tissue Train tensile stress stimulation three-dimensional hydrogel stent cell tissue culture system.

Functional highlights of the three-dimensional cell tissue augmentation culture model: after burning three-dimensional cell tissue culture and stretching three-dimensional cell tissue culture in the true sense of three-dimensional culture - the system with a variety of coated surfaces (Amino, Collagen (Type I Or IV), Elastin, ProNectin (RGD), Laminin (YIGSR) collagen hydrogels for extracellular matrix scaffolds in biomaterial scaffold studies, compared to traditional nanofiber scaffolds and porous scaffolds, hydrogel scaffolds The network contains a lot of water, which can supply cell nutrients well, and can also cross-link bioactive factors to regulate cell growth and differentiation. Therefore, hydrogel scaffolds can better simulate the tissue-like physics required for cell growth. The spatial structure has high plasticity, relatively simple manufacturing process and convenient clinical application.

  1. Three-dimensional cell tissue pressure-carrying culture system model of American three-dimensional cell tissue after-force culture model

Three-dimensional cell culture in three-dimensional cell culture

1) The system provides periodic or static pressure loading of various tissue, three-dimensional cell cultures;

2) based on the deformation of the flexible film substrate, uniform force;

3) Real-time observation of the reaction of cells and tissues under pressure;

4) can selectively block stress loading on cells;

5) Simultaneously have multi-channel cell pull force loading function;

6) Up to 4 channels, 4 different programs can be run simultaneously, and multiple different pressure deformation rate comparison experiments are performed;

7) Multiple frequencies (0.01-5 Hz), multiple amplitudes and multiple waveforms can be operated in the same program;

8) Better control of waveforms under ultra-low or ultra-high stress;

9) A variety of waveform types: static waveform, positive rotation waveform, cardiac waveform, triangular waveform, rectangle and various special waveforms;

10) computer system for pressure loading cycle, size, frequency, duration precise intelligent control typical application range: detection Biochemical reactions of various tissues and cells under pressure.

  1. Three-dimensional cell tissue augmentation culture model. Three-dimensional cell tissue stretchestensile force loading culture system model of American flexcell company Three-dimensional cell culture three-dimensional cell tension loading culture

1) The system provides axial and circumferential stress loading on two-dimensional, three-dimensional cells and tissues;

2) based on the deformation of the flexible film substrate, uniform force;

3) The reaction of cells and tissues under stress can be observed in real time;

4) can selectively block stress loading on cells;

5) Simultaneous multi-channel cell pressure loading function;

6) In combination with the Flex Flow parallel slab flow chamber, fluid shear stress can be applied while pulling the cells;

7) Up to 4 channels, 4 different programs can be run simultaneously, and multiple different tensile deformation rate comparison experiments are performed;

8) Multiple frequencies, multiple amplitudes and multiple waveforms can be operated in the same program;

9) Better control of waveforms under ultra-low or ultra-high stress;

10) Multiple waveform types: static waveform, positive rotation waveform, cardiac waveform, triangular waveform, rectangle, and various special waveforms;

3 Three-dimensional cell cultures in life applications:

Customized 3D cell culture services are common in our lives, and there are also 3D cell culture related products on the market.

  • Creative Bioarrayoffers 35 human cell systems with over 160 different cell types. Moreover, we also provide our customers primary cells from over 13 types of other animals.
  • These cells taken from living tissue are extremely accurate as they are literally coming from the source and are available from many sources on the human body. These living samples can give extremely accurate information about the cells in vivo and give relevant information regarding the living systems.
  • Not only canCreative Bioarray offer such a wide range of primary cells of humans, but we also have a selection of primary cells of animals for comparative testing.

