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And so far, all the gear I’ve seen has been rather well-balanced in terms of advantages and drawbacks, meaning that overpowered god-builds feel unlikely, and creative stat-juggling should be quite the fun challenge. However, for those who’ve been waiting five years for another hunter x hunter web game adventure, I don’t think x hunter online will be good enough. An ancient robot who devoted his life to give birth to his children?
If you’re in the mood for a more competitive battle, x hunter game’s “hunter x game” player-versus-player (PvP) mode, much like the battlegrounds in hunter x hunter game online, pits two teams against one another in a battle to the death. But hunter x hunter games has a loot system. hxh game involves gathering piles of loot, something which is addictive for veteran RPG gamers.The portable screen magnifies small details that are lost when the console is docked.
 Be prepared to play the hunter x game beyond the first main ending; that’s simply the end of the first part, and the full Hunter X Online plays out over five different endings. Canonically strong team combinations.Though Nintendo’s limits on full Excel-spreadsheet nerdery may be a shortcoming in the eyes of those who revel in such systems, if the idea of an RPG is to role-play then shouldn’t I be able to slay the final boss if I, the player, role-playing as the hero, am skilled enough? It’s odd that hunter x hunter browser game and now Zelda champion such outside-the-box thinking when it ought to be role-playing hunter x hunter mmorpg games that consider such matters the most heavily. Because while the traditional – and less obvious – fighting hunter x hunter online game archetypes are present and correct, from all-rounder hunter x online game, to nimble, acrobatic hunter x hunter mmorpg online, to tricksy, technical, trap-setting Dr.
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Conventional antibody

Conventional antibodies or fully sized antibodies are glycoproteins called immunoglobulins that are produced by the reaction of plasma cells with foreign molecules or antigens. The most basic function of antibodies is to bind specific antigens and stimulate immune responses to protect the body from infection. Antibodies include several subtypes, and here IgG and IgM subtypes are mainly described. Antibodies to IgG and IgM subtypes are widely used in research, diagnosis and treatment.

  1. Structure of conventional antibodies

The basic structure of an intact antibody consists of four peptide chains, including two heavy chains and two light chains, joined together by disulfide bonds. The antibody is shaped like a letter Y, and the hinge region of the Y structure is elastic. Each peptide chain has a constant region (very conserved across all antibodies) and a variable region (specific in antibody). The symbol of the light chain variable region is VL, and the symbol for the light chain constant region is CL (Fig. 1 left). Similarly, the variable and constant regions of the heavy chain are designated as (VH) and (CH), respectively. Carbohydrates usually bind to the CH2 region of the heavy chain. The Fc segment includes only the constant region (CH) of the heavy chain, but the Fab segment (Fab) that binds to the antigen includes a variable region of the constant region and the heavy chain and a variable region (VH and VL) of the light chain. The Fv region (variable fragment) contains only two variable regions.

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Fig. 1 The basic structure of a complete conventional antibody (left) and usual antibody fragment (right)

  1. Application of conventional antibodies

Conventional antibodies have been used in research to detect target proteins by Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA) for decades. Full-size antibodies are also used in clinical tests, such as pregnancy tests and the detection of HIV in the blood by ELISA. In addition, conventional intact antibodies are also used in the treatment of diseases. For example, infliximab is an antibody that recognizes tumor necrosis factor and is used to treat intestinal diseases and rheumatoid arthritis. Trastuzumab or Herceptin is an antibody that binds to epithelial growth factor II and is used to treat metastatic breast cancer. In addition, there are many antibodies, including Muromomab, that are used in basic therapies after organ transplantation to prevent graft rejection.

Advantages of using conventional antibodies include the ability of the Fc region to activate the body's immune response and bind to the target molecule to destroy it. Disadvantages of using intact antibodies include the inability to penetrate into certain tissues due to their large size. The ability of the Fc segment to activate some immune responses that are harmful to the patient is a disadvantage of clinically applied intact antibody therapy. The Fc region usually causes some non-specific binding and is detrimental to the application of antibody detection.

  1. Antibody fragment

A fragment of an antibody can be obtained by chemical reagents and genetic engineering methods. The fragments obtained by chemical reagent are achieved by disrupting the disulfide bond in the hinge region or by digesting the antibody with proteases, including pepsin and papain. Genetically engineered fragments provide a large number of fragments, each with specific binding regions and functional traits.

Fab, Fab', (Fab') 2, and Fv

An antigen-binding fragment (Fab) can be obtained by chemical treatment and protease digestion, which is derived from the variable regions of the IgG and IgM. The antibody portion from which the Fabs fragment is removed is the Fc fragment and consists of the constant region of the heavy chain. Antigen-binding fragments include Fab, Fab', (Fab') 2, and Fv. These fragments are capable of binding antigen, but they lack the Fc segment, which includes constant regions 2 and 3 of the heavy chain. When the antibody was digested with papain, two separate F(ab) fragments were isolated from the Fc region. However, after digestion with pepsin, a F(ab')2 fragment with a small portion of the Fc hinge region was isolated from the antibody. Although the separation of antibody fragments by chemical means can produce many useful diagnostic and therapeutic tools, it is very time consuming and requires a large amount of antibody as a raw material.

The monovalent F(ab) fragment has only one antigen-binding region, whereas the multivalent F(ab')2 fragment has two antigen-binding regions that are joined together by disulfide bonds. The F(ab')2 fragment produces two monovalent Fab' fragments and a free thio group that can be used for the binding of other molecules.

The Fv fragment is the smallest fragment of the product after enzymatic analysis of IgG and IgM type antibodies. Fv fragment antigen binding region, which consists of VH and VC regions, but they lack the CH1 and CL regions (see Figure 1 right panel). VH and VL are combined in the Fv fragment by non-covalent bonds.

ScFv, bispecific antibody, trispecific antibody, tetraspecific antibody, double-scFv, mimi antibody, Fab2, Fab3

The genetic engineering method is capable of producing a single-chain variable region (ScFv), which is an Fv-type fragment that includes VH and VL regions linked together by a flexible polypeptide (see Fig. 1 right panel). If the binding region has a length of at least 12 residues, the ScFv fragment is the monoclonal antibody. Different forms of Fv molecules can be created by manipulating the length of the V-domain and the hinge region. The resulting scFv molecule whose linker is a 3-11 residue cannot be folded into a functional Fv domain. Together with other scFv molecules, these molecules create a bivalent, bispecific antibody. If the length of the linker is less than 3 residues, the scFv molecules can interact to produce a trispecific or tetraspecific antibody. Multivalent scFvs have a stronger affinity for antigen binding than corresponding monovalent antibodies. The Mini antibody is a scFv-CH3 fusion protein that is loaded into a bivalent dimer. Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can be produced by two different variable regions, allowing these Bis-scFv molecules to simultaneously bind two non-existing epitopes. Genetic methods can be used to generate bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are capable of binding to 2 (Fab2) or 3 (Fab3) different antibodies at the same time.

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Section I: Overview

Recombinant protein drugs, also known as recombinant DNA protein drugs, and genetically engineered protein drugs, refer to the use of recombinant DNA technology to optimize the modification of the gene encoding the protein of interest, and use a certain vector to introduce the target gene into an appropriate host cell. A biologically active protein preparation obtained by expressing a protein of interest and subjected to extraction and purification techniques for the treatment or prevention of human diseases.

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First, the development of recombinant DNA technology and recombinant protein drugs

  1. The establishment of molecular biology theory

On April 25, 1953, James Watson and Francis Crick, working at the Cavendish Laboratory at Cambridge University, published a paper entitled "Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid" in the journal Nature and found double helix structure of DNA. At the same time, Nature published two papers by Rosalind Franklin and Maurice Wilkins, which proved the DNA structure model and jointly opened the era of molecular biology. Watson, Crick and Wilkins shared the 1962 Nobel Prize in Physiology and Medicine.

In 1958, at the 12th Symposium on Experimental Biology in the United Kingdom, Francis Crick presented the Central Dogma of Molecular Biology and published it in the proceedings of the seminar (Symp. Soc. Exp. Biol. 12).

In January and April 1965, Marshall Nirenberg published a paper, "RNA Codewords and Protein Synthesis" in Science and PNAS, deciphering the nucleotide genetic code, and thus achieved his collaboration with Har Khorana and Robert Holley in 1968 who got the Nobel Prize in Physiology and Medicine.

  1. Discovery of multiple tool enzymes

In 1956, Arthur Kornberg of the University of Washington isolated DNA polymerase and shared the 1959 Nobel Prize in Physiology and Medicine with Severo Ochoa.

In 1967, DNA ligase was discovered almost simultaneously in laboratories such as Gellert, Lehman, Richardson, and Hurwitz.

On July 28, 1970, Hamilton Othanel Smith of Johns Hopkins University published a paper "A Restriction enzyme from Hemophilus influenzae *1I. Purification and general properties" in the journal J. Molecular Biology, confirming restriction enzymes. And thus achieved the 1978 Nobel Prize in Physiology and Medicine with Werner Arber and Daniel Nathans.

  1. Establishment of DNA recombinant technology

On October 1, 1972, Paul Berg of Stanford University published a paper "Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli" in PNAS. This indicates that the recombination of DNA molecules was completed in vitro and shared the 1980 Nobel Prize in Chemistry with Fredrick Sanger and Walter Gilbert.

On November 15, 1973, Stanley Norman Cohen of Stanford University and Herbert Boyer of the University of California, San Francisco collaborated in the publication of the paper "Construction of biologically functional bacterial plasmids in vitro" in the journal PNAS, which announcd the arrival of recombinant DNA technology and genetic engineering.

  1. Recombinant protein drugs come out

On August 24, 1978, David Goeddel of Genentech and others used E. coli to express and synthesize the world's first recombinant human insulin.

On May 14, 1982, Eli Lilly submitted a request for the listing of human insulin, the world's first recombinant protein drug, to the U.S. Food and Drug Administration (FDA). On October 28, 1982, the FDA approved the market for the drug (Humulin®). In 2017, the series still ranked the ninth in the global diabetes drug market with sales of US$1.34 billion/year.

Second, the expression system and basic procedures of recombinant protein drug preparation

The preparation of recombinant protein drugs mainly includes prokaryotic expression system and eukaryotic expression system. The prokaryotic expression system is mainly bacteria, and the most commonly used are Escherichia coli and Bacillus subtilis. The eukaryotic expression system is complex and has eukaryotic cell expression, including yeast cells, baculovirus-insect cells, mammalian cells, and transgenic animal and plant bioreactors.

Like most drug development processes, recombinant protein drug development mainly includes three parts: pharmaceutical research, pharmacology research and clinical research. However, unlike traditional small molecule drugs, even if the drug preparation process has been determined, the preparation process of the recombinant protein drug will largely determine the quality of the drug and directly affect the safety and effectiveness of the drug. Therefore, analysis and discussion from the development process of recombinant protein drugs will help us better understand the preparation process.

