The Guardian of Human Skeleton: FGFs

Fibroblast growth factor (FGFs) is a polypeptide composed of about 150-200 amino acids and exists in two closely related forms, namely, basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF). Its receptor (FGFR) belongs to the receptor protein tyrosine kinase, currently known FGFR mainly includes four types, FGFR1, FGFR2, FGFR3 and FGFR4. Clinically, FGFR overexpression and activation in tumor tissues are found to be associated with the occurrence of a variety of cancers, which can promote tumor angiogenesis and tumor cell proliferation. Therefore, FGFR is widely considered as an important drug target for anti-tumor, which has attracted extensive attention from pharmaceutical scientists in various countries.

In vitro experiments, both types of FGF stimulated the DNA synthesis of osteoblast-like cells isolated from rat cranial cap. Meanwhile, bFGF stimulated the colony formation of differentiated chondrocytes in AGAR, playing a role in mitogen and morphology. AFGF can be formed by macrophages, cartilage and chondrocytes, which can stimulate the proliferation of immature chondrocytes and their progenitors. In vivo studies, FGF has been observed to promote the repair of articular cartilage. The expression of FGF was also found in the mouse fracture model by immunohistochemistry.

In addition, fibroblast growth factor has the following main effects on the human body:

  1. Effects on the skeletal system: It promotes the generation of a large number of osteoblasts and inhibits osteoclasts. To treat osteoporosis, femoral head necrosis, arthritis, rheumatism and diseases caused by calcium deficiency.
  2. Effects on the digestive system: It can strengthen gastrointestinal function, promote the digestion of enzymes, increase appetite, treatment of chronic gastritis.
  3. Effects on the blood system: It can strengthen the hematopoietic function of bone marrow, promote the generation of stem cells, and then generate a large number of red blood cells and white blood cells. Strengthening left ventricular thickness, enhancing myocardial elasticity, effectively treating heart disease. Effectively removing low density protein in the blood, preventing deposition in the blood vessel wall, and treating thrombosis.

Here are some important FGFs members and their main functions below:

  1. FGF2

Fibroblast growth factor 2(FGF2), also called the basic fibroblast growth factor(bFGF), is widely involved in cell growth, differentiation, migration, angiogenesis and tumorigenesis. The FGF2 gene is located at 4q26 of human chromosome, the length of which is 38kb, contains 3 exons and 2 introns. The active FGF2 can be combined with the fibroblast growth factor receptor (FGFR) which has the nature of tyrosine kinase receptor (FGFR) through heparin sulfate proteoglycans (HSPG), activate PKC, Ras /Raf /MEK/ERK, P13K, JAK/STAT and other signaling pathways, and participate in the regulation of cell proliferation, differentiation and malignant transformation. In the cardiovascular system, studies have shown that FGF2 is involved in cardiac hypertrophy caused by stress load and angiotensin. However, some studies also have shown that FGF2 can cause cell chromatin concentration, inhibit proliferation and cause cell apoptosis. Clearly, the cardiovascular role of FGF2 is not fully understood.

  1. FGF18

FGF18 is a highly conserved protein composed of 207 amino acids, with 30% to 70% homology with other members of FGFs found. FGF18 exists in a variety of cell types, and its function is not limited to skeletal development, but can stimulate the proliferation of numerous mesenchymal cells, epithelial cells and tissues, including lung, kidney, heart, testis, spleen, skeletal muscle and brain. As a member of the growth factor family, FGF18 plays an important role in morphogenesis, tumor growth and other cellular and tissue development. Compared with other growth factors except wound surface repair, FGF18 has potential clinical value in the treatment of skeletal diseases such as chondrodysplasia, chondroplasia and bone repair, with a wider range of effects. The decreased expression of FGFR3 was also observed by studying the FGFR3 and STAT1 signaling pathways, suggesting that FGF18 may play a role independent of FGFR3.

  1. FGF23

FGF23 is an endocrine protein synthesized by osteoblasts and osteoblasts. It has been confirmed that the main role of FGF23 is to regulate the level of blood phosphorus as a hormone, which plays an important role in the metabolism of calcium and phosphorus, parathyroid gland, as well as participates in the regulation of bone mineral metabolism. FGF23 is a protective factor for kidney, promoting urinary phosphorus excretion and maintaining stable blood phosphorus metabolism.

In addition, FGF also has powerful functions and deep repair effect. The study of FGFs plays an immeasurable role in modern clinical medicine, surgery and cosmetic surgery.

Advances in vaccine adjuvants research (part one)

  1. 1.Definition of vaccine adjuvant?

What is a vaccine adjuvant? Before answering the question, let’s see what is an adjuvant. Adjuvant, also known as immunomodulator or immunepotentiator, originated from the Latin word “Adjuvare” and is meant to aid or enhance. An adjuvant is an additive of a vaccine. When it is mixed with an antigen and injected into the body, it can enhance the body’s immune response to the antigen or change the type of immune response. It is a non-specific immunopotentiator and itself has no antigenicity. The ideal adjuvant not only enhances the immune response, but also provides the body with optimal protective immunity.

  1. 2.Application of vaccine adjuvant

(1) Enhancing the immunogenicity, immune response rate and tolerance of purified or recombinant antigens;

(2) Reducing the amount of antigen or the amount of inoculation required to achieve immunoprotection;

(3) Improve the immune efficacy of vaccines in infants, the elderly or people with impaired immune systems;

(4) As an antigen delivery system that takes up antigen through the mucosa, it can promote the absorption of the vaccine by the gastrointestinal mucosa. The concept of adjuvants is derived from ulcers formed at the site of inoculation and promotes the production of high levels of specific antibodies, even those produced by inoculation of unrelated substances can induce the production of highly specific antibodies;

(5) Adjuvants can increase the infiltration of cells, prevent antigen degradation, transport antigens to specific antigen-presenting cells, enhance antigen presentation or induce cytokine release.