Bone cells, which are found within the bone tissue, are responsible for bone production, maintenance and modeling. There are three different types of cells that found only in the bone. The osteoblasts are derived from mesenchymal stem cells and its function is bone matrix synthesis and its subsequent mineralization. The osteoclasts are large cells that dissolve the bone and osteocytes are cells inside the bone. At Creative Bioarray, we offer 6 types of human primary bone cells including: Human Bone marrow-derived endothelial Cells, Human Osteoblast Cells, Human Osteoblasts (HOB), Human Osteoblast Cells (Postnatal), Human Calvarial Osteoblasts (HCO) and Human Osteoblasts-femural (HO-f). The method we use to isolate endothelial cells was developed based on a combination of established and our proprietary methods. These cells are pre-coated with PECAM-1 antibody, following the application of magnetic beads pre-coated with secondary antibody. Human osteoblasts may be used for various types of in vitro, in vivo, or regenerative medicine studies in normal or diseased systems. In addition, they may be used in bone development studies.

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Many people regard designer brand for their footwear as the only way to go and businesses that offer those brands tend to get patronized more often. Wholesale shoes and boots is the way for retail businesses to go to take advantage of the latest fashion trends on the market at discounted prices. Even for those who have a liberal spending budget for their business and gets the styles that they want when they first come out, let's face it, that getting a discount on the quality that shows off business sense, good taste, and stylish sensitivity leaves more money in your pocket for other needs such as more inventory or bonuses to employees for exceeding sales quotas.

There are those who believe that what you put on your feet can make or break the entire look of a clothing choice and in part, there is some truth to this. Poorly fitting shoes or those that are worn with the heel lifts wearing out can make a brand new suit look dingy. With wholesale boots or wholesale shoes, especially designer names that people readily recognize, a retailer can give people the option to create a perfectly polished look that speaks volumes about taste, style, and fashion sense, and no one has to know that the designer boots or shoes did not cost the full price; including the customer making the purchase.

A wide variety of wholesalers offer great choices in wholesale shoes and boots, and not just designer names, for those who do not have to have a brand name label on their feet to reflect their personal fashion sense. A properly fitted shoe or boot is essential to foot comfort, whether it is for a busy day at work or a dazzling night out on the town. Getting stylish and trendy bargain wholesale shoes in flats, platforms, stacked heels, clogs or whatever style is desired, is the ticket to avoiding paying a fortune to resell footwear to the public with a handsome profit for your business.

Whether your business is attempting to appeal to those with a shoe fetish or just has a reputation for providing the public with a variety of shoes to choose from, wholesale shoes are fantastic choices. Many wholesalers provide instant access to the wholesale boots and shoes market and make it possible take advantage of great prices whether for low quantities or high volume stocking up. When it comes to designer quality, some big names have remained constant over the years, and at the same time the introduction of newcomers to the industry has broadened the selection and made it possible for retailers who swear by designer selections to help their customers stay outfitted from head to toe in their ideal choices.

Getting wholesale boots and wholesale shoes, whether design or everyday style, can be the ticket to providing customers with the luxury and comfort their feet deserve in every season. Whether the quest is for women, men or children's selections, a little due diligence can put these discounted designer brands and others right at the ready for those who want to have a big name in their footwear selections on the premises, but don't want to have to spend a fortune to get them. Wholesale shoes and boots in designer varieties are the sensible way to stay trendy and maintain your establishment's spending budget at the same time.

 

View More: Wholesale Shoes China 

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Neutron scattering research at the Department of Energy's Oak Ridge National Laboratory has revealed clear structural differences in the normal and pathological forms of a protein involved in Huntington's disease.

Huntington's disease, an incurable neurodegenerative disorder, starts as a genetic mutation that leads to an overabundance of "huntingtin" protein fragments, which form clumps in the brain.

Valerie Berthelier of the University of Tennessee Graduate School of Medicine, who co-led the study published in Biophysical Journalwith ORNL's Chris Stanley, said the goal was to establish a baseline understanding of huntingtin's structure in order to eventually determine the true structural basis of Huntington's disease.

"This is a very first step -- the hope is that we do this basic research to shed light on the structures of the protein," Berthelier said. "If we can start identifying any of these structures as toxic or potentially toxic, and then think about how drugs could interact with them, then we might be getting to the point of rationally designing therapeutics that would target those specific structures."