The pharmaceutical research of recombinant protein drugs mainly includes two stages: laboratory research and pilot research. The laboratory research is generally divided into the construction of recombinant engineering cells (including animal and plant bioreactors), cell culture and protein expression, and separation and purification of target proteins, protein pharmaceutical preparations, and quality research and quality control modules throughout. The pilot study mainly includes modules such as pilot process amplification, quality standard establishment and stability test. Pharmacological research mainly includes modules such as major pharmacodynamics, pharmacokinetics and safety evaluation.

When the target protein is isolated and purified to obtain a certain purity of the protein stock solution, after determining the preliminary formulation to prepare the pharmaceutical preparation, or after the laboratory basically determines the drug preparation process and produces at least one batch of samples that have passed the preliminary test (small test), it should be timely to carry out the stability study of protein and its preparations, the research data can be used as an important basis and content of the stability test; however, in the application of clinical materials, at least one batch of three consecutive trial samples must be completed under the pilot scale amplification process and the stability data of the sample should last for 6 months.

Samples prepared in laboratory tests can be used to carry out major pharmacodynamics and pharmacokinetic studies, while samples for safety evaluation studies in principle use samples should be prepared by pilot studies. This can better reflect the safety of actual production products and only samples prepared on a pilot scale can meet the sample size required for the test.

When drug research and development enters pharmaceutics pilot research and pharmacology research, it can be called into the preclinical research phase. In the laboratory pharmaceutical research stage, or only the initial pharmacodynamic or pharmacological test, it can not be called the preclinical research stage.

To be continued in Part Two…

 

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Camel/shark source antibodies and Nanobodies

In addition to conventional antibodies, camelids and sharks also contain specific heavy chain antibodies (hcAbs) that are composed entirely of heavy chain homodimers lacking the light chain. The Fab portion of these antibodies is referred to as VHH (the variable domain of the heavy chain antibody) and is the smallest antigen binding region found in nature.

Nanobodies are VHH-derived recombinant domains and are capable of binding antigen. They are very stable and can be easily mass produced by traditional simple systems, such as bacteria (but conventional antibodies with light and heavy chains are difficult to express in bacterial systems) and are therefore promising tools for research and therapeutic applications, especially in the field of super-resolution microscopy, mass spectrometry and targeted protein degradation. Nanobodies can be delivered to living cells by covalent attachment to the polypeptide, or can be expressed and recognized directly in vivo, but conventional antibodies with both light and heavy chains cannot be used for living cells. For example, anti-GFP Nanobodies are used to develop electromagnetic control systems that study neuronal activity in vivo. When the anti-RFP or GFP Nanobody binds to the far red dye Atto, it can obtain a fluorescence signal amplification of 118 times that of GFP or RFP, which can be used to generate whole body mouse neuronal connections. They can also be used in structural studies to stabilize the active state of proteins. AAV expression vectors have been shown to produce universal influenza vaccines with linked Nanobodies against four different influenza strains. Recombinant anti-mouse and anti-rabbit IgG secondary Nanobodies have the potential to replace widely used polyclonal secondary antibodies. Nanobodies have a unique ability to cross the blood-brain barrier; however, they are often processed and cleared out of the body very quickly. Nanobodies can be used for specific purposes, such as (co)immunoprecipitation or real-time fluorescent protein tracing of intracellular targets in living cells.

Cattle Long CDR3H

Approximately 10% of bovine immunoglobulins contain a long CDR3H region with multiple cysteine residues that are believed to contribute to antibody diversity.

Applications of antibody fragments

In some applications, fragments are more advantageous than intact antibodies. This topic was recently disscussed by Nelson. One of the advantages is that the fragment is smaller than the intact antibody, and can enter the tissue in which the intact antibody cannot enter and exert therapeutic effects and immunohistochemical staining. Antibody fragments are smaller than conventional antibodies and are generally not glycosylated, allowing their products to be expressed in prokaryotic expression systems, saving time and money. However, fragments lacking the Fc domain are faster in vivo than conventional antibodies and are unable to elicit Fc-mediated cytotoxicity unless they bind to a valid original for better therapeutic purposes. While the lack of an Fc domain is advantageous for immunohistochemistry and other assays because of the reduced non-specific binding of antibodies to Fc receptors. Antibody fragments without an Fc region have the advantage of being able to reduce non-specific binding. The anti-influenza neuraminidase antibody is a ScFv that is widely used in diagnosis. The anti-epithelial cell adhesion molecule Ep-CAM antibody MOC-31 is a ScFv for cancer therapy. Bispecific antibodies, trispecific antibodies and tetraspecific antibodies have potential applications in radioimmunotherapy and in vivo imaging diagnosis.

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Although various antibody fragments have certain advantages, they are generally not used in experiments. Of more than 45,000 articles surveyed, only a few articles were related to the application of antibody Fab fragments.

Fc fragment receptor

The Fc receptor (FcR) is a molecule expressed mainly on/in innate immune cells that recognizes and binds to the Fc domain of an antibody, thereby providing them with a cellular system to elicit an immune response. The multiple functions of FcR reflect the broad protection or regulation of antibodies, including mediation of targeting substrates for neutralization and clearance, and adaptive immunity. The biological function of FcR is regulated by an immunoreceptor tyrosine-based activation motif (ITAM) and an immunoreceptor tyrosine-based inhibition motif (ITIM) as receptor interfaces for activation and inhibition of signaling pathways, respectively. Thus, signaling by ITAMs can trigger cell activation, phagocytosis and endocytosis, while signaling by ITIMs has an inhibitory effect on cell activation. A description of the FcR of all classes of immunoglobulins is available, some of which are discussed below.

IgG receptor

This family includes FcγRI, FcγRII, FcγRIII and subtypes thereof. They are responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ACDP).

Another IgG-binding receptor is the neonatal Fc receptor (FcRn), which is involved in the transfer of passive humoral immunity from the mother to the fetus. FcRn also protects IgG from degradation in vivo, which is why they have a long half-life in serum. The Fc-FcRn interaction is promoted by changes in the Fc region, which leads to the development of better therapeutic antibodies.

IgE receptor

They include a high affinity FcεRI capable of binding to monomeric IgE and a low affinity C-type lectin FcεRII capable of preferentially interacting with the IgE complex. FcεRI mediates immediate hypersensitivity responses to many allergic reactions by stimulating cell degranulation and releasing a range of inflammatory mediators on mast cells and basophils. FcεRII can exist in both membrane-bound forms for the delivery of down-regulated IgE synthesis and in the presence of soluble fragments to produce an up-regulation effect. Its role in the endocytic process of IgE allergen complexes in human airways and intestinal epithelial cells is being actively studied and may be a potential target for the treatment of allergic airway inflammation caused by food allergies.

IgA receptor

FcαRI is the only member of this type and is expressed only in bone marrow lineage cells. It plays a role in pro-inflammatory and anti-inflammatory responses depending on the IgA binding state. Although secretory IgA (SIgA) binding to mucosal sites has anti-inflammatory effects, including prevention of pathogen invasion, binding of serum IgA leads to an inflammatory response. FcαRI also regulates neutrophil viability based on the inflammatory microenvironment.

TRIM21

TRIM21 shows a very broad range of antibody specificities and can therefore be distinguished from other FcRs. It can bind IgG, IgM and IgA. In addition, it is expressed in most cells that produce tissue. TRIM2 is involved in antibody-mediated viral replication interference by targeting cytosolic virus-antibody complexes for proteasomal degradation.

Binding of the Fc domain to FcR may have negative effects in monoclonal antibody-based assays such as immunohistochemistry (IHC), flow cytometry (FACS), and chromatin immunoprecipitation (ChIP). Non-specific binding to FcR may introduce background noise, resulting in false positives. Solutions to this problem include the use of (i) an isotype control for gating, (ii) serum to compete extensively for non-specifically binding receptors, or (iii) purified IgG to specifically block Fc receptors .

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FHA is encoded by the fhaB gene and synthesized as a precursor of 367 kDa, the C-terminal portion of which is cleaved to yield the mature 220kDa FHA protein28. Mature FHA is exported and expressed on the bacterial surface, and is also released from the cell surface into the environment. FHA has several different binding activities that implicate it as an important adhesin. An Arg–Gly–Asp (RGD) amino acid motif promotes adherence to monocytes by the leukocyte-response integrin/integrin-associated protein complex and complement receptor type 3 (CR3). A carbo-hydrate-recognition domain allows adherence to cilia, and a lectin-like binding domain promotes adherence to sulphated carbohydrates and heparin, which are found at the surface of the respiratory tract. Despite these binding activities, in vivo studies have failed to define clearly the role of FHA in Bordetella pathogenesis. Some have failed to observe a difference between wild-type and an FHA-mutant B. pertussis in a mouse model of infection, whereas others have observed B. pertussis FHA mutants to be deficient in tracheal colonization. A protease, SphB1, was recently shown to be required for the secretion of FHA. B. pertussis SphB1 mutants were impaired in their ability to colonize the mouse respiratory tract. Instillation of purified FHA into the respiratory tract before inoculation with the SphB1 mutants, or co-infection with wild-type bacteria alleviated the attenuation of the mutants. This indicates that release of FHA from the bacterial surface contributes to the colonization process. Such contradictions might derive from the use of the human-specific pathogen B. pertussis in a mouse model. Studies using B. bronchiseptica indicated that FHA is required, but is not sufficient, for colonization of the rat trachea.

It has been proposed that FHA forms a hairpin-like rod. Its amino acid sequence contains two regions of different, imperfect direct repeats of 19 residues, known as repeat regions 1 and 2 (R1 and R2), which are proposed to form β-sheet structures which comprise much of the hairpin shaft. Both the RGD and carbohydrate recognition domains are located at the tip of the hairpin shaft, and might be ideally placed for interaction with host structures. Study of the amino-terminal segment of FHA led to the proposal of an alternative structure in which the overall shape of the molecule is a rod, but in which the shaft comprises β-helical segments that are derived mainly from the repeat regions. In this model, the CR3-binding domain, the RGD motif and the carbohydrate-recognition domain are located in the middle of the shaft.

Genome sequence information revealed that the fhaB sequences of the three bordetellae are highly conserved. The B. bronchiseptica FhaB protein is predicted to contain copies of R1, whereas the B. pertussis and B. parapertussis FhaB proteins are predicted to each contain only 38 copies. The FhaB protein of B. bronchiseptica might therefore be expected to form a longer shaft than the FhaB protein of B. pertussis or B. parapertussis. Previous studies have also determined that B. bronchiseptica FhaB contains more copies of R1 than the FhaB of B. pertussis; however, the B. bronchiseptica strain that was studied (strain GP1) contained only 40 copies of R1. So, the repeat number of R1 could vary not only between species, but also between strains of the same species. The consequences of this variability should be further investigated. An FHA homologue was recently characterized in Bordetella avium. Although the amino acid sequence of this FhaB is clearly different to those of the three mammalian-adapted species, B. avium FHA seems to have a similar role in the infection biology of this species. The completion of the B. avium genome sequence project will enable a full evaluation of B. avium FHA-like genes and their similarity to those described here. Two other genes, fhaS and fhaL, which are predicted to encode FHA-like proteins, have also been identified in the Bordetella genome sequences.