  1. 3.Classification of adjuvants

At present, there is no uniform standard for the classification of adjuvants in the world. According to the chemical composition, it can be divided into aluminum salt adjuvant, protein adjuvant, nucleic acid adjuvant, lipid-containing adjuvant and mixed adjuvant.

(1) Aluminum salt adjuvant

Aluminum salt has been used clinically for more than 80 years and is the first classic adjuvant approved by the US FDA for human use. Many vaccine ingredients contain aluminum salts such as DTP vaccine and Haemophilus influenzae vaccine. Depending on the preparation process, vaccines with aluminum salts as adjuvants can be divided into two types: aluminum adsorption vaccines and aluminum precipitation vaccines. The aluminum adsorption vaccine is to add an antigen to an aluminum hydroxide or aluminum phosphate solution; and the aluminum precipitation vaccine is to add an aluminum suspension to the antigen solution. Aluminum hydroxide or aluminum phosphate is an aluminum adjuvant that is often used. The study found that aluminum adjuvant vaccine can reduce the amount of antigen used and enhance the strength and durability of the body’s immune response. The mechanism of action of aluminum salts is still not clear. It is generally believed that the antigen-adsorbed aluminum salt particles form a gel state and are injected into animals to form an antigen reservoir. These insoluble particles can adsorb antigenic substances and increase the surface area of the antigen. In addition, the adjuvant can form a macrophage-rich granuloma at the injection site, delaying the absorption of the antigen, thereby prolonging the stimulation time of the antigen, and retaining the antigen for several days under normal conditions for a few weeks, and the antigen is taken at the injection site. Ability is enhanced. Studies have shown that aluminum hydroxide as an adjuvant can also activate Th2 cells to secrete IL-4, induce the expression of CD83, CD86 and MHC-II molecules, and then produce a Th2 type humoral immune response. Aluminum salts have many advantages as vaccine adjuvants, but there are also deficiencies. Although it can effectively induce a humoral immune response, it does not act on cellular immunity and cannot induce a cellular immune response.

(2) Protein adjuvant

Most of protein adjuvants belong to small molecule polypeptides or glycoproteins. A class of biologically active substances synthesized and secreted by immune cells and certain non-immune cells, generally cytokines, play an important role in the differentiation of Th cells. It can also enhance the function of NK cells and T lymphocytes, and has a wide-ranging effect on the immune response of the body. The use of a protein adjuvant in combination with an antigen enhances the immunogenic efficacy of the vaccine, and it can also be assembled into a plasmid and mixed with the antigen for injection. IL-12 is produced by monocytes and B cells and has a variety of biological activities. It can significantly reduce the number of bacterial invasions and increase the expression levels of IgG2a and IgA in the mucosa and immune system. It is a cytokine with broad application prospects. Adjuvant. It induces a Th1-type immune response, and the treatment of tumors and AIDS is in clinical trials.

 (3) Nucleic acid adjuvant

In the process of researching vaccines, some nucleic acid substances are also found to have the characteristics of adjuvants. The most representative one is CPG DNA, in which the unmethylated cytosine deoxynucleotides and guanine deoxynucleotides are Unit oligomers, agonists of TLR9, are currently a hot spot in adjuvant research. It plays an important role in enhancing specific immune responses, inducing non-specific immune responses in the body, and regulating the type of immune response. The characteristic sequence of CPG-ODN can activate a variety of immune effector cells, such as T cells, B cells and NK and other immunocompetent cells, so CPG-ODN is used in more experimental studies. Bacterial DNA is a source of CPG-ODN, and its role includes enhancing both humoral and cellular immunity. It is more likely to be used in tumors and infectious diseases. It has not been reported that CPG-ODN has serious side effects on humans, but it has been found in animal models that CPG-ODN can induce autoimmune diseases.

(4) Lipid-containing adjuvant

Lipid-containing adjuvants include lipopolysaccharide (LPS) and liposomes. LPS is a Gram-negative bacterial outer membrane lipopolysaccharide, and lipid A is the main component of adjuvant action in LPS. The researchers co-immunized mice with LPS as an adjuvant to pertussis vaccine. The results showed that LPS not only improved the immune efficiency of the vaccine, but also reduced the occurrence of type I hypersensitivity. Liposomes are similar to biofilms. Ultrafine spherical preparations, usually formed by bilayers of phospholipids and cholesterol coated with antigens, are capable of transporting antigens and as adjuvants for vaccines. Liposomal adjuvants are not toxic and can reduce the toxicity of the antigen and can degrade in the host itself. Studies have shown that liposome vaccine delivery can enhance the body’s humoral and cellular immune responses, and the structure of the liposome facilitates the presentation of antigens to antigen-treated cells. Studies have also shown that liposomes combined with Freund’s reagent or aluminum hydroxide have a multiplier effect. However, it also has shortcomings, such as poor stability, easy oxidation, and high production costs. Therefore, current research on the application of liposomes has been suspended for medical research.

(5) Mixed adjuvant

MF59 is an oil-in-water emulsion which is a uniform droplet emulsion formed by mixing Tween 80, sorbitol trioleate and squalene under high pressure conditions. This mixed adjuvant can induce a local immune stimulating environment at the injection site, increase chemokines, cytokine levels, and accumulate MHC+ cells in the muscle. In addition, MF59 is also able to enhance the ability of dendritic cells to take up antigen. Because MF59 enhances the immunogenicity of influenza in people with low immunity, it was certified as an adjuvant to influenza vaccines in the 1990s. A large amount of data shows that MF59 is safer for influenza vaccines.