The researchers conducted a side-by-side study of model protein systems in solution using a time-resolved small-angle neutron scattering technique at ORNL's High Flux Isotope Reactor. The use of neutrons, a non-damaging but highly penetrating particle, allowed the team to study the biological materials over time without degrading the samples' structural integrity.

"We compared the normal and disease versions of the protein to see how they change over time," Stanley said. "You can see there's a discrepancy all the way from the early stages to the end-state fibrils."

The study's results showed key differences in the ways mutant and normal huntingtin proteins take shape. The disease protein, for instance, initially forms aggregates of one to two peptides, whereas the normal version makes bigger aggregates, gathering seven or eight peptides together.

These data on the very early stages of protein aggregate formation support a growing focus of the research in the amyloid field. Amyloid disorders, such as Parkinson's, Alzheimer's and Huntington's, all involve protein aggregation phenomena leading to a disease.

"There is no strong correlation between neuronal cell loss and the amount of protein aggregates found in the brain," Stanley said. "You could have a case with a lot of aggregates but minimal symptoms -- and you can find the converse. Researchers think there must be something happening at the earliest stage that's giving rise to toxicity."

Stanley says the team hopes to continue its research to obtain higher-resolution structural data and refine their understanding of the huntingtin protein.

"We'd like to use this small-angle neutron scattering technique in combination with others to get a better idea for how these early structures are forming and also ask the question -- are they toxic or not?" Stanley said.

The research is published as "Investigating the Structural Impact of the Glutamine Repeat in Huntingtin Assembly." Coauthors are Helen McWilliams-Koeppen, Erica Rowe and Tatiana Perevozchikova.

Related Tags:Protein Structure, Electron Microscope, VLP Construction 

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Overworked and stressed out? Look on the bright side. Some stress is good for you.

"You always think about stress as a really bad thing, but it's not," said Daniela Kaufer, associate professor of integrative biology at the University of California, Berkeley. "Some amounts of stress are good to push you just to the level of optimal alertness, behavioral and cognitive performance."

New research by Kaufer and UC Berkeley post-doctoral fellow Elizabeth Kirby has uncovered exactly how acute stress -- short-lived, not chronic -- primes the brain for improved performance.

In studies on rats, they found that significant, but brief stressful events caused stem cells in their brains to proliferate into new nerve cells that, when mature two weeks later, improved the rats' mental performance.

"I think intermittent stressful events are probably what keeps the brain more alert, and you perform better when you are alert," she said.

Kaufer, Kirby and their colleagues in UC Berkeley's Helen Wills Neuroscience Institute describe their results in a paper published April 16 in the new open access online journal eLife.

The UC Berkeley researchers' findings, "in general, reinforce the notion that stress hormones help an animal adapt -- after all, remembering the place where something stressful happened is beneficial to deal with future situations in the same place," said Bruce McEwen, head of the Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology at The Rockefeller University, who was not involved in the study.

Kaufer is especially interested in how both acute and chronic stress affect memory, and since the brain's hippocampus is critical to memory, she and her colleagues focused on the effects of stress on neural stem cells in the hippocampus of the adult rat brain. Neural stem cells are a sort of generic or progenitor brain cell that, depending on chemical triggers, can mature into neurons, astrocytes or other cells in the brain. The dentate gyrus of the hippocampus is one of only two areas in the brain that generate new brain cells in adults, and is highly sensitive to glucocorticoid stress hormones, Kaufer said.

Much research has demonstrated that chronic stress elevates levels of glucocorticoid stress hormones, which suppresses the production of new neurons in the hippocampus, impairing memory. This is in addition to the effect that chronically elevated levels of stress hormones have on the entire body, such as increasing the risk of chronic obesity, heart disease and depression.

Less is known about the effects of acute stress, Kaufer said, and studies have been conflicting.