The B. bronchiseptica genome contains a locus that seems to encode the bio-synthesis of Type IV secretion system protein ptlF homolog (ptlf processs) Tfg and which is absent from the B. pertussis and B. parapertussis genomes. Tfg are distinguished from other pili by their polar location. They have several functions, including adherence of pathogens to host cells, twitching and social motility and DNA uptake in Neisseria gonorrhoeae and Bacillus subtilis. The Tfp secretion machinery has homology to the type II secretion apparatus, which forms the general secretory pathway. Tfp fibres are formed from multimers of the major pilin subunit, although minor subunits might also be present. The identity of the major pilin subunit gene in the putative B. bronchiseptica tfp locus is not obvious. However, there are several genes that could encode either the major or minor pilin subunits on the basis of a highly conserved amino-terminal leader sequence that is characteristic of Tfp pilins and is cleaved by the prepilin peptidase PilD (BB0792). After cleavage, the N-terminal residue is often phenylalanine, which subsequently becomes N-methylated. Many of the predicted proteins of the tfp locus are also homologous to type II secretion apparatus proteins. We propose that this locus encodes Tfp biosynthesis on the basis that the predicted protein product of BB0791 is approximately 70% identical to PilT proteins of other Tfp secretion systems throughout the entire protein. PilT is specific to tfp loci and is responsible for pilus retraction, which is required for twitching motility56. There are no reports of Tfp secretion systems in the bordetellae. Future work is likely to investigate the ability of this locus to direct expression of the Tfp secretion apparatus and characterize its role in the adherence of B. bronchiseptica to host structures.

B. bronchiseptica encodes a type III secretion system (TTSS). A TTSS is an export apparatus that delivers specific effector proteins into host cells with subsequent alterations in host-cell behaviour. The B. bronchiseptica TTSS is involved in the alteration of host immune cell function, which reduces the effectiveness of the host response to B. bronchiseptica infection. Previously, the locus had been shown to be present in B. parapertussis and B. pertussis. An ovine isolate of B. parapertussis was shown to transcribe some TTSS genes, but information regarding the expression of the TTSS locus in B. pertussis is contradictory. One study failed to demonstrate expression of some genes of the locus, whereas another study demonstrated the transcription of TTSS genes. The genome sequence information showed that, in B. parapertussis, two of the gene— one regulatory (BPP2241) and one structural (BPP2262)— are pseudogenes, which might indicate that the locus is non-functional in B. parapertussis, at least in this human isolate. However the B. pertussis locus is intact, which might indicate that there are mutations in unlinked regulatory genes that are required for TTSS expression. Some of this confusion might stem from the fact that studies of the expression of the B. pertussis TTSS locus are incomplete, and have only examined a small number of the genes in the locus. Future work is likely to investigate the expression of the locus as a whole, at both the translational and transcriptional levels.

Reference

[1] Bishop R E. The lipid A palmitoyltransferase PagP: molecular mechanisms and role in bacterial pathogenesis [J]. Molecular Microbiology, 2005, 57(4):900-912.

[2] The bordetellae: lessons from genomics. Nat Rev Microbiol. 2004 May; 2(5):379-90.

[3] Cherry, J. D. Pertussis — the trials and tribulations of old and new pertussis vaccines. Vaccine 10, 1033–1038 (1992). 2.

[4] Cherry, J. D. Historical review of pertussis and the classical vaccine. J. Infect. Dis. 174 (Suppl.), S259–S263 (1996).

[5] Heininger, U. et al. Clinical characteristics of illness caused by Bordetella parapertussis compared with illness caused by Bordetella pertussis. Pediatr. Infect. Dis. J. 13, 306–309 (1994).

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The Autoimmune disease models

1. Childhood Asthma

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

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

Specific immunoassay is an important indicator for evaluating allergic conditions in children with asthma. Most children's asthma is associated with allergies, and allergies can increase the persistence and severity of asthma. The original test is very important for guiding allergen immunotherapy and preventing asthma attacks. The skin prick test is the most basic test method to help clinicians find allergens and assist clinicians in the diagnosis of asthma. Skin tests are usually carried out using a variety of common inhaled allergens, including indoor dust, cockroaches, pollen, fungi, animal skins, and silk. Usually, the above-mentioned allergen immersion liquid is diluted into a skin test solution in a certain ratio, and a skin pricking needle is used for percutaneous test on the forearm palm side, and 0.01% of histamine phosphate and physiological saline are used as controls to eliminate false negatives and false positives. Antihistamines, corticosteroids and other drugs should be discontinued before the skin test. This method is safe, simple fast, economical, and has been popularized and applied internationally, and it is painless, so it is very suitable for children. The specific IgE (sIgE) assay is one of the most important detection methods for in vitro diagnosis of allergies when the asthmatic children are allergic to an allergen, and the sIgE assay for the allergen is performed in vivo. The classical detection method is Radioallergosorbent test (RAST), which is a CAP method. There are two reporting methods for the CAP system to detect sIgE results. The method is sensitive, specific, accurate and absolutely safe, avoiding the severe side effects that may be induced by skin tests or systemic responses (very few SPT), and the test is not subject to operator proficiency, medication, and skin conditions (such as severe skin disease, skin scratches). The detection of total serum IgE has been widely used as a screening test for the diagnosis of allergies for many years. However, in addition to allergic diseases, many factors such as race, gender, age, parasitic infection and seasonality can affect IgE levels in serum. In addition, the total IgE level of serum in 20-30% of allergic patients is normal, or even lower than normal level. And the total IgE is not specific, because allergic reaction is not determined by the body, but by the specific IgE corresponding to the allergen. Therefore, the detection of total IgE by serum alone is not perfect for judging the allergic condition of children with asthma, nor does it indicate which allergen is allergic. However, IgE and IgG can be used as indicators to evaluate the efficacy of specific immunotherapy. If the treatment is effective, the former decreases and the latter increases. The Phadiatop screening test is to embed more than 90% of the common allergens in the air in the same CAP and measure them using the CAP system. For example, one or several sIgE phadiatops in serum indicate an allergic reaction, but a negative result can’t exclude allergies because phadiatop only includes common but not all inhaled allergens, and the types of allergens are regional.

2. Mucous membrane pemphigoid

Mucous membrane pemphigoid (MMP) is a heterogeneous group of chronic, autoimmune subepithelial blistering diseases which predominantly affects the mucous membranes and occasionally involves the skin. In vivo, MMP is characterized by linear deposition of IgG, IgA, or C3 along the epithelial basement membrane zone. Although the oral and ocular mucosae are the most common sites affected, the nasopharynx, esophagus, larynx and anogenital region may also be involved. This disorder results in mucosal and/or skin blistering, ulceration, and subsequent scarring. The severity and distribution of the disease is highly variable from mild cases involving only oral mucosa to severe cases involving the eye, genital and esophageal mucosa. The disease may be implicated in the larynx or esophagus, causing stenosis, and may even be life-threatening. Since the consequences of this disease can be severe and limited therapeutic options are available once scarring is developed, early diagnosis of this disease is critical. However, as the disease is rare and the early symptoms are non-specific, MMP is often unrecognized in the early inflammatory stage. Other nomenclatures for MMP include cicatricial pemphigoid, oral pemphigoid, ocular cicatricial pemphigoid (OCP), ocular pemphigoid, and benign mucous membrane pemphigoid. Autoantibodies to one or several autoantigens in the mucosal or epithelial BMZ have been identified in MMP patients. The association of MMP with human leukocyte antigen (HLA) major histocompatibility class II HLA-DQB1*0301 has been demonstrated. The cause is usually unknown, but there are some reports say that MMP can be triggered by medications such as methyldopa, clonidine and D-penicillamine.

The pathogenesis of MMP is complex. MMP has been found to be heterogeneous and involves several different antigens. The pathogenicity of autoantibodies in MMP has been demonstrated in vivo and in vitro.   

Circulating IgG and/or IgA autoantibodies against components of the basement membrane zone found in MMP patients’ serum indicate that MMP is mediated by a humoral immune response. Loss of immunologic tolerance to structural proteins in the BMZ results in development of MMP antibodies. By using immunoblotting and immunoprecipitation techniques, a variety of autoantigens including the bullous pemphigoid antigen 1 (BPAg1) (a 230-kDa protein, BP230), the bullous pemphigoid antigen 2 (BPAg2) (a 180-kDa protein, BP180), integrin subunits α6/β4, laminin-332 (also called epiligrin and laminin-5), laminin-6, and collagen type I have been identified. BPAg1 is an intracellular protein, whereas BPAg2 and α6/β4 integrins are transmembrane proteins. The most frequently targeted autoantigen in MMP is BPAg2. Laminin-5 is thought to be the major ligand between the transmembrane proteins and the anchoring filaments. Anchoring fibrils, composed of type VII collagen, are located deeper in the lamina densa. These autoantigens are not exclusive to MMP. Although BPAg2 is more common, autoantibodies to both BPAg1 and BPAg2 can be present in BP, and autoantibodies against type VII collagen are also found in epidermolysis bullosa acquisita.

An antibody-induced complement-mediated process results in epithelial detachment. Passive transfer studies in newborn mice have shown that antibodies to BPAg2 induce subepidermal blisters by an inflammatory mechanism. This interaction triggers immunologic events that result in the expression of inflammatory mediators that induce migration of lymphocytes, eosinophils, neutrophils, and mast cells to the BMZ. The separation of epithelium from the underlying tissue within the BMZ results from either direct cytotoxic action or the effect of lysosomal proteolytic enzymes. Passive transfer of antibodies against laminin 5 induced non-inflammatory subepidermal blisters, indicating that anti-laminin 5 IgG is pathogenic, although the mechanism is not clear. Anti-α6 antibody produced separation of epithelium from basement membrane. Fibroblasts can also be activated by the process of producing inflammatory cytokines such as transforming growth factor beta (TGF-β), which is known to induce fibrosis. The collagen produced may lead to scarring. Several studies have shown a predominance of CD4+ T cell and Langerhans cell infiltrates in the conjunctiva of MMP patients, indicating the involvement of cellular immunity in the pathogenesis of MMP. The presence of Th17 lymphocytes in conjunctival biopsies was significantly increased in patients with OCP.

MMP can affect multiple mucosal sites, occasionally involve the skin. It is a chronic, progressive disease that most frequently involves the oral mucosa (85% of patients), followed by ocular conjunctiva (65%), nasal mucosa (20–40%), skin (25–30%), anogenital area and/or pharynx (20%), larynx (5–15%), and esophagus (5–15%). Lesions occurring at any site may heal with scarring. There is a great variability in the presentation and severity among patients with both localized and extensive involvement. Localized disease can progress to extensive disease. Those who have the disease affecting only the oral mucosa and/or the skin with less tendency of scarring and with minimal clinical significance are defined as “low-risk” MMP patients. On the contrary, “high-risk” patients are those who have disease occurring in any of the following sites: ocular, nasopharyngeal, esophageal, laryngeal, and genital mucosa. Despite medical treatment of MMP, the high likelihood of scar formation at these sites is associated with a poor prognosis. The ocular involvement can result in blindness, scarring of the laryngeal mucosa can result in sudden asphyxiation, scarring of the esophagus can influence food taking, and scarring of the anogenital mucosa can seriously affect the patients’ daily activities.