(6) Aggregate structure adjuvant

The researchers compared the immunopotentiating effects of three novel molecular aggregate formula adjuvants [RAM1, Glycolamide (RAM2) and 5th generation dendrimer (RAM3)] and evaluated these adjuvants ability of an adjuvant to enhance a Th1 or Th2 response when applied with a soluble protein antigen. In this study, ovalbumin (OVA) was used as an antigen, and the above three new aggregates were adjuvants, and tuberculin, Al (OH)3, and Freund’s incomplete adjuvant were used as controls. Results RAM1 was superior to other adjuvants in the three adjuvants, and the induced cytokines were mainly Th1 type, and induced a Th2-type response in the late stage of inoculation. In this study, ovalbumin (OVA) was used as an antigen, and the above three new aggregates were adjuvants, and tuberculin, Al (OH)3, and Freund’s incomplete adjuvant were used as controls. Results RAM1 was superior to other adjuvants in the three adjuvants, and the induced cytokines were mainly Th1 type, and induced a Th2-type response in the late stage of inoculation.

To be continued in Part Two.

Reference

[1] Aguilar J C, Rodríguez E G. Vaccine adjuvants revisited[J]. Vaccine, 2007, 25(19):3752-3762.

[2] Coffman R L, Sher A, Seder R A. Vaccine Adjuvants: Putting Innate Immunity to Work[J]. Immunity, 2010, 33(4):492-503.

[3] Mata-Haro V, Cekic C, Martin M, et al. The Vaccine Adjuvant Monophosphoryl Lipid A as a TRIF-Biased Agonist of TLR4[J]. Science, 2007, 316(5831):1628-1632.

[4] O’Hagan D T, Valiante N M. Recent advances in the discovery and delivery of vaccine adjuvants[J]. Nature Reviews Drug Discovery, 2003, 2(9):727-35.

Introduction to TCR

What is TCR?

T cells are the main functional cells in the acquired immune system, which are responsible for identifying antigens and directing other immune cells to carry out immune responses. T cell antigen receptor on T cell surface plays a key role in antigen recognition. T cells play an important role in the immune system and can attack pathogens and cancer cells. T cell receptor (TCR) can recognize different ligands with wide affinity and participate in activating various physiological processes. TCR cell therapy customizes functional TCR, which has the best antigen recognition characteristics and uses the human immune system to fight cancer.

The structure of TCR

 

Figure 1. The structure of TCR

T cells are the main components of adaptive immune response. The structure of their antigen recognition receptors has been confirmed. The cloned TCR consists of alpha and beta chains as heterodimers. TCR heterodimers mainly bind to several signal transduction subunits of CD3, CD3gamma, CD3delta and CD3E heterodimers and CD3delta homologous dimers. Different subunits of CD3 contain the activation sequence of tyrosine, the immune receptor, ITAM, but the number of each subunit is different. CD3gamma, CD3delta and CD3epsilon contain one, respectively, while CD3delta contains three tandem ITAMs, so that each T cell receptor can produce 10 ITAMs. Tyrosine phosphorylated ITAM can couple TCR with intracellular signal transduction pathways and recruit SH2 domain proteins, such as tyrosine kinase ZAP70, from TCR. However, there are still two hypotheses to explain why TCR complex contains so many signal transduction subunits and ITAM. One is that CD3 or ITAM alone may perform different signal transduction functions by recruiting unique effector molecules, and the other is that the main function of multiple TAMs is to amplify TCR signals.

TCR signaling pathway

TCR recognizes a peptide that binds to MHC molecules presented by antigen presenting cells (APCs). Single TCR can recognize different ligands (auto-peptide and foreign-peptide) with wide affinity. TCR is involved in triggering different functional outputs. In thymus, the binding intensity of pMHC and TCR signals determines the process of cell development and differentiation. When the binding force is between the minimum and the maximum, it promotes the survival of thymocytes and transforms into the mature stage of CD4*CD8 or CD4CD8*. If the TCR and pMHC are too low or too high, cell apoptosis will occur. Peripherally, the low affinity binding of autologous pMHC to TCR provides the strong survival signal necessary to maintain the initial T cells, and also promotes the full activation of autologous pMHC in high affinity encounters with foreign antigens.

The intensity of TCR signal is crucial for the production of appropriate T cells. TCR signal transduction response guides the successful differentiation of CD4* T cells into different T helper cell subsets, and plays a key role in specific T helper cell subsets. The intensity and duration of TCR cells are related to the differentiation of memory T cells, and are also the basic determinants of T cell impotence or depletion. TCR signal is regulated by biochemical and molecular mechanisms, leading to signal amplification or attenuation. The mechanism of TCR regulation is complex and diverse, but it can be divided into three basic levels: early signal transduction effector molecule; signal molecule development stage; and dynamic regulation of TCR signal intensity.

TCR binds to the MHC complex expressed on antigen presenting cells (APC) to activate TCR signaling pathway. SRC family protein tyrosine kinase LCK binds to the cytoplasmic domain of CD4 and CD8 common receptors, and is recruited to TCR through the co-binding of CD8 or CD4 with MHC or MHC class I complexes, respectively. LCK phosphorylation enables protein tyrosine kinase ZAP70 to bind to the CD3 chain. T cell activation cohesion factor LAT is phosphorylated, activated T cells, recruited multiple cohesion factors and effector molecules, forming LAT signal transducers. Activation of LAT-related effector molecules leads to signal transduction through three main signaling pathways: calmodulin, MAPK and NF-KB signaling pathways. Calmodulin signal transduction activates T cell nuclear factor (NFAT) for nuclear translocation; MAPK signal transduction leads to actin aggregation and activation of transcription factors FOS, JUN, AP-1; and NF-K B signal transduction causes nuclear translocation of REL and NF-K B transcription factors: These transcription factors synergize to cause T cell proliferation, migration, cytokine production and effect function. TCR is also usually associated with the activation of its common receptor CD28 or cytokine receptors (such as PI3K, AKT, PIP3, PTEN, JAK, STAT) and other signaling pathways.