To clear up the confusion, Kirby subjected rats to what, to them, is acute but short-lived stress -- immobilization in their cages for a few hours. This led to stress hormone (corticosterone) levels as high as those from chronic stress, though for only a few hours. The stress doubled the proliferation of new brain cells in the hippocampus, specifically in the dorsal dentate gyrus.

Kirby discovered that the stressed rats performed better on a memory test two weeks after the stressful event, but not two days after the event. Using special cell labeling techniques, the researchers established that the new nerve cells triggered by the acute stress were the same ones involved in learning new tasks two weeks later.

"In terms of survival, the nerve cell proliferation doesn't help you immediately after the stress, because it takes time for the cells to become mature, functioning neurons," Kaufer said. "But in the natural environment, where acute stress happens on a regular basis, it will keep the animal more alert, more attuned to the environment and to what actually is a threat or not a threat."

They also found that nerve cell proliferation after acute stress was triggered by the release of a protein, fibroblast growth factor 2 (FGF2), by astrocytes -- brain cells formerly thought of as support cells, but that now appear to play a more critical role in regulating neurons.

"The FGF2 involvement is interesting, because FGF2 deficiency is associated with depressive-like behaviors in animals and is linked to depression in humans," McEwen said.

Kaufer noted that exposure to acute, intense stress can sometimes be harmful, leading, for example, to post-traumatic stress disorder. Further research could help to identify the factors that determine whether a response to stress is good or bad.

"I think the ultimate message is an optimistic one," she concluded. "Stress can be something that makes you better, but it is a question of how much, how long and how you interpret or perceive it."

The eLife paper was coauthored by UC Berkeley colleagues Sandra E Muroy, Wayne G. Sun and David Covarrubias of the Department of Molecular and Cell Biology; Megan J. Leong of the Helen Wills Neuroscience Institute; and Laurel A. Barchas of the Department of Integrative Biology. Kirby is now a post-doctoral fellow at Stanford University.

Kaufer's research was funded by a BRAINS (Biobehavioral Research Awards for Innovative New Scientists) award from the National Institute of Mental Health of the National Institutes of Health (R01 MH087495) and the National Alliance for Research on Schizophrenia and Depression. Kirby was supported by fellowships from the California Institute for Regenerative Medicine and the U.S. Department of Defense.

Related Tags:FGFR4, FGF23, FGF2

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Often times in the summer you might lose your appetite or feel too full because of the heat. Or maybe you're running late in the morning and still need to eat breakfast. A quick and easy protein shake is a great way to get all the nutrients your body needs without feeling like you're stuffing yourself.

MYTH: Protein shakes are for bodybuilders and will make me bulky.

REALITY: If you read my last article about Toning Your Muscles, you now know that nobody becomes a bodybuilder by accident. Everybody's body needs protein and adding a supplement to ensure you get enough is only going to help in the effort to reshape your body.

MYTH: Protein shakes will make me fat.

REALITY: If you are eating too much and then you add in a protein shake on a regular basis, you may see some weight gain. In that case, use the shake to replace a meal or snack. If you're one of my clients, however, that is used to hearing from me "you're not eating enough!" this may be a good way to get some extra calories in.

MYTH: It's not healthy to eat too much protein.

REALITY: It's actually very difficult to eat so much protein that it has an adverse effect on your system if you're a healthy individual. The American College of Sports Medicine recommends every person eat 0.8x their body weight in kilograms of protein every day. For a 150lb person, this is about 68g protein every day. If you're concerned about your protein intake, consult your physician. If your physician expresses no concern then you're likely in the clear.

Making sure you get enough protein in your diet is essential if you want to change the shape of your body. One of my favorite analogies is protein as the bricks to build the house that is your body. This analogy is detailed in my article Dieting Fads and Why They Don't Work.