To be continued in Part Three…

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The physical properties of metal powder are mainly reflected in the following aspects:

  1. 1.Theparticle size and composition

The particle size and composition of the metal powder depends on the preparation conditions of the metal powder, and it has a great influence on the behavior of the metal powder during pressing and sintering as well as the properties of the product.

Using particle diameters to characterize metal powder particle sizes is only accurate for ideal spherical metal powders, while metal shapes for other shapes can only be approximated. The metal powder used in the metal powder metallurgy porous material has a particle size mainly between several micrometers and 500 micrometers.

  1. 2.Shapes

The shape of the metal powder particles is an important indicator of the properties of the metal powder, which has a great influence on the process performance of the metal powder. The strength, permeability, and uniformity of properties (isotropy) of the article are related to the shape of the metal powder particles.

Both spherical metal powder and non-spherical metal powder can be used to produce a porous material, but it is desirable that the metal powder be spherical in order to improve the pore uniformity and permeability of the article. For the convenience of describing the spherical metal powder, we cite a characteristic coefficient such as the ratio of the major axis to the minor axis of the spherical metal powder, and consider the metal powder having a coefficient of less than 1.2 as a spherical metal powder.

For complex shaped metal powders, this can be represented by deviation from the equivalent volume of spheres or by the length of the particles: the ratio of width to thickness. In the production of a highly permeable porous material, the sphericity of the metal powder (i.e., the percentage of the number of spherical metal powder particles to the total number of metal powders) is required to be 60% or more.

  1. 3.Metal powder specific surface

Most reactions start on the surface of the particles, so the ratio of the surface area of the metal powder particles to their volume or weight - specific surface is one of the important parameters in the metal powder metallurgy process, which directly affects the pressing properties and sintering properties of the metal powder.

For most metal powders, the specific surface value can range from 0.01 square meters per gram to tens of square meters per gram. The specific surface of the metal powder depends not only on the particle size and shape of the metal powder, but also on the surface state of the particles (or the degree of development of the surface).

The finer the particle size of the metal powder, the more complicated the shape, and the rougher the surface, the larger the specific surface of the metal powder; on the contrary, the coarser the particle size of the metal powder, the more regular the shape (such as a sphere), the smoother the surface (no unevenness) of metal powder, the smaller the specific surface. The particle size, shape and surface state of the metal powder are determined by the conditions and methods for preparing the metal powder.

  1. 4.True density and microhardness of metal powder

The density of the metal powder particles is usually smaller than the theoretical density of the raw materials from which it is produced because many of the metal powders produced by the method have considerable internal pores and a large number of lattice vacancies. The true density of the so-called metal powder refers to the density of the metal powder, including only the closed pores inside the particles. The true density of the metal powder varies with the method of milling, and the true density of the metal powder is also strongly dependent on the oxide content.

The microhardness of metal powder particles is an indicator of the plasticity of metal powder. The microhardness value depends to a large extent on the content of various impurities and alloying elements in the matrix metal, and is related to the degree of lattice twist. When preparing a porous material, the metal powder has a certain hardness, which is beneficial for ensuring high permeability of the product. Therefore, for a plastically good metal (such as sodium titanium powder), in order to achieve a certain hardness value, it is often performed before pressing.

  1. 5.Lattice state of metal powder

The metal powder particles are usually composed of crystal grains of various sizes, and the size and orientation of the crystal grains also depend on the manufacturing method of the metal powder. In many cases, there is a correlation between metal powder particle size and grain size. In the process of atomizing the metal powder, the droplets are cooled from the molten liquid phase, and the smaller particles are cooled rapidly, so that the crystal grains of the atomized metal powder fine particles are usually smaller than the coarse particles.

In general, metal powders are produced under non-equilibrium conditions. Therefore, the metal powders obtained by various methods have crystal defects to varying degrees. For example, in the reduction of oxides, the crystal structure of the oxide is converted into a metal crystal structure, but in practice this transformation is incomplete; the atomized metal powder is crystallized from the liquid state quickly, and oxides are present, of course, there may be lattice defects.

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Incidence of lung cancer

Lung cancer has the highest incidence of cancer in the world. According to WHO’ statistics, there are an estimated 18.1 million new cancer patients worldwide in 2018, of which lung cancer accounts for about 2.094 million, accounting for 11.6%. Lung cancer treatment drugs are also the focus of research and development of major pharmaceutical enterprises. From earlier afatinib and gefitinib to now osimertinib and PD-1/L1 inhibitors, it can be found that although these drugs have been approved for lung cancer, but the specific types of lung cancer are different.

Classification of lung cancer

The classification of lung cancer is a very complicated problem. To define exactly what kind of lung cancer is, it needs to be combined with the three aspects of typing, staging and molecular information to understand.

Cancer typing mainly describes what lung cancer cells look like. According to the morphological characteristics of lung cancer cells under microscope, lung cancer cells can be initially divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The growth characteristics, diffusion risk and treatment regimen of these two types of lung cancer are different, so we should first distinguish between the two types of lung cancer. The vast majority of lung cancer is NSCLC, which accounts for about 85%. NSCLC can be further divided into three types: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Adenocarcinoma is the main type, accounting for about 50% of NSCLC.

Staging mainly describes the diffusion degree of lung cancer cells. After lung cancer is diagnosed, doctors use a series of tests to determine the staging of lung cancer by examining whether the cancer is only locally or has spread to lymph nodes or other organs of the body. NSCLC and SCLC are slightly different in the professional staging system, but for the convenience of communication, they can be summarized into 0, I, II, III or IV. I, II, III will sometimes be divided into A and B, such as IIA, IIIB and so on. Stage 0 and I have the best prognosis and the highest cure rate, while stage IV is commonly referred to as advanced cancer, indicating that the cancer has metastasized to other tissues or organs.

Molecular information is genotyping. According to the type of gene mutation, lung cancer can be divided into EGFR mutation, ALK mutation and ROS1 mutation. Different types of gene mutations are suitable for different targeted drugs.

Classification of drugs for lung cancer

At present, targeted drugs are mainly aimed at the types of gene mutations in NSCLC. According to the staging of lung cancer, surgical treatment, chemotherapy, radiotherapy and other traditional methods are generally used in stage I, II and III. In addition, stage IV has different treatment methods according to the type of gene mutation.

Targeted therapeutic drugs and immunotherapeutic drugs are the two mainstream therapeutic drugs for lung cancer, in which targeted therapeutic drugs are divided into small molecules and macromolecules. Small molecule targeted drugs are mainly used in the treatment of NSCLC, such as mutation of EGFR and ALK, while macromolecular targeted drugs mainly refer to VEGF/VEGFR monoclonal antibodies. Immunotherapy is a good choice for patients without obvious driving gene, mainly refers to PD-1/PD-L1.

Genotyping and treatment of lung cancer

EGFR-TKI: EGFR mutation is a type of gene mutation with high incidence in lung cancer. In view of this mutation, erlotinib, gefitinib and afatinib have been approved one after another. In the FLAURA trial, the median progression-free survival time of osimertinib was 18.9 months, which was much higher than that of gefitinib / erlotinib group (10.2 months). However, there is no solution to the treatment after first-line drug resistance of osimertinib.

ALK-TKI: ALK-TKI is mainly used to treat NSCLC with ALK positive mutation. The first, second and third generations of ALK-TKI have been approved, which are crizotinib, ceritinib and alectinib, respectively. The National Comprehensive Cancer Network (NCCN) guidelines recommend alectinib as the first line of treatment for NSCLC with ALK positive mutation. Among them, crizotinib and alectinib are used in the treatment of locally advanced or metastatic NSCLC patients with ALK positive mutation. Ceritinib is used in patients with locally advanced or metastatic NSCLC who have previously progressed after treatment with crizotinib or who are intolerant to crizotinib.

VEGF/VEGFR monoclonal antibodies: bevacizumab combined with chemotherapy, EGFR-TKI and PD-L1 immune checkpoint for the treatment of NSCLC has been approved by FDA. At the same time, bevacizumab combined with ALK-TKI is also in the clinical trial stage.

PD-1/L1: PD-1/L1 is mainly aimed at lung cancer without obvious driving gene. FDA has approved multiple PD-1/L1 therapies for various indications of lung cancer.

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Chromatographic analysis is one of the active branches of analytical chemistry and an important means of material separation analysis. It is increasingly used in environmental, biochemical, and fine chemical product analysis. Application of chromatography related technologies is involved in almost all fields. From classical plate chromatography to column chromatography, from gas chromatography, liquid chromatography, supercritical fluid chromatography to capillary electrophoresis and electrochromatography, it plays an important role in scientific research and industrial production.     

  1. Originof chromatographic technology

The ancient Romans used a piece of cloth or a piece of paper to analyze dyes and pigments. More than 100 years ago, German chemists improved the method to make it more reproducible and quantitative, which later became today's paper chromatography technology. In 1901, Russian botanists used calcium carbonate as an adsorbent to separate plant pigments. Then in 1903, a new method for separating plant pigments by adsorption principle was proposed. In 1906, this method was officially named as chromatography, but it was not taken seriously due to the slow and inefficient separation. Until 1931, German scientists used a similar method to separate more than 60 kinds of pigments such as carotene, and chromatographic methods were then widely used.

  1. Development and application of gas chromatography technology

Since the first creation of practical gas-liquid chromatography in the world in 1952, gas chromatographs, as a representative of modern analytical instruments, have developed into an industry with considerable production scale and formed a discipline with considerable knowledge of detection technology. Gas chromatography is widely used in environmental samples for pollutant analysis, drug quality testing, natural product composition analysis, pesticide residue determination in food and industrial products, quality control and other fields due to its high separation efficiency, fast analysis speed and good selectivity. With the advent of new gas chromatography instruments, detectors, and data analysis methods, the application fields of gas chromatography will become more and more extensive.

The combination of gas chromatography and other technologies has also developed very rapidly in recent years. It is mainly used in combination with mass spectrometry, spectroscopy, etc., combined with chemical reactions, and computer. Gas chromatography is an effective tool for separating complex mixtures, but it is not possible to qualitatively identify unknowns; mass spectrometry, spectroscopy, and nuclear magnetics are effective tools for identifying unknown structures, but require that the samples analyzed be as simple as possible, not complex mixtures. The combination of chromatography and these techniques is therefore a recognized and effective tool for dissecting the structure of unknowns in complex mixtures. In recent years, the development of the two technologies is directly combined, eliminating the need to collect condensation in the middle, so that the analysis time is shortened and the sample consumption is reduced.

Organic mass spectrometers, magnetic mass spectrometry, quadrupole mass spectrometry, ion trap mass spectrometry, time-of-flight mass spectrometry (T0F), and Fourier transform mass spectrometry (FTMS) are currently available on the market to be combined with gas chromatography. With the continuous updating of interface technology, the interface devices are smaller and simpler, and the shape is lighter. The function of GC-MS is more powerful. The resolution of GC-TOFMS can reach about 50M. The combination of GC-MS plays an important role in many areas of analytical testing and scientific research, especially as a necessary tool in the routine testing of many organic compounds. It is widely used in environmental protection, health, food, agriculture, petroleum, chemical and other industries.