Application of TCR

Tumor therapy

T cells were modified by TCR gene to exert specific immune regulation or cytotoxic activity. It has great potential in the treatment of malignant tumors and other diseases. Tumor-associated antigen-specific TCR gene was transduced into T lymphocyte for the treatment of tumors, starting with melanoma.

In addition to melanoma, some scholars have recently attempted to extend TCR gene therapy to the treatment of colorectal cancer, synovial cell carcinoma, neuroblastoma, lymphoma and other tumors.

TCR DNA vaccine

TCR gene can be used as a target gene for therapy besides being a guide gene for targeted therapy. In recent years, the use of TCR DNA vaccine to treat some diseases has been gradually carried out. TCR DNA vaccine is a kind of gene vaccine. Through the expression of antigen protein in the body, the body can induce specific immune response, selectively kill pathogenic T cells or inactivate them to play a therapeutic role.

The future of TCR

At present, two research hotspots in the field of TCR gene therapy are: (1) mutation of CDR region to improve the affinity of TCR transduction; (2) pairing of TCR A and beta chains. There are three main problems in TCR gene therapy in the future: (1) in vivo environment of T cells after transfusion; (2) quality problems of T cells after transfusion; (3) safety of transduction of TCR.

In TCR gene therapy, antigen-specific TCR gene modified T cells in tumors and infectious diseases have achieved exciting results, which has proved to be a promising therapeutic strategy. Antigen-specific TCR gene-modified T cells have replaced the traditional effector cells and become the hotspot of this field. Although there is still some distance from large-scale clinical application and becoming a conventional treatment method, it is believed that TCR gene therapy will become more and more perfect and play a greater therapeutic role with the deepening of research.

Construction and Evaluation of Single-chain Antibody Library

Abstract: As a key genetically engineered antibody, single-chain antibody consists of three parts, the antibody light chain variable region, the heavy chain variable region and a segment of the bridge polypeptide chain. The single-chain antibody not only has strong targeting, but also is not easy to dissociate, has a simple structure, is easy to operate and transform, and is advantageous for antibody library screening. At present, there are two main ways to construct single-chain antibodies: 1. Amplification of antibody light chain and heavy chain variable regions from hybridism cell lines, but the construction of single-chain antibodies has a rejection reaction. 2. RNA was extracted from canine spleen B lymphocytes, reverse transcribed into cDNA, and antibody variable region was obtained. The phage display antibody library technology is a new technology currently available for the preparation of human monoclonal antibodies. It uses the RT-PCR method to amplify the human immunoglobulin full set variable region gene and then recombine into the prokaryotic expression vector, and forms a fusion protein with the phage coat protein to express the antibody fragment on the surface of the phage, and screens by panning. The specific variable region gene can conveniently and efficiently perform high-throughput screening of antibodies, and opens up a simple and rapid production route for the preparation of human monoclonal antibodies. A key aspect of this technology is the construction of a library of human phage antibodies.

Keywords: antibody library, single-chain antibody, construction

Phage single-chain antibody library for pancreatic cancer

Pancreatic cancer is one of the most malignant tumors known to date. Pancreatic cancer is the fourth leading cause of cancer death in Europe and America due to the insidious pancreatic cancer and lack of specific symptoms and signs. The phage antibody library technology has opened up new fields for the research of genetically engineered antibodies, and has been used in the diagnosis and prevention of infectious diseases, the identification of autoimmune diseases and viral diseases, imaging analysis of tumors and targeted therapy or gene therapy. The field shows great potential and broad application prospects. Compared with the traditional hybridism technology, the technology is simple, easy to produce, low in production cost, large in screening capacity, and can be prepared in large quantities by fermentation.

The total RNA of peripheral lymphocytes of patients was extracted, and the H chain and L chain variable region genes were amplified by RT-PCR. The VL fragments were randomly spliced ​​into ScFv fragments by SOE-PCR. The ScFv fragment was then cloned into a specific vector and electroporated into a competent strain, rescued by the helper phage M13K07, and a human phage single-chain antibody library of pancreatic cancer was obtained, which aims to lay a foundation for the biological treatment of pancreatic cancer.

Pneumoconiosis phage single-chain antibody library

At present, pneumoconiosis is still the most common type of occupational disease in China with the highest incidence rate, the risk of death, and the most serious harm to workers. So far, there is no cure for it, which can only be eliminated or reversed according to the condition. Delaying the progression of the disease, therefore, active prevention and early diagnosis are particularly important to improve the quality of life and prolong life. The construction method is similar to the above and will not be described again.