There are different kinds of whey protein powder that you can buy but be aware: a lot of them taste nasty! I've tried a lot of them and my some of favorites are Designer Whey, Pure Protein and Syntrax Nectar. Pure Protein you can actually find at Stop & Shop for $21 for a 2lb container. This should last you at least a month. You can click STORE on my website and then click Protein Powders to see what they look like or to order from Amazon. Whatever you decide to buy, make sure there is less than 5g of fat, carbs, and sugar, and more than 15g of protein per 1 scoop.

And finally, adding a protein shake is merely an idea if you're looking for something quick and easy to get your protein in. By no means do you HAVE to include it. You can get all the nutrients your body needs from "real" food.

Related Tags:Protein Expression, Bacillus Expression, Yeast Expression Systems 

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This article is intended to provide several analysis methods of a few important substances that they are well known. The first is bile acids analysis.

  

  Bile acid is an important component of bile and plays an important role in fat metabolism. Bile acid mainly exists in the intestinal and liver circulation system and acts as a protective role during recycling. Only a small amount of the bile acid enters the peripheral circulation.

 

  Bile acid analysis is of great importance to the research and development of clinical chemistry and bile acid drugs. As early as the ancient Chinese " Shennong's Herbal Classic " recorded the efficacy of carp gall. In addition, many important prescriptions, such as Bezoar powder and liushen pill, contain animal bile acids. Studies on bile acids in western countries began in the early 19th century, and quantitative studies began in the 1970s.

 

  The second is Catecholamines acids also known as pyrocatechol. Catecholamine is a neurotransmitter including catechins and amines. Catechins and amines are bound by an enzymatic process of l-tyrosine with sympathetic nerve, adrenal medulla and chromaffin cells. In general, catecholamine refers to norepinephrine (NA), adrenaline (Adr) and dopamine (DA). All three catecholamines are converted from tyrosine as precursors.

 

  As a neurotransmitter and hormone, catecholamine is closely related to people's health and disease. It is not only directly involved in behavioral activities or the regulation of neural functions such as blood pressure, heart rate, breathing and sleep, but also associated with some functional diseases such as schizophrenia and depression. Catecholamines Analysis began in the 1940s.With the deepening of the research on catecholamine, the analytical methods have been developing and improving all the time. At present, the research methods of molecular electrochemistry have become the leading one

 

  The third one is eicosanoids. Eicosanoids is a kind of bioactive unsaturated fatty acids, it is an important inflammatory factor, widely exists in body fluids and tissues, regulating many physiological and pathological processes. There are many kinds of eicosanoids in vivo, the content is low, and there are a lot of isomers. Therefore, the separation and analysis of eicosanoids in vivo are facing great challenges.

 

  As for eicosanoids analysis, Although there have been a lot of research on analytical methods, but still many problems waiting to solve. Neither extraction method has formed a standard extraction scheme for different biological samples. Therefore, the establishment of standardized extraction methods for different biological samples will greatly facilitate the eicosanoids analysis. The most powerful tool for analysis is the combination of chromatography-mass spectrometry. The characteristics of high sensitivity, high selectivity, high flux and high speed provide a good technical platform for the establishment of fast analytical methods. With the continuous development of various technologies, analytical methods will be continuously optimized and play a greater role in the research of biomarkers discovery, pathological mechanism and drug action mechanism.

 

  The last one is vitamins. There are two parts of vitamins analysis: water soluble vitamins analysis and fat soluble vitamins analysis.

 

  Vitamin is one of the important elements of human growth and development. At present, many foods are added with water-soluble vitamins, but there is a serious question whether they meet the requirements. Therefore, the development of research on it has certainly guiding significance for people's scientific intake and scientific control of the additive amount in food.

 

  Fat soluble vitamins are insoluble in water but soluble in fats and some organic solvents, including vitamin A, vitamin D, vitamin E and vitamin K. Fat soluble vitamins can be stored in large amounts in the body, mainly in the liver, so excessive intake will cause poisoning. Fat-soluble vitamins are becoming increasingly popular in clinical field. Thus, it is necessary to give reasonable attention to the analysis of the content and function of fat-soluble vitamins so that ensure the safety of use, maintain physical health.

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