  1. Development and application of liquid chromatography

At the beginning of liquid chromatography, a large diameter glass column is used to transport the mobile phase with a liquid level difference at room temperature and pressure, called classical liquid chromatography. Liquid chromatography is a type of separation and analysis technique characterized by a liquid as a mobile phase. The stationary phase can take many forms, such as paper, sheets, and packed beds. The mobile phase of classical liquid chromatography relies on gravity to flow slowly through the column, so the particle size of the stationary phase cannot be too small. The separated samples are collected after classification and analyzed, so that the classical liquid chromatography not only has low separation efficiency, slow analysis speed, but also complicated operation. It was not until the 1960s that a stationary phase with a particle size of less than 10 μm was developed, and a high-pressure infusion pump and an automatically recorded detector were used to overcome the shortcomings of classical liquid chromatography and to develop into liquid chromatography.

Liquid chromatography (LC) is a widely used field in various chromatographic modes. About 80% of the compounds in the world are analyzed by different modes of HPLC (such as normal phase HPLC, reverse phase HPLC, ion exchange chromatography and ion chromatography, volume exclusion chromatography, affinity chromatography, etc.) , including high molecular compounds, ionic compounds, heat labile compounds and biologically active compounds.

Liquid chromatography is developed from gas chromatography and liquid chromatography, and its structure and operation process are perfect. New research and development of liquid chromatography and its combined technologies have been widely used in chemical production, pharmaceutical industry, food industry, biochemical, medical clinical testing and environmental monitoring. Compared with the chromatography method, many other detection methods are ultimately not as stable, reliable and eliminated as liquid chromatography after long-term repeated verification, their feasibility, accuracy, precision and efficiency. It can be seen that liquid chromatography is not only of extraordinary significance to the present, but also has considerable development prospects in the near future.

  1. Development and application of supercritical fluid chromatography

Supercritical fluid chromatography (SFC) is a chromatographic method in which a supercritical fluid is used as a mobile phase. Supercritical fluids are substances that are neither gases nor liquids. Their physical properties are between gas and liquid. Supercritical fluid chromatography is a new chromatographic technique developed in the 1980s. It has advantages not found in the gas phase and liquid phase, and it can separate and analyze some objects that cannot be solved by gas and liquid chromatography. The application is very extensive and the development is very rapid. According to industry estimates, about 25% of all separations have involved difficult-to-handle substances, and supercritical fluid chromatography can achieve satisfactory results.

Supercritical fluids have physical properties that are extremely advantageous for separation, which are just between the gas and the liquid. The diffusion coefficient and viscosity of the supercritical fluid are close to that of gas chromatography, so the mass transfer resistance of the solute is small, and rapid separation can be obtained. On the other hand, its density is similar to that of liquid chromatography, which facilitates the separation and analysis of substances with high thermal instability relative to molecular mass at lower temperatures. In addition, the physical and chemical properties of supercritical fluids, such as diffusion, viscosity, and solvent forces, are functions of density. Therefore, as long as the density of the fluid is changed, the properties of the fluid can be changed, and it is not necessary to pass the gas-liquid equilibrium curve from a similar gas to a similar liquid. Supercritical fluid chromatography is widely used in environmental protection, medicine, food and agriculture.

  1. Development and application of capillary electrochromatography

Capillary electrochromatography (CEC) is a new separation technique developed on the basis of capillary electrophoresis and microcolumn liquid chromatography. Due to technical limitations, until the 1990s, the research of capillary electrochromatography was rapidly developed.

At present, capillary electrochromatography is the leader tool in drug analysis, focusing on the separation of drug-related impurities and the separation of chiral drugs. The analysis targets mainly neutral drugs and polycyclic aromatic compounds. With the continuous improvement and improvement of capillary electrochromatography technology, it will be applied in the analysis of biotechnology, environmental protection, agricultural chemistry, fine chemical products, food industry and other fields. According to the current development trend of capillary electrochromatography technology, CEC as a developing separation technology will have wider application prospects in the foreseeable future.

Since the establishment of chromatography, whether it is the development and improvement of basic theory of chromatography, new separation mode, new stationary phase equipment, chromatography or capillary electrophoresis instruments, it has been rapidly developed in its practical application. In terms of column type, classical columns are currently commonly used. The capillary column is very suitable for trace analysis and has fast analysis speed and low sample consumption, which is one of the future development directions of the column type. It is foreseeable that future chromatographic technologies will be developed in conjunction with other technologies, to high precision, high sensitivity and miniaturization.

About  Creative Proteomics

Established in 2004, Creative Proteomics has gradually developed into an integrated company that provides proteomics, metabolomics, glycomics, and bioinformatics analysis services to researchers in the pharmaceutical, biotechnology, agriculture and nutrition industries, as well as academic and government organizations. With a continuing focus on quality, we are proud of satisfying the needs of our clients both at home and abroad covering more than 50 countries and districts.

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Chemokines are small protein families that play a central role in inflammation. During the inflammatory process, chemokines are locally produced at the site of noxious stimuli and act as central agents to recruit immune cells expressing their cognate receptors, 7 transmembrane G protein-coupled receptors (GPCRs). CCL20, an alternatively named liver and activation-regulated chemokine, is a soluble chemokine expressed by epithelial cells. Epithelial keratinocytes and synovial-lining cells are known to produce large amounts of CCL20 during homeostatic as well as inflammatory and pathological conditions such as cancer, psoriasis and rheumatoid arthritis. The homologous receptor for CCL20 is CC chemokine receptor 6; CCL20 antibody is the only chemokine known to interact with CCR6. In response to CCL20 signaling, immune cells with CCR6, such as immature dendritic cells, effector/memory T-cells, and B cells, migrate and infiltrate surrounding tissues, thereby activating the inflammatory cascade.

 

Because ccl20 cancer expression is significantly enhanced in inflammation induced by inflammatory cytokines such as interleukin-1 and tumor necrosis factor alpha, CCL20-CCR6 interaction is thought to be pathologically inflammatory. The process plays a role.

 

Rheumatoid arthritis is one of the most common autoimmune diseases. The first sign of RA is often synovitis, which appears as a swollen, painful joint. Although specific factors that initiate synovitis are not known, synovial lining epithelial cells and synovial fibroblasts are considered to be major inducers of the inflammatory response. Synovial fluid from RA patients is effective chemistries - attracting human monocytes and pro-inflammatory T helper 17 cells, which then induce and worsen the RA inflammatory process. Because reactive synoviocytes produce large amounts of CCL20, and CCR6 is the major receptor for Th17 cells, the CCL20-CCR6 interaction is thought to play during the inflammatory process. play a key role in.

 

The CCL20-CCR6 interaction can also play an important role in certain types of dermatitis. Psoriasis, for example, a harmful psoriasis event that begins in the skin is followed by infiltration of Th17 cells. Because CCR6 is expressed on the surface of Thl7 cells, B cells, dendritic cells, and tissue damage effector T cells, CCL20 can represent the major chemoattractant of these cell types in psoriasis. Further evidence for the importance of the CCL20-CCR6 interaction can be found in the study of the psoriasis mouse model induced by interleukin 23. In this model, injection of IL-23 resulted in inflammation of interleukin 22 (IL-22)-dependent psoriasis. However, Ccre+ mice did not present psoriasis-like symptoms when injected with IL-23, indicating that CCR6 requires the development of psoriasis.

 

Human keratinocytes produce large amounts of CCL20, particularly under the influence of the cytokine interleukin-17, IL-22 and TNF-α derived from Th17. While CCL20 and CCR6 are rarely detected in normal skin, both present increased expression levels in atopic dermatitis and pustular psoriasis. Strong induction of CCL20 and accumulation of CCR6+ cells can be observed in microscopic immunohistochemical analysis of human dermatitis lesions. These observations provide additional evidence for the role of CCL20 and CCR6 in the inflammatory process of dermatitis.

 

MAb biologics currently available for the treatment of immune disorders can be roughly divided into three groups: immunostimulatory cytokine inhibitors, immune cell depletion, and A subsidiary molecular blocker. These biological products may be useful for treating inflammatory diseases; however, due to the gradual decrease in the rate of non-response or response to these treatments, for patients with new mechanisms of action to cater for, for example, CCL20/CCR6-mediated disorders There is an urgent need for alternative biological products for medical needs. The antibodies of the subject invention represent the alternative biological product.

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Background and overview

Whole-genome bisulfite sequencing (WGBS) is considered as the "gold standard" for methylation sequencing. The principle is that it is treated with Bisulfite, and the C base which is not methylated in the genome is converted into U, and after PCR amplification, it becomes T, which is distinguished from the C base which has methylation modification, and is combined with high throughput sequencing technology, which can be used to determine whether a CpG/CHG/CHH site is methylated, namely to be used to map a single-base resolution genome-wide DNA methylation map.

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The application

Mapping of epigenetic features of tumor microenvironment using whole genome bisulfite sequencing:

Research Background

The development and progression of solid tumors involves dynamic interactions between tumor epithelial cells and their microenvironment. Currently, most epigenetic studies on tumors focus on epithelial tumor cells. There are few studies on the molecular characteristics of the tumor microenvironment that maintains the tumor phenotype. Tumor-associated fibroblasts (CAF) are a major group of tumor microenvironments. CAF can promote the migration and proliferation of prostate cancer cells. The epigenetic signature of CAF is unclear.

Research content

In this paper, WGBS technology was used to detect methylation of tumor-associated fibroblast CAF and non-malignant prostate fibroblast NPF, and the differential methylation region DMR between the two was screened and DMR annotation was performed. The results show that DMR enrichment in CAF occurs in the gene regulatory region, and the combined analysis with RNAseq demonstrates that these DMRs regulate the transcription of genes. In addition, the DMR gene annotation results in CAF show that DMR is enriched for tumor-associated signaling pathways. These DMRs screened in CAF can be used to diagnose prostate cancer.

Research results

  1. Non-malignant prostate fibroblast NPF was isolated from 4 cases of prostate cancer isolated from CAF and 4 adjacent tissues. Whole genome methylation sequencing (WGBS) was performed, and the average sequencing depth of each sample was > 7x. The results show that NPF and CAF have similar methylation profiles.
  2. A comparison of NPF and CAF showed that there were 7,534 DMRs (differential methylation regions) between the two groups, with CAF having more hypomethylated regions (5038).
  3. It is found that the DMR identified in CAF is enriched in the regulatory region of the gene. The main features include: most of it is outside the CpG island region; the hypomethylated DMR is more located in the promoter region and closer to the transcription initiationSite (TSS); DMR is enriched in the enhancement sub-region.
  4. Gene annotation of DMR, 30% of DMR is enriched for tumor-associated signaling pathways.
  5. To further investigate the role of DMR in the regulatory region, RNAseqwas used to detect gene expression levels. The results showed that 445 MDRs were significantly associated with 220 DEGs (differentially expressed genes), of which 162 DMR were associated with up-regulation of DEG. And this correlation can be verified by BSP and RT-PCR.
  6. Compare methylation data in prostate cancer epithelial cells and prostate cancer tissues in the TCGA database, of which 50 hypermethylated DMRs and 15 hypomethylated DMRs can be considered tumor-specific DMRs for prostate cancer Diagnosis (AUC > 0.8).