Ribosomal display single-chain antibody library

Ribosomal display is a new technology for the screening and identification of functional proteins completely isolated, avoiding the drawbacks of traditional in vivo screening techniques, resulting in increased library capacity and enhanced molecular diversity. It binds the correctly folded protein and its mRNA to the ribosome, forming an mRNA-ribosomal-protein trimer that links the genotype and phenotype of the protein of interest. In recent years, ribosome display technology is built on a single-chain antibody library and applications have made great progress. The main processes of ribosome display technology include:

  • Construction of ScFv ribosome display template
  • ScFv single-chain antibody in vitro transcription and translation
  • Affinity screening and enrichment of ribosomal complexes
  • Evaluation of screening efficiency
  • In vitro evolution of ScFv molecules

The ribosome display technology is completely in vitro, and has the advantages of simple database construction, large library capacity, and simple screening method, no need to select pressure, and the like, and the introduction of mutation and recombination technology to improve the affinity of the target protein, so it is a large-scale construction. Because it can screen high-affinity protein molecules in a short period of time, its emergence opens up a new way to prepare small molecule antibodies. It is believed that with the continuous solution of shortcomings such as system stability in ribosome display technology, it will have great application value for the construction of antibody libraries and the screening of small molecule antibodies.

Anti-canine parvovirus single-chain antibody library

According to the larger bacterial display technology of bacterial particles, antibodies can be displayed on the surface of bacteria and the binding of antigen and antibody can be monitored by flow cytometry (FCM) in real time by fluorescent labeling, and the antibody-expressing strain with high affinity can be sorted. Using bacterial endometrial display technology, the foreign gene is expressed together with the NlpA signal peptide, and the target protein is displayed in the bacterial inner membrane. The NlpA signal peptide can transport the fusion gene of interest to the bacterial periplasmic space, and utilizes 6 specific amino acids to form a serotonin bond with the outer membrane of the bacterial endometrium during the transmembrane process, anchoring to the outside of the bacterial inner membrane to form a bacterial display antibody library. The outer membrane of the bacteria is treated with lysozyme, and the antibody library is screened by fluorescent labeling specific antigen binding to FCM. The method utilizes a 6 amino acid short peptide to anchor the foreign protein, has no effect on the conformation of the expressed antibody protein, and maintains genotype and phenotypic association. The aim is to screen recombinant antibodies with affinity anti-CPV gene, and lay a foundation for neutralizing active antibodies in subsequent studies. The neutralizing antibodies can bind to neutralizing epitopes on the virus, prevent the virus from binding to the corresponding receptors on the cell surface, and prevent diseases.

Evaluation

The ultimate goal of building a library is to screen out various specific antibody molecules. Whether the antibody of interest can be screened, in addition to the affinity of the antibody of interest and the target antigen, the quality of the library is also a determining factor. A library of antibodies with greater capacity and diversity will ensure the success of the screening. In general, the storage capacity is positively correlated with the affinity of the antibodies screened. High-affinity antibodies can only be screened in a high-capacity antibody library. At the same time, it must have good diversity. For natural antibody libraries, a wider range of antibody gene sources can better ensure the diversity of antibody libraries.

References

[1] Baca M, Presta LG, O ‘Connor SJ, et a1. Antibody humanization using monovalent phage display [J].  J Biol Chem, 1997, 272:10678-10682.

[2] Lowenfels A B, Maisonneune P. Epidwminology and prevention of pancreatic cancer [J], Jpn J Clin Oncol, 2004, 349(5):238-242

Top 5 Reasons Why You Should Adopt a Pet

A pet is an animal that brings many benefits to the owner. Being a pet owner will give you a sense of fulfillment and satisfaction that is why having a pet around you at home can be a wonderful addition to one’s life. If you do not own a pet yet and is contemplating to have one, then you may want to consider the following reasons why you should adopt a pet. Below are the top 5 reasons why owning a pet will positively affect your life.

  1. Pets can give you unconditional love and companionship. Pets make you always feel accepted and loved. Just like genuine love; they offer constant companionship and friendship. Your favorite animals give you one thing better than respect and loyalty. They can give you unquestionable, unbridled, and unwavering love. Treat your friend with the respect they deserve and they will repay you with lifelong attention and affection, especially when you need it most.
  2. Pets can uplift a person’s mood. No matter how sad, angry, depressed, or lonely you may be, spending some time with your pet will help you forget your problems. Recent study shows that having your favorite pet around you can reduce stress. You can count on your pets to be there for you in many ways that other people can’t. They can give you love and friendship, and can also enjoy comfortable silences. Animals can be the best antidote to loneliness.
  3. A pet is a prescription for good health. How can these friendly creatures help you live longer? A per can increase a person’s physical activity and improve the overall health status. Medical studies revealed that having a pet has many health benefits especially for the pet owners. Owning a pet can help lower the blood pressure, fight loneliness, and conquer depression. In addition to this, exercise is a great side effect of owning an active pet. This is a great excuse to get physical.
  4. Pets will help you meet new friends. There is no doubt that people gravitate towards other people who have their pets with them in the park or other places. This will increase your chances of meeting other people, giving you the opportunity to increase your network of friends and acquaintances.
  5. A pet teaches you to have a sense of responsibility. If you do not care enough for your pet, they will surely lose their life. Owning a pet is a great way to learn the value of responsibility. You will learn that life doesn’t just revolve around you, that there are other beings depending on you too. Owning a pet is similar to parenthood, it teaches you discipline and responsibility. Raising a pet entails a lot of effort. It is vital to provide your pets with high quality wholesale pet products to save your time in going to the pet store and money in providing supplies for them.

 

3 Essential Tips To Successfully House Train Older Dogs

House training older dogs is a more delicate process than with younger dogs. If you are looking to adopt an older dog that was not previously exposed to any house training at a young age, it is going to bring along its bad habits such as chewing, excessive barking, making holes in the yard or “marking” the territory.

Furthermore, you will also experience more difficulties in house training older dogs as they tend to respond and progress much slower to most training methods and commands than younger dogs.

Here are 3 useful tips that can help you in house training older dogs successfully.

