Battling infectious diseases with 3-D protein structures

An international team of scientists including the Computation Institute has determined the 3-D atomic structures of more than 1,000 proteins that are potential targets for drugs and vaccines to combat some of the world’s most dangerous emerging and re-emerging infectious diseases.

These experimentally determined structures have been deposited into the World-Wide Protein Data Bank, an international archive freely available to the scientific community. The 3-D structures help expedite drug and vaccine research and advance the understanding of pathogens and organisms causing infectious disease.

“Almost 50 percent of the structures that we have deposited in the Protein Data Bank are proteins that were requested by scientific investigators from around the world,” said Wayne Anderson of Northwestern University Feinberg School of Medicine, who is director of the project. “The NIH has also requested us to work on proteins for potential drug targets or vaccine candidates for many diseases, such as the Ebola virus, the Zika virus and antibiotic-resistant bacteria. We have determined several key structures from these priority organisms and published the results in journals such as Nature and Cell.”

This milestone effort, funded by two five-year contracts from the National Institute of Allergy and Infectious Diseases (NIAID), totaling a budget of $57.7 million, represents a decade of work by the Center for Structural Genomics of Infectious Diseases (CSGID). CSGID is led by Anderson in partnership with the University of Chicago, the University of Virginia School of Medicine, the University of Calgary, the University of Toronto, the Washington University School of Medicine in St. Louis, the UT Southwestern Medical Center, the J. Craig Venter Institute, the Sanford Burnham Prebys Medical Discovery Institute and University College London.

Before work begins on a targeted protein, a board appointed by the NIH examines each request. Once approved, the protein must be cloned, expressed and crystallized, and then X-ray diffraction data is collected at the U.S. Department of Energy’s (DOE) Advanced Photon Source, a DOE Office of Science User Facility at Argonne National Laboratory. This data defines the location of each of the hundreds or even thousands of atoms to generate 3-D models of the proteins’ structures that can be analyzed with graphics software.

Each institution in the Center has an area of expertise it contributes to the project, working in parallel on many requests at once.

The University of Chicago team focuses on antibiotic resistance factors such as metallo β-lactamases, essential enzymes that are potential drug candidates, as well as transcription factors responsible for virulence from pathogenic bacteria.

Argonne Distinguished Scientist Andrzej Joachimiak, the co-principal investigator of CSGID, said the initiative was a highly collaborative effort. The team works closely with microbiologists and chemists from the biology community to identify chemicals that will inhibit protein activity; then they produce and crystallize proteins with those inhibitors bound to determine their structures using X-ray crystallography.

The University of Chicago team has recently characterized protein crystal structures with novel inhibitors from two important agents that cause human disease: Mycobacterium tuberculosis and Cryptosporidium parvium, responsible for tuberculosis and the parasitical infection cryptosporidiosis, respectively.

In their research, the team uses community resources in the Advanced Protein Characterization Facility and data collection facilities at the Advanced Photon Source.

Until recently, the process of determining the 3-D structure of a protein took many months or even years — but advances in technology, such as crystallization methods, the powerful X-rays from the Advanced Photon Source, upgrades to computational hardware and software, and automation of many of the steps have dramatically accelerated the process.

The Seattle Structural Genomics Center for Infectious Disease, a similar center funded by NIAID, is also on track to complete 1,000 3-D protein structures soon. Interested readers can browse all of the structures deposited by the CSGID; anyone in the scientific community interested in requesting the determination of structures of proteins from pathogens in the NIAID Category A-C priority lists, or organisms causing emerging and re-emerging infectious diseases, can submit requests to the Center’s web portal. As part of the services offered to the scientific community, the CSGID can also provide expression clones and purified proteins, free of charge.

Related Tags:Protein StructureElectron MicroscopeVLP Construction

Replacing The Cells Lost In Parkinson Disease

Replacing these cells is considered a promising therapeutic strategy. Although DA cell–replacement therapy by transplantation of human fetal mesencephalic tissue has shown promise in clinical trials, limited tissue availability means that other sources of these cells are needed.

Now, Ernest Arenas and colleagues at the Karolinska Institue, Sweden, have identified a new source for DA cells that provided marked benefit when transplanted into mice with a PD-like disease.

In the study, DA cells were derived from ventral midbrain (VM) neural stem cells/progenitors by culturing them in the presence of a number of factors — FGF2, sonic hedgehog, and FGF8 — and engineering them to express Wnt5a.

This protocol generated 10-fold more DA cells than did conventional FGF2 treatment. Further analysis revealed that these cells initiated substantial cellular and functional recovery when transplanted into mice with PD-like disease.