Further reading: Whole Genome Bisulfite Sequencing (WGBS) Data Analysis Pipeline and Principles and Workflow of Whole Genome Bisulfite Sequencing

Reference

  1. Pidsley R, Lawrence MG, Zotenko E, et al. Enduring epigenetic landmarks define the cancer microvironment. Genome Res 2018; 28(5): 625-638. (IF: 10.101)
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Cytokines are proteins or small molecular peptides that transmit information between cells and have immune-regulating and effecting functions, including hemopoiesis, cell growth and repair of damaged tissues. Cytokines can be divided into Il34, interferon, tumor necrosis factor super family, colony stimulating factor, chemokine, growth factor, IRF1, etc. Many cytokines play roles in the body through paracrine, autocrine or endocrine, and have multiple physiological characteristics such as pleiotropy, overlap, antagonism and synergy, forming a very complex cytokine regulatory network that participates in a variety of important physiological functions of the human body.

The realization of cytokine function mainly plays a biological role by binding to the corresponding cytokine receptors on the cell surface. The combination of cytokines and their receptors initiates complex intramolecular interactions that ultimately lead to changes in cell gene transcription. Participate in immune response and immune regulation, regulate innate and adaptive immune responses, stimulate hematopoietic function, stimulate cell activation, proliferation and differentiation, induce or inhibit cytotoxic effects, and finally induce apoptosis.

As the main cytokine, Il34 is a cellular substance produced by various cells and plays a role in white blood cells. Interleukin originally refers to the cytokines produced by white blood cells and playing a regulatory role in white blood cells, but now it refers to a group of cytokines with basically clear molecular structure and biological functions and an important regulatory role that are uniformly named. It belongs to the same category of cytokines as hemocyte growth factor. They coordinate and interact with each other to complete hematopoietic and immune regulation functions. Interleukins play an important role in the transmission of information, activation and regulation of immune cells, activation, proliferation and differentiation of T and B cells, and inflammatory response.

A substance similar to the function of cytokines is kinin release-related peptidase, which is the main rate-limiting enzyme in the kinin system. Serine protease, which is found in most tissues and body fluids, is also a kind of endopeptide enzyme, of which KLK3 is the most important component. Different from the above-mentioned cytokines, the kinin release-related peptidase specifically clews the substrate peptide at the carbon terminal, which can lyse the kinin and release the active kinin, which plays a regulatory role in the cardiovascular system and renal function. KLK is divided into two major groups: plasma KLK and tissue KLK, which are converted from prekallikin and KLK precursors, respectively. They differ greatly in molecular weight, substrate, immunological properties, gene structure, and kinin types released. Plasma KLK, also known as Fletcher factor, is a high molecular weight glycoprotein that is specifically expressed in hepatocytes.

In addition, IRS1 is an important anti-nutrient factor in soybean and some other plant feeds, which is closely related to KSR1. Trypsin inhibitors are widely found in beans, cereals, oil crops and other plants. Ras kinase inhibitors are distributed in all parts of these crops, but mainly exist in the seeds of crops. In seeds, ras kinase inhibitors are mainly distributed in protein-rich tissues or organs, located in protein bodies, vacuoles or in cell fluids. The activity of ras kinase inhibitor iron was different in different crop seeds. Ras kinase inhibitor activity is usually highest in soybean. It protects the yolk and egg embryo by preventing protein breakdown and preventing bacteria from reproducing in the egg.

Cytokine research has very important theoretical and practical significance, it helps to clarify immune regulation mechanism of the molecular level, contribute to disease prevention, diagnosis and treatment, especially the use of genetic engineering technology in the production of recombinant cytokines have been used in the treatment of tumor, infection, inflammation, hematopoietic dysfunction, etc, and received good curative effect, has very broad application prospects. Generally speaking, cytokines are a large group of substances with certain tissue distribution specificity, which usually depends on the tissue distribution or cell expression specificity of their corresponding receptors. By binding to receptors, downstream signaling pathways, such as the stat pathway, are initiated, and eventually active transcription factors are formed to enable the expression of proteins that enhance phagocytosis, promote differentiation, and regulate immunity.

Of course, in addition to IRF1, many factors derived from lymphocytes or macrophages are also involved in the expression and regulation of immune responses related to cell function. Lymphocyte cytokines are derived from lymphocytes, and the biological activities of various factors are different, such as macrophage activation and T cell reproduction. However, according to the current research level, the physical and chemical properties of the factors themselves are not clear. In addition, during the study of the immune response, many bioactive molecules were found in the supernatant of mitogen stimulated cell culture, and the researchers named them after the activity measured by themselves. Nearly 100 kinds of factors were reported over a decade. Later, by means of molecular biology, it was found that many factors named after biological activities were actually the same substance with pleiotropic effects.

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As a supplier in producing leading protein products and providing efficient protein services, Creative BioMart has been always striving great efforts to satisfy its clients’ needs and fulfil its goal to set up a standard for protein production as well as to accelerate the development of biochemical science and technologies. Creative BioMart combines innovative technologies, cost-effective products and services with simple procurement and fast-speed delivery, achieving a comprehensive laboratory solution with great values to scientists and researchers.

 

Why are you suggested to choose Protein Labeling service provided by Creative BioMart? To be subjective and specific, here are the advantages of the company’s protein labeling service that you could not ignore and could benefit your research development:

 

Custom Service Upon Your Needs

Creative BioMart provides custom protein labeling and conjugation service in accordance with personalized needs and requirements. The company claims to supply a variety of labels, protocols and conjugates on the basis of high quality, simple, time-saving yet efficient approaches, including but not limited:

· Enzyme/Antibody conjugation

· Fluorescent Labeling in Vitro

· Fluorescent Tagged Protein in Vivo

· Protein Biotinylation

· Stable Isotope Labeling

 

To take Stable Isotope Labeling as an example, Creative BioMart has contracted over 500 protein production projects with an overall success rate of 90 %. The purity of those isotopic labeled proteins is beyond 95% and the isotope enrichment c.a. is 98%.

 

Professional Analysis and Full Report of Variables Affecting Labeling and Conjugation Behaviors

 

Creative BioMart pays great importance to generate a full report along with the product delivery. It provides a thorough analysis to some critical variables affecting the labeling and conjugation behaviors.

· In vivo labeling behavior

· Immunogenicity and In vitro stability of the linker group

· Types of labeling or conjugation reaction: specific site vs. random coupling

· Labeling/Conjugate stoichiometry

· Sites of labeling/conjugation: to reduce steric hindrance and minimize negative impact on folding and activity

 

Creative BioMart also provides structural analysis of the labeled proteins. The company’s NMR laboratory provides an accurate structural analysis on the labeled proteins. Normally the analysis will be done after the appropriate labeled proteins have been produced and purified on scale.

 

Since 2005, Creative BioMart has provided thousands of high quality proteins featured in recombinant proteins, native proteins and GMP proteins to support researchers and scientists in biochemical and pharmaceutical industry. With years’ development, the company has updated a wide range of protein techniques to guarantee the product and service quality, including protein expression, protein purification, protein labeling, etc. To learn more about the company and its protein labeling service, visit https://www.creativebiomart.net/Protein-Labeling.htm or send your inquiry to contact@creative-biomart.com.

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In the long-term natural selection and evolution, the organization and properties of organism constituent materials have been continuously optimized and improved, so that simple mineral and organic materials are used to meet complex mechanical and functional requirements. The principle is to make best adaptation to the living environment. Nature is a mentor of mankind.

The superior properties of natural biomaterials can provide useful insights into the optimal design of man-made materials, especially the development of high-performance biomimetic materials. Among them, functional gradient design is one of the basic performance optimization strategies that are commonly used in biomaterials. Revealing the gradient design criteria in nature and the corresponding performance optimization mechanism are of great significance for guiding the design and promotion of high performance biomimetic gradient materials.

Recently in a study, Zhang Zhefeng, Department of Materials Fatigue and Fracture Research, Shenyang Institute of Materials Science, Chinese Academy of Sciences, collaborated with Robert O. Ritchie, professor at the University of California at Berkeley, and Marc A. Meyers, professor at the University of California, San Diego, revealed that the gradient structure orientation features are widely existed in biological tissues and materials. They also proposed a new idea of bionic design to improve the contact damage resistance of materials. The mechanical properties of gradient changes are obtained by controlling the orientation of microstructures to achieve local stiffness. The optimal distribution and matching of strength and toughness improve the overall mechanical properties, as shown in Figure 1. Through mechanical analysis and numerical simulation, they established the quantitative relationship between structural orientation and various mechanical properties, clarified the mechanism of material damage resistance improvement, and pointed out the design method of corresponding biomimetic gradient structure.

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Figure 1.

On this basis, they further summarized the basic functional gradient material design forms and principles commonly found in nature, and took typical biological materials as examples, according to composition and composition, organizational structure (including the arrangement of structural units, spatial distribution, scale and orientation), interface and different types of gradients in the multi-level structure scale combination and matching ideas to concretely describe and analyze the gradient in biomaterials, summed up the role and corresponding mechanism of gradient design in material performance optimization.

At the same time, they summarized the recent progress in the design and application of biomimetic gradient materials, especially the application of new material preparation technologies, such as 3D printing in the field of biomimetic gradient materials, and pointed out the future scientific issues and challenges that need to be addressed in natural and biomimetic gradient materials studies.

The research was funded by the Institute of Metals, the introduction of outstanding scholars, and the Multidisciplinary University Research Initiative project of the US Air Force Scientific Research Office. Related results were recently published in Acta Biomaterialia 44 (2016) 31-40 and Progress in Materials Science (doi: https://doi.org/10.1016/j.pmatsci.2017.04.013).

About  natural materials

Biomaterials can be classified into two main groups: synthetic and natural biomaterials. The latter exhibit several advantages over the former, such as biocompatibility, inherent biodegradability, remodeling and critical biological functions. Therefore, natural biomaterials are usually applied in the repair or replacement of damaged human tissues and organs. They have the ability to adequately support cell adhesion, migration, proliferation and differentiation. For example, natural polymers have been used to make natural hydrogels as extracellular matrix that mimic the biological milieu to bridge the gap between conventional cell cultures and complex native in vivo environments. Cellulose, a polysaccharide mainly found in plants can promote bone regeneration. Another example is the silk fiber produced by spiders, its biodegradability is ideal for applications like surgical suture and drug delivery.

In Matexcel, our selection of naturally biomaterials and their derivatives includes DNA and protein-based biomaterials (collagen, gelatin, fibrin, silk, elastin) and polysaccharide-based biomaterials (cellulose, chitin/chitosan, glucose, alginate, hyaluronic acid).