* First and foremost, a complete check-up at the vet is necessary in order to eliminate any existing health problems or complications such as urinary tract or flea infections. Older dogs may also experience some form of diarrhea if there is a sudden change from their previous dietary habits. Do not punish him for these “mistakes”. Instead be patient and allow your dog a week to adapt to the changes gradually. If conditions did not improve, consult your vet.

* Always establish a routine schedule for eating and drinking for your dog. This helps you to easily monitor your dog’s toilet habits and decide when to bring your pet out for a walk every time he or she finishes its meals. Most adult dogs tend to eliminate 30 – 45 minutes after eating or drinking. Learn the signs and watch out for clues of circling and sniffing the floor.

* Older dogs have shorter attention spans therefore more repetitions are needed during the house training process to reinforce the desired habits. Never use violence on them if they make a mess in the house. Scolding the dog is pointless unless you actually see it eliminating indoors. Use a firm voice command “No” and guide the dog to its designated spot. Praise or reward your dog when it relieves itself outdoors.

Therefore, house training an older dog successfully depend a lot on your consistency, discipline and perseverance. Treating the dog with patience and understanding, using the appropriate house training methods are the main ingredients to developing a well behaved adult dog with good toilet habits.

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What is Tetramer technology

Tetramer technology: a technique in which MHC monomer molecules are tetramerized to increase their affinity and can be combined with multiple TCRs on T cells.

Introduction to tetramer technology

 

Since the soluble MHC monomer molecule has a low affinity with the TCR, the dissociation is fast, and the multivalent molecule can bind to multiple TCRs on one specific T cell, so that the dissociation speed is greatly slowed down. To this end, Altman et al. proposed the construction of MHC class I molecular tetramers by means of the biotin-avidin cascade amplification principle. The method uses a genetic engineering technique to add a 15 amino acid residue of Bio A substrate peptide (BSP) to a MHCI-like molecule such as the carboxy terminus of the HLA-A2 heavy chain to form a fusion protein, which is pressed in vitro. A certain proportion is incubated with beta microglobulin and specific antigenic short peptides to fold into the correct conformation and become pMHC complexes. Biotin is labeled on the lysine residue of the substrate peptide such that a fluorescein-labeled streptavidin binds to four biotinylated pMHC complexes to form a tetramer, MHC-antigen peptide tetramer and After binding of TCR on antigen-specific CFI-, antigen-specific CTLs in vivo can be quantified by flow cytometry and sorted for in vitro culture amplification and functional analysis.

 

Effect

The tetramer technology enables specific, efficient and direct quantification of antigen-specific CTI-activity assays, and can be applied to immunological research and detection, specific immunotherapy, and vaccine efficacy monitoring.

 

  • As a clinical diagnostic tool, quantitatively measure the ratio of antigen-specific CTI in peripheral blood and tissues, and perform phenotypic and functional analysis. Altman et al. have detected antigen-specific CTI in a large number of asymptomatic AIDS patients. The detection rate is as high as 2%. Zerbini et al. used tetramer technology and immunohistochemistry to detect peptide-specific CD8 CTLs in tumor tissues of hepatocellular carcinoma patients for the first time, providing broad prospects for the application of immunotherapy to MAGE antigens in HCC. In situ staining of tetramers in autoimmune diseases was first used in TCR transgenic mice. Through the quantitative detection, phenotypic and functional analysis of antigen-specific CTL, it laid the foundation for elucidating the pathogenesis of viral infectious diseases, tumors and autoimmune diseases.

 

  • For adoptive tumor immunotherapy: Mei-denbauer et al. administered a large number of Melan-A peptide-specific CTLs (42.1%) to 8 patients with refractory malignant melanoma, and found peripheral blood singles. The ratio of antigen-specific CTL in nuclear cells is determined by infusion. 01% to 0.07% rose to 2% after infusion. Moreover, these cells can survive in vivo for several weeks, have the function of secreting INF-7, and preferentially aggregate to the tumor to play a role.

 

(3) Monitoring of vaccine efficacy: In recent years, the use of antigen peptide pulse treatment as a vaccine to immunize the body to induce an effective CTL response has become a research hotspot. The efficacy of the vaccine can be monitored by tetramers constructed with homologous peptides.

 

About us

Creative Peptides is specialized in the process development and the manufacturing of bioactive peptides. We are dedicated to offering custom peptide synthesis, process development, GMP manufacturing as well as catalog products for customers in industry and research area. Here are some our products like: Glycopeptides SynthesisGhrelin PeptidesGlucagon Like PeptidesChelate Peptides, etc.

A brief talk on the Sodium Butyrate

Sodium butyrate is a chemical substance with a molecular formula of C4H7NaO2 and a molecular weight of 110.0869. The character is white or white like powder, and has special cheese rancidity like odour and hygroscopicity. The density is 0.96g/mL (25/4 C), the melting point is 250~253 C, and it is soluble in water and ethanol.

About the enzyme activity of the sodium butyrate, there are mainly four aspects: 1, buffering capacity of feed ingredients is the main factor affecting the free acid content in the stomach. The greater the buffer capacity, the more free acid can be absorbed in the stomach, which makes the free acid in the stomach decreased, the pH value in the stomach increased, and then affect the activity of protease and protein digestion and decomposition. Generally speaking, the higher the contents of protein, calcium, phosphorus and trace minerals, the higher the buffer capacity of the feed. 2, the dissociation degree is small, acidity is weak, to achieve the same acidification capacity is much larger than inorganic acid, adding cost is high; Sodium butyrate as acidifier, added in the feed diet is direct, added acid is easy to be neutralized by alkali, loss of acidification; if can not reach the small intestine, can not effectively reduce the intestinal acidification effect PH value can not inhibit the growth of harmful bacteria and promote the proliferation of beneficial bacteria. 3. Sodium butyrate is stable at low temperature and under sealed condition, but it can not be mixed with substances, dust, superheated materials and strong oxidizing agents. It is easy to dilute and agglomerate during storage or cause feed moisture. 4, the absorption and metabolism of short chain fatty acids in the colon are very strong, and butyric acid is the fastest. Because of the rapid metabolism of butyric acid, the serum half-life of butyric acid in the body is only 6 minutes. In the use of sodium butyrate as a therapeutic drug, in order to achieve effective blood concentration, the commonly used method is to use sustained-release technology, sodium butyrate is coated into tablets, so that butyric acid can reach the colon without previously being absorbed and metabolized by the small intestine.