Importantly, the mice did not develop tumors, a potential risk that has precluded the clinical development of embryonic stem cells as a source of DA cells. These data led the authors to suggest that Wnt5a-treated neural stem cells might be an efficient and safe source of DA cells for the treatment of individuals with PD.

The article Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice is published in the December 3 issue of the Journal of Clinical Investigation.

Related Tags:FGFR4FGF23FGF2

Protein Array Spreads Most Areas of Your Research

What are protein arrays?

Protein array, a high-through detection system, is used to track proteins’ interactions and activities, therefore, it can determine their function and determining function on a large scale.

The basic theory of it

The Detection object of array is protein instead of DNA. It is different from the gene array, which is used to detect gene expression on the level of mRNA. It detects the model of expression on the level of protein.

Array format

There are two formats of it: antibody arrays and target protein arrays. The first is used to recognize the protein and other molecules from samples by using the antibody on the format. The other one is used for detecting protein or other molecules with the protein on format. Such as, the function of drugs, antibody, nucleic acids, lipids or other proteins.

With the advantage of high-through detection, protein array is the best tool to study the function of protein. What’s more, because of the diverse proteins and complex function of protein in creature cells, this product with the versatile capacity in handling samples and high-through detection utilizes to simplify and accelerate progress in the study of its function. The most used method for its detection is fluorescence labeling. Of course, other labels also can be used in this detection. Such as, affinity, photochemical or radioisotope tags.

In the study of biochemical activities of proteins, three types of them are widely used.

The first one is analytical microarrays. It is also called capture arrays. In this field, a library of antibodies, aptamers or affibodies is arrayed on the support surface. Because each of them will bind to specifically to a particular one, so they are used as capture molecules.

The second one is called functional one, also known as target one. They are different from analytical arrays. It is mainly formed with arrays, which contains full-length functional proteins or its domains. These are mainly used to study the biochemical activities of the entire proteome in a single experiment.

The last one is reverse phase protein arrays (RPPA). In this field, it is mainly including complex samples like tissue lysates. And RPPA allow for the determination of the presence of altered proteins or other agents, which might be the result of disease.

These are main functions and features of this useful product. It is widely used in many fields. So it is very useful for scientists.

Related Tags:Protein ExpressionBacillus ExpressionYeast Expression Systems

Introduction to Nutrition Elements-Inorganic Nutrition

We know that there are six essential nutrients for people’s lives: water, inorganic salts, vitamins, carbohydrates, fat and protein. The first three are inorganic nutrients, and the last three are organic nutrients.

As a college student who majors in bio-chemistry, today, let me introduce the first three inorganic nutrients for you. So let’s start.

Water is the source of life. In any biochemical reaction, water acts as a catalyst and solvent. Water provides a place for chemical reactions. And it also increases the reaction rate extremely. What is more importantly, water and human metabolic reactions are closely related. It is the famous TCA Cycle that is carried out in the human body fluid environment. Nutrition can be dissolved in water and transported to the whole body with blood. It can be said if there is no water, then there is no life. There is nothing important than water.

Also it has been found that inorganic salts have a great relationship with hormone synthesis and some special protein synthesis in human body. For example, human hemoglobin contains negative ferrous elements (creative proteomics provides excellent bioinformatics analysis ). Human bones and teeth are rich in calcium. And the substances that control human neurons, synapses that release neurotransmitters, are sodium and potassium salts. Lack of these two inorganic salts can lead to symptoms of muscle weakness. Dangerous words can even lose the ability to breathe. In addition to metal salts, other non-metallic salts are also very important. N, P and S are necessary for the composition of human genetic material DNA. Lack of these three elements is fatal to human beings.

In addition to the above two accidents, vitamins are also very important nutrients and vitamins are divided into two kinds, Water Soluble Vitamin and Fat Soluble Vitamin. There is a difference between the two. Water-soluble vitamins are soluble in water but not in non-polar organic solvents. They need not be digested. After being absorbed directly from the intestine, they are circulated to the tissues needed by the body. Most of the excess vitamins are excreted by urine and stored very little in the body. Water soluble vitamins are easily soluble in non-polar organic solvents, but not in water. They are absorbed in the small intestine through bile emulsification and enter the various organs of the body from the lymphatic circulation system. A large number of fat-soluble vitamins can be stored in the body. Lack of vitamins can have many effects on the human body. For example, vitamin C deficiency can lead to scurvy. Lack of vitamin D may lead to calcium deficiency. And the most effective way to supplement vitamins is to eat more vegetables and fruits, balanced nutrition. Children can get more sunshine. Sunlight can promote the production of vitamin D in children, which is very helpful for children’s growth.

What have you learned from today’s lecture? If you like my presentation, please pay attention to our website https://www.creative-proteomics.com. That’s all for today’s introduction. See you next time. Thank you very much. Good bye.

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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