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A promising new approach to treating some types of cancer is to program the patient’s own T cells so that they can destroy the cancer cells. This approach, called CAR-T cell therapy, is currently available against certain types of leukemia, but so far it does not treat solid tumors such as lung or breast tumors well.

Now, in a new study, researchers from the Massachusetts Institute of Technology have developed a new approach to improve this therapy, making it a weapon against almost any type of cancer. Specifically, they developed a vaccine that significantly enhanced the anti-tumor T cell population and allowed these T cells to aggressively invade solid tumors. The results of the study were published in the July 12, 2019 issue of Science, titled “Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.”

In a study of mice, these researchers found that they were able to completely eliminate 60% of the solid tumors in mice that received CAR-T cell therapy but also received booster vaccination. In treating solid tumors, these genetically modified T cells alone have little effect.

Darrell Irvine, author of the paper and deputy director of the Koch Institute for Comprehensive Cancer Research at the Massachusetts Institute of Technology, said, “By adding a vaccine, CAR-T cell therapy, which had no effect on survival, resulted in complete remission in more than half of the mice treated with the vaccine.” In addition, the first author of the paper was Leyuan Ma, a postdoctoral fellow at the Massachusetts Institute of Technology.

Targeting tumors

So far, the FDA has approved two types of CAR-T cell therapy, and both of them are used to treat leukemia. In both therapies, T cells extracted from the patient’s blood are programmed to target proteins or antigens found on the surface of B cells. (“CAR” in CAR-T cell therapy is an abbreviation for chimeric antigen receptor.)

Scientists believe that one reason such therapies do not work well for solid tumors is that tumors often create an immunosuppressive environment that loses function before T cells reach their targets. To solve this problem, given that lymph nodes have a large population of immune cells, Irvine and his team decided to try to provide a vaccine that enters lymph nodes in order to stimulate CAR-T cells there.

“Our guess is that if these T cells can be boosted through the CAR receptor in the lymph nodes, they will receive the correct set of priming signals to make them more functional, so that they can resist shutdown and still function when they enter the tumor,” said Irvine.

To develop such a vaccine, the Irvine team used techniques they had discovered several years earlier. They found that by linking the vaccine together with fat molecules called lipid tails, they were able to deliver this vaccine more effectively into the lymph nodes. This lipid tail binds to a protein found in the blood called albumin, which allows the vaccine to hitchhike directly to the lymph nodes.

In addition to this lipid tail, this vaccine contains an antigen that stimulates CAR-T cells once they reach the lymph nodes. This antigen can be the same tumor antigen targeted by these T cells or an arbitrary molecule of choice for these researchers. For the latter case, CAR-T cells must be re-engineered so that they can be activated by both this tumor antigen and this arbitrary molecule.

When tested in mice, these researchers found that either type of vaccine significantly boosted T cell responses. When mice were given approximately 50,000 CAR-T cells but were not vaccinated, CAR-T cells were barely detectable in their bloodstream. In contrast, when the booster vaccine was given the day after the T cell infusion and again one week later, CAR-T cells proliferated until they constituted 65% of the total T cell population in these mice after two weeks of treatment.

This dramatic enhancement of the CAR-T cell population results in complete clearance of glioblastoma, mammary adenoma, and melanoma in many mice. In the absence of vaccination, CAR-T cells had no effect on tumors, whereas after vaccination, CAR-T cells cleared tumors in 60% of mice.

Long-term memory

Irvine said the technique is also expected to prevent tumor recurrence. Approximately 75 days after the initial treatment, these researchers injected these mice with the same tumor cells as the cells that formed the original tumor, and they were subsequently cleared by the immune system. Then about 50 days later, they injected slightly different tumor cells that did not express the antigen targeted by naïve CAR-T cells, and these mice were also able to eliminate these tumor cells.

This suggests that once these CAR-T cells begin to destroy the tumor, the immune system is able to detect other tumor antigens and generate a “memory” T cell population that also targets these tumor antigens.

“If we select mice that appear to be cured and rechallenge them with tumor cells, then they will completely eliminate these tumor cells. This is another exciting aspect of such a strategy. One needs to let T cells attack many different antigens to succeed, because if CAR-T cells recognize only one antigen, then the tumor only needs to let this antigen mutate to escape the immune attack. If this therapy induces new T cell priming, then this escape mechanism will become more difficult.” Irvine added.

Although most of this study was performed in mice, these researchers found that human cells coated with CAR antigens also stimulate human CAR-T cells, suggesting that this same approach may also work in human patients. This technology has been licensed to a company called Elicio Therapeutics, which is seeking to test it using CAR-T cell therapies already in development.

Irvine said, “There is really no barrier to doing this quickly in patients, because if we take CAR-T cells and equip them with an arbitrary peptide ligand, then we don’t have to change these CAR-T cells. I hope that within the next one to two years, this method can be tested on patients anyway.”

About the author

Starting from a small supplier of proteins and enzymes for academic institutes and biotech companies, Creative BioMart has always been focusing on developing high quality protein products including recombinant protein, GMP proteins, native proteins, assay kit, etc. and efficient protein service. Over the past decade, our products and services are proved to have served our customers well and our brand has become one of the most trustworthy in the market.

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Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with “two-target” functionality can interfere with multiple surface receptors or ligands associated.

1. Development history of three generations of bispecific antibodies

The advent of hybridoma technology in the 1970s reminded people whether the same antibody could be used to target two different targets, namely Bispecific Antibody (bsAb).

Most of the earliest bispecific antibodies are developed by chemical coupling methods. Although the method is much simple, the resulting products are complex and heterogeneous and difficult to use in industrial production. Until the 1980s, scFv-based recombinant bispecific antibodies appeared and gradually became the focus of research with the popularization of recombinant DNA technology.

Amgen/Micromet’s BiTE (Bispecific T cell Engager) is the archetype of the first generation of bispecific antibodies, characterized by a simple structure and two scFvs connection. But it also has obvious disadvantages such as short half-life and low expression. A pump for continuous administration is clinically necessary due to the short half-life with only two hours. Fortunately, the clinical dose of BiTE is less than one tenth of the amount of common antibodies, which virtually solves the problem of mass production. The first recombinant bispecific antibody Blinatumomab (CD3-CD19 BiTE) was finally approved by the FDA in December 2014 for marketing in the United States after various difficulties.

Genentech’s Paul Carter team invented the knob-into-holes bispecific antibody in the 1990s as the second-generation bispecific antibodies, with a structure and stability similar to that of natural IgG. Roche/Chugai’s Emicizumab (factor IX and factor X bsAb) using the knob-into-hole technology was approved by the FDA in November 2017 as the second bispecific antibody marketed in the United States.

There are currently about 120 bispecific antibodies in different clinical development stages around the world, including third generation bispecific antibodies. If taking T cell-targeted therapy as an example, Roche’s CEA-TCB (CD3-CEA bispecific antibody) is a typical representative of third-generation bispecific antibodies, which utilizes the knob-into-hole technology of second-generation bispecific antibodies, and Roche engineers invented CrossMab technology shortly after the acquisition of Genentech and successfully solved the bottleneck of knob-into-hole in common light chain. In addition, CEA-TCB achieves bivalent binding of tumor antigens and monovalent binding of CD3, enabling the bispecific antibody to produce avidity effects when combined with tumor antigens while reducing CD3 antibody binding toxicity. At present, CEA-TCB has completed Phase 1 clinical trials and achieved satisfactory results.

2. The challenges for bispecific antibodies development

Since 1986, the first antibody drug, Muromonab (OKT3), has been approved for marketing, the FDA has approved the listing of 73 antibody drugs by the end of 2017, and there are only two bispecific antibodies listed above. The development of bispecific antibodies lags significantly behind therapeutic monoclonal antibodies, which mainly results from the following reasons: Early bispecific antibody has a difficult expression and poor stability, as well as a complicated production process; The early development cost of bispecific antibodies is significantly higher than that of monoclonal antibodies; Bispecific antibody project involves target biology, structural biology, antibody engineering, and screening strategies.

In 2017, the market capacity of antibody drugs broke through the $100 billion mark for the first time. Antibody drugs have become one of the fastest growing areas in the pharmaceutical industry. Bispecific antibodies at the forefront of antibody drug development have long been the development direction of major pharmaceutical companies around the world. It is believed that bispecific antibody will have a considerable market share in the field of solid tumor treatment. Compared with CAR-T, bispecific antibody drugs have the advantages of dose controllable and flexible drug administration period. At the same time, bispecific antibody drugs can also target other immune cells, such as NK, Macrophage, etc.

Author Bio

Creative Biolabs was established in 2004 by scientists who are dedicated to conquering of cancer. Over the past 10 years, Creative Biolabs has grown into a recognized world leader in antibody (rAb) discovery, engineering, production, and analysis. Standing on the shoulder of a giant, the bispecific antibody (BsAb) team has a collective of experienced scientists committed to providing high-quality services to customers all over the world. Now, with the cutting-edge platforms and methods (quadroma development, chemical conjugation, and genetic engineering), a comprehensive list of bispecific antibody products is available to customers in academia and industry fields.

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Brief Introduction to Short Tandem Repeats

Microsatellite DNA, also known as short tandem repeats (STR) or simple repeat sequences (SRS or SSR), is widely found in prokaryotic and eukaryotic genomes, consisting of a unit of two to thirteen nucleotides repeated hundreds of times in a row on the DNA strand which is about 5% of the eukaryotic genome, the basic unit (core sequence) is 1-6bp. The most common of these is (CA) n and (TG) n, and the human genome has about 5 × 104 ~ 1 × 105 (CA) n repeats which take 10% of the genome. Each microsatellite DNA has the same core sequence structure, the number of repeating units is about 10 to 60 times, and its length is generally not more than 300bp, mostly located in the non-coding region of the gene, intron or untranslated region. which may be present in the Alu sequence or Satellite sequence, but in the coding sequence and exon also can find the presence of microsatellite DNA.

 

The high polymorphism of microsatellite DNA is mainly due to the difference in the number of tandem numbers. There is a big difference in the distribution for microsatellite DNA in different races and populations due to the number of repeat units and repetition, which constituted STR genetic polymorphism. And the number of repetitions between different individuals at a homologous STR site is also different so that STR loci analysis can identify individuals that are similar to fingerprint recognition. It is possible to create a personal gene file by identifying a specific sequence of genomes at particular loci. Currently, there are more than 10,000 STR loci are available. STR analysis has become an important analytical method for individual identification and paternity testing in the field of forensic science. It can be applied to judicial case investigation, that is, genetic fingerprint analysis.

 

The Causes of STR

The replication slip caused by mismatches between DNA strands during the mitotic process is considered to be the most common cause of the occurrence of STR, and in general, there will be an average of one-thousandths of microsatellite DNA will undergo replication slippage. The study showed that the rate of tandem duplication at repeat sequences was higher than the probability of point mutations occurring elsewhere in the genome. Most of the replication slides only cause a change in the repeat unit, and the probability of replication slip is different due to the size of the different copy units and different species.