For example. Breast cancer is a common malignant tumor, which seriously affects the health of women. In recent years, the incidence of breast cancer in the world is increasing year by year and the age is gradually younger [. Sodium butyrate is a sodium salt of short-chain fatty acid butyric acid. It mainly changes chromatin structure by changing the acetylation degree of histone and participates in the expression of many genes. Sodium butyrate can be produced by anaerobic bacteria after digestion and fermentation of carbohydrates and proteins in the above foods. Sodium butyrate accounts for a large proportion of energy sources in colon epithelial cells, which stimulates the release of colon peptides or growth factors, regulates the blood supply of colon mucosa and promotes the growth of colon epithelial cells. Epithelial cell proliferation, but some reports have shown that sodium butyrate can also inhibit the proliferation of tumor cells and induce cell senescence and apoptosis.

The industrial enzyme production of the Sodium Butyrate is a complex access, so for more information, you can click here to visit our website.

A brief talk on the invertase

Invertase, also known as sucrase or beta-D-fructofuran glucosidase, is the key enzyme in sugar metabolism in organisms. It catalyzes the following reactions in sucrose metabolism: sucrose + H2O fructose + glucose. Sucrose is the main product of photosynthesis in higher plants. It is an important factor in carbon transport, sink metabolism, sugar accumulation, fruit quality formation, and also a regulator of cell metabolism. Sucrose may play a role by influencing gene expression. Therefore, transaminase, which is closely related to sucrose metabolism and accumulation, has become one of the hotspots of papain enzyme, biochemical, physiological, ecological and molecular biology research in recent years.

Invertases are highly polymorphic, including acid invertases (AI), neutral invertases (NI) and alkaline invertases. Many reports regard neutral invertase and alkaline invertase as the same invertase. Acidic invertase mainly exists in enzyme urease or bound to the cell wall, and its optimum pH is 3.0-5.0; neutral and alkaline enzymes are located in the cytoplasm, and the optimum pH is about 7.0. The molecular weight of the transformed enzyme is 50~80 kD, which is monomer or two dimer. There are vacuolar invertase expression in reproductive organs. Studies on cultivated tomatoes, wild tomatoes and transgenic tomatoes showed that the expression of vacuolar invertase determined the soluble sugar content of the fruit at the late ripening stage. In situ hybridization showed that cell wall invertase might exist in serrapeptase enzyme. Tissue and sub-tissue localization of invertase showed that high activity of invertase in cell wall was associated with rapid tissue growth, while high activity of vacuolar invertase was often associated with fruit development or rapid expansion of storage organs. This phenomenon was found in seedlings, young leaves, young roots and young fruits. Fast growing tissue always has a high level of AI activity, while NI activity is much lower than AI activity. In melon, the increase of AI activity provides hexose as a carbon source for rapid tissue growth. Invertase from melon fruits hydrolyzes sucrose to glucose and fructose to maintain osmotic pressure of cells.

Invertase is related to fruit development, maturation and sugar accumulation. As fruit ripening and sucrose accumulation, AI activity decreased. This change has been found in netted melon, sugar beet and other plants. Schaffer et al. concluded that the decrease of soluble AI (in vacuole) activity was a primary condition for sucrose storage by comparing sweet and non-sweet melon varieties. Sucrose hydrolase activity (insoluble AI and soluble AI, NI) of peach was higher in young fruit stage. Sucrose accumulation and hydrolytic enzyme activity decreased with fruit development. As a cytosolic enzyme, NI has lower AI level in mature tissues, so NI is more important for sucrose hydrolysis. NI was found to regulate sucrose metabolism in mature tissues of sugarcane, beet, citrus and carrot, but not in immature tissues. The activity of NI in immature tissues of Melon reticulata was higher than that of AI. With fruit development, the decreasing trend was the same as that of AI. Gao et al. thought that the function of NI in sucrose metabolism of Melon fruits might be different from other sucrose storage papaya papain

Sucrose metabolism in higher plants is a complex process. The distribution of key invertases in plants and their changes in sucrose accumulation have been studied. It is a research direction to study the direct effect of invertase genes on sugar accumulation in fruit by molecular biological means. In addition, environmental stress and invertase regulation have also been studied. The study provides a good foundation, but the adversity area involved and the accumulation of information are very limited. Because most of the growth cycles of plants and crops are in adverse ecological conditions, it is still of great practical significance to study the polymorphism expression and physiological regulation of invertase under various stresses.

How does DNA vaccine work?

DNA vaccines are a type of nucleic acid vaccines, which are also called genetic vaccines. The plasmid vector containing the encoded protein gene sequence is introduced into the host by intramuscular injection or microprojectile bombardment, and the antigenic protein is expressed by the host cell, thereby inducing the host cell to generate an immune response to the antigen protein to prevent and treat the disease.

Nucleic acid vaccines are developed by modern biotechnology means, including immunology, biochemistry, molecular biology, etc. They are divided into DNA vaccines and RNA vaccines. At present, the research on nucleic acid vaccine is mainly based on DNA vaccine. DNA vaccines are also known as naked vaccines. After the DNA vaccine is introduced into the host, it is taken up by the cells, and the protein antigen of the pathogen is expressed in the cells, and the cellular immunity and humoral immunity are stimulated by a series of reactions.

The immune mechanism of the nucleic acid vaccine is illustrated as follows:

  1. Nucleic acid vaccine is a nucleic acid-mediated immunization vaccine developed in recent years.

The essence is that the eukaryotic expression vector containing the pathogen antigen gene can be taken up by the body cells and express the antigenic protein of the pathogen when it is introduced into the body, thereby inducing the body’s immune response to the protein. A systemic or local immune response can be triggered as the route and location of the introduction differs. In a systemic immune response, both humoral and cellular immunity can be induced.

  1. Nucleic acid vaccine can trigger a comprehensive immune response

When a protective antigen gene with a highly expressed regulatory sequence is introduced into an animal’s somatic cell, only a small amount is taken up by the cell and enters the nucleus. Under the control of the promoter on the vector, the antigen gene mRNA is transcribed, and the latter enters the cytoplasm and is translated. Corresponding antigenic protein.

 

  1. Nucleic acid vaccine can also induce local immune response and immune memory

If a gold granule coated with a nucleic acid vaccine is introduced into the mucosa by a gene gun, it may be taken up and expressed by lymphocytes or mucosal epithelial cells in the mucosa-associated lymphoid tissue under the mucosa, and the produced antigen protein is easily localized by the antigen. The presenting cells (APC) recognize, ingest, process and present to TH cells, further activating B cells in local lymphoid follicles to differentiate into plasma cells and Bm cells, which produce immune memory, the former can synthesize IgA, and the IgA monomer has a J chain linked together. When passing through the mucosa, the secretory sheet produced by the mucosal epithelial cells is linked to the dimeric IgA, and the stable secretory type IgA is discharged together with the mucosal secretion, distributed on the mucosal surface, in the mucosal part. It plays a very important role in defense against infection.

So, here comes with the question: how does the DNA vaccine work?

 

The mechanism of action and influencing factors of DNA vaccine

The DNA vaccine is a new vaccine in 1990, also known as a nucleic acid vaccine. The DNA vaccine clones a foreign gene encoding an antigenic protein into a eukaryotic plasmid expression vector, introduces the recombinant plasmid directly into the animal cell, and allows the foreign gene to express the antigenic protein in the animal through the transcriptional system of the host cell. Thereby inducing the host to produce an immune response to the antigenic protein for the purpose of preventing and treating the disease.

DNA vaccine can stimulate the body-specific immune response, has a long immunization period, has the advantages of low production cost, easy mass production and preservation, and is a new generation vaccine with application prospects. However, compared with traditional vaccines, the immune response stimulated by DNA vaccines is relatively weak, which is not enough to cause sufficient immune protection, especially for humans and large animals. Therefore, studying the mechanism of action and influencing factors of DNA vaccines is crucial for the development and application of the vaccine.

The mechanism of action of DNA vaccine

Current studies have shown that the immune response induced by DNA vaccines includes both humoral immunity (specific antibodies) and cellular immune responses with longer memory times and cell killing power. It is generally considered that after the nucleic acid vaccine is introduced into the body, it is taken up by surrounding tissue cells, antigen-presenting cells or other inflammatory cells. The plasmid DNA molecules taken up by tissue cells such as muscle cells are then transcribed into mRNA in the nucleus and then transferred to the cytoplasm for translation into antigenic protein molecules. An antigenic protein molecule secreting cells released into the interstitial space is captured by an antigen-presenting cell, processed into an antigen peptide, and presented to the T cell to initiate an immune response. APCs in peripheral lymphoid organs directly ingest nucleic acid vaccines, express antigens and present them to T cells, triggering an immune response. Dendritic cells are the most important antigen-presenting cells in the process of nucleic acid immunization, while B cells do not participate in antigen presentation during nucleic acid immunization. After eliciting an immune response, the cytotoxic T cell response recognizes and kills the muscle cells expressing the foreign antigen, causing the myocyte to dissolve and release the intracellular antigen, and APC directly acquires the antigen from the injection site, and then initiates the subsequent immune response. The combined action of several pathways allows DNA vaccines to stimulate T lymphocytes via the histocompatibility complex MHC I and MHC II pathways, as well as activate B lymphocytes. Tissue cells such as muscle cells may play a role in storing plasmids and releasing them during the immunization process.

DNA vaccines are a relatively new development in the field of vaccinology, and this approach is now moving toward rational DNA vaccine design. Strategies include optimizing vector backbones, transgenic sequences, co-expression stimuli, introduction systems for vectors, and targeting vectors for obtaining appropriate immunostimulation. Another consideration is the use of design methods to optimize gene expression. Since been discovered, DNA vaccine has entered a new stage after more than 10 years of development. Due to its ease of use and wide application, it has become a trend in the development of new vaccines in the 21st century. Although the DNA vaccine itself is relatively less immunogenic, recent studies have shown that DNA vaccines are primed, enhanced with recombinant viral vectors or recombinant protein vaccines, and are extremely effective in activating the immune system and inducing a strong immune response. This prime-boost immunization program has been used as a new vaccine model. The effects of DNA vaccines are determined by factors such as the plasmid itself, vaccine adjuvants, and immunization protocols, and their interactions. There are many strategies for enhancing the immunological or therapeutic effects of DNA vaccines, including optimization of antigen expression, selection of immune pathways, delivery vehicles, and selection of adjuvants.