 

STR Detection Method

STR analysis is one of the most useful methods in molecular biology which is used to compare specific loci on DNA from two or more samples. There are two common methods for STR detection: capillary electrophoresis (CE) and gel electrophoresis, which can be used to determine the specific amount of microbes Satellite sequence and draw the STR map. Typically, each allele is shared by about 5-20% of people. And the advantages of STR analysis will be reflected in the simultaneous identification of multiple STR loci. Each individual can be identified accurately by the resulting STR map. In theory, if there were 16 STR loci being used in combination, the recognition rate will be 0.999999999998.

 

The Importance of STR Analysis

It is still common for cell lines to be misidentified and cross-contamination, although scientists use a variety of traditional methods to identify cells, there are still dozens of cross-contamination happened. And even some researchers found that cell lines were misidentified or cross-contamination while cell identification reports for the higher score study articles publication, resulting in erroneous conclusions. All of which will lead to the waste of research funding and time, and resulting in a large number of invalid or erroneous data that will mislead other researchers. Based on the statistics data, around 20% of the cell lines were misidentified and cross-contamination in the labs so that it is a serious concern for researchers to provide accurate cell line identification and prevent cell lines from cross-contamination. Currently, several STR loci have been developed to analyze cross-contamination and cell types at the same time, that can detect up to 0.1 ng of DNA (about 15 Diploid genomes) with high sensitivity for the trace pollution.

All the mentioned above is the complete guide for the Short Tandem Repeat and its importance!

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Firstly, let us see two antibodies:

  1. 4G2 antibody

This product is a mouse monoclonal antibody that specifically recognizes E protein from DENV-2. The E protein-binding antibody 4G2 is an epitope-specific antibody that can be used in ELISA.

  1. A11 antibody

Recombinant Human Antibody (EDE2 A11) is capable of binding to DENV E proteins, expressed in HEK 293 cells as the combination of a heavy chain (HC) containing VH from anti-DENV E proteins mAb and CH1-3 region of human IgG1 and a light chain (LC) encoding VL from anti-DENV E proteins mAb and CL of human kappa light chain.

So, what is DENV?

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Figure1. Dengue virus

Dengue virus (DENV) belongs to a serotype subgroup of flaviviridae. Its morphological structure is similar to that of Japanese encephalitis virus (JEV), but its size is small, about 17-25 nm. It can be divided into four serotypes according to antigenicity, 1, 2, 3 and 4 serotypes. There are antigenic differences among different viral strains in the same type.

Viral characteristics and physicochemical properties

DENV has a capsule and is a single positive strand RNA virus without segments. The viral genome is about 11 KB long. It encodes three structural proteins (C-prM-E) and seven non-structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). Among them, E protein is the main structural protein of the virus, which determines the histophilicity of the virus and mediates the binding of the virus to cell receptors. At the same time, it is also an important component of influencing virulence, causing protective immune response and immunopathological damage. The virus is sensitive to heat and can be inactivated at 56 ℃ for 30 minutes. Lipid solvents such as chloroform, acetone, lipase or sodium deoxycholate can inactivate the virus by destroying its envelope. Viruses are sensitive to gastric acid, bile and protease, and to ultraviolet and gamma rays.

Antigenicity and serotype

According to the antigenicity of virus E protein, it can be divided into four serotypes: DENV1, DENV2, DENV3 and DENV4. The antigenicity of these viruses was cross-linked, but not with other antigens of flaviviridae. Antigenic determinants of viral envelope protein E can induce protective neutralizing antibodies and hemagglutination inhibitory antibodies, and may be involved in the occurrence of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). NS1 and NS3 are both immunoreactive and immunogenic, which can induce protective immunity against dengue virus homology in mice.

Clinical symptoms

Dengue virus often causes asymptomatic recessive infection. The main clinical manifestations of the patients are dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The main clinical manifestations of DF include protruding fever, severe headache (mostly frontal pain), post-ocular pain (aggravation of eyeball movement), general pain and joint pain, nausea and vomiting, lymphadenopathy, leukopenia and thrombocytopenia. DF not only causes heart, liver, lung, kidney, brain and other system damage, but also leads to myocardial damage. The clinical manifestations of DHF/DSS are more serious. In addition to the above DF symptoms, severe and persistent abdominal pain, nasal, oral and gingival bleeding, skin bleeding and ecchymosis, black stool, extreme thirst, pale skin and mucosa, chills, restlessness or sleepiness, hematocrit > 35% and other manifestations can occur. Serious cases can cause death. DENV infection can also cause encephalitis.

Pathogenesis

The pathogenesis of dengue fever and dengue haemorrhagic fever has not been fully elucidated so far. It is generally believed that the complex interaction between virus and host causes severe risk factors, including patient's age, virus serotype and genotype, and host's genetic background. Recent studies have shown that there are several hypotheses about the pathogenesis: viral affinity, viral toxicology, host susceptibility, antibody-dependent enhancement (ADE), cross-reactive T cell response and cytokine storm. This paper briefly introduces ADE and cytokine storm.

Scholars believe that there are group-specific determinants and type-specific determinants on the surface of DENV. The antibodies produced by the former have a strong enhancement effect on DENV infection, called enhanced antibodies, while the antibodies produced by the latter are called neutralizing antibodies. When the body is re-infected with different types of DENV, serum neutralizing antibodies cannot completely neutralize the virus. Enhanced antibodies can bind with the virus to form immune complexes. These immune complexes promote the replication of the virus in these cells through Fc receptors on the membrane of monocytes or macrophages, resulting in ADE effect, leading to clinical outcomes in patients. Symptoms worsen, blood concentration and shock occur, resulting in DHF/DSS. However, DHF/DSS can also occur in many patients with primary infection, indicating that there are other pathogenic factors.

Cytokines are small molecular weight soluble proteins or polypeptides secreted by immune cells and other types of cells. They have the functions of transmitting information between cells, regulating immune response, and participating in inflammatory damage and other pathological processes. However, excessive inflammation can cause systemic inflammatory response syndrome and make the body in a state of hypermetabolism and hyperdynamic circulation. Over-release of inflammatory cytokines may lead to multiple organ dysfunction syndrome and even death. The control of inflammation is essential in the treatment of many diseases. It was found that the levels of IL-1, IL-4, IL-6, IL-13 and TNF-alpha were significantly increased in severe patients with dengue fever. There is a significant correlation between TNF-alpha and thrombocytopenia. Studies have shown that the transition from Th1-type response to Th2-type response is an important mechanism of DHF/DSS. Cytokines in severe patients are mostly Th2-type cytokines. After virus infection, excessive release of cytokines and inflammatory factors increases vascular permeability, leading to hemorrhage and shock.

Diagnosis

The diagnosis of dengue virus is mainly a combination of clinical symptoms and laboratory diagnosis. Laboratory diagnosis of dengue virus includes virus isolation, antigen and antibody detection, gene detection and so on.

  1. Virus isolation

Virus isolation is the golden standard for the diagnosis of viral diseases. The duration of dengue viremia is relatively short. Samples should be collected within 4 to 5 days of the onset of fever. Virus isolation and culture can be carried out by living mosquitoes, cells or animals.

  1. Antigen detection

NS1 protein is the main marker antigen of dengue virus infection. NS1 protein appears earlier than IgM antibody and can be used for early diagnosis. At present, several commercial kits have been used to detect the NS1 antigen of dengue virus.

  1. Serological detection

The serological method is more convenient because the humoral immune response of patients infected with dengue virus lasts for at least several weeks after the onset of the disease and the sample can be collected for a relatively long time. Detection of IgM, IgG capture ELISA, IgM, IgG indirect ELISA and hemagglutination inhibition test are the most commonly used serological methods for diagnosis of dengue virus infection.

  1. Gene diagnosis

In recent years, the molecular biology diagnostic methods of dengue virus have been enhanced. Nucleic acid amplification has become the most important method for rapid diagnosis of dengue virus. Various RT-PCR methods try to detect the RNA of dengue virus. The nucleic acid amplification technique can be used to identify the serotype of dengue virus as well as to detect the virus.

Owing to its high throughput and parallel processing, gene chip technology can detect and analyze pathogens comprehensively. Its simple and fast operation, low cost and small size provide wide application prospects in the fields of molecular biology, disease diagnosis and treatment. Scientists used PCR to amplify the nearly full-length dengue virus 2 gene, digested the amplified products by enzyme-digestion-PCR, and constructed the dengue virus 2 DNA library to obtain microarray probes. The probe was fabricated into a DNA chip for detection of type 2 dengue virus by dot analyzer. The results showed that the hybridization of the sample with type 2 dengue virus gene chip was highly sensitive and specific.

Dengue fever, as an important vector-borne infectious disease, has been increasing year by year, seriously endangering human health. Although some achievements have been made in the related research, there are still many problems to be solved urgently. There is still no consensus on the pathogenesis of dengue fever, especially in severe cases. There is no effective prevention and treatment measures. Future work should start with the mechanism of virus and host, obtain effective treatment of dengue fever as soon as possible, and todevelop a protective multivalent vaccine.

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August 16, 2019, as a leading supplier and manufacturer in antibody field, Creative Biolabs recently has updated its new product types for therapeutic antibody, which has at least three types of mechanisms. For instance, the neutralizing effect of the therapeutic antibody on its ligand directly binds its ligand, so that its ligand does not act on the target tissue and cells to trigger an undesired reaction; antibody-mediated cytotoxicity of the antibody binds to the target cell, Cytotoxicity that triggers immune cells; complement-mediated cytotoxicity antibodies have a complement-binding site that, upon binding of the antibody to the target cell, triggers the complement effect and kills the target cell.

 

These new items of Creative Biolabs are including but not limited to:

Anti-CD34 Therapeutic Antibody

Anti-CD55 Therapeutic Antibody

Anti-CD81 Therapeutic Antibody

...

 

those newly-updated products in therapeutic antibodies will bring a lot to our customers in their research programs. Our experienced scientists can guarantee the high quality of them and they can perform well during all kind of research items to assist researchers to achieve success. The anti-34 therapeutic antibody is a kind of recombinant humanized monoclonal antibody expressed in CHO binding to human CD34. It can recognize the human cd34 antibody membrane-protein in the cell membrane and perform well in the clinical treatment of hematologic malignancies and solid tumors, etc. The anti-CD55 therapeutic antibody is binding to human CD55. cd55 antibody is built with strong potential to be applied as an additive agent to treat gastric cancers. Studies show that a single 20mg dose can induce the cell killing in human primary gastric tumors, which results to tumour cell regression in over half of PAT-SC1-treated patients. And we do believe patients with gastric cancers can be totally free in the nearly future. The target of anti cd81 antibody is human CD81, which is generated by genetic immunization. It is very promising in the virus-host interactions. All in all, newly-updated products will bring Creative Biolabs another shine. Dr. Carlose Smith explained, a senior scientist in R&D of Creative Biolabs.

 

with the faith of providing high-quality products and professional services, Creative Biolabs is always trying its best to develop products and services needed by our customers. And we do offer the custom services and products for some specified items. Of course, Creative Biolabs has gained a good fame among our customers and other biological companies.Dr. Carlose added.

 

We do believe that Creative Biolabs will be much more better in the near future due to its dutiful spirit of enterprise. Now, it has been well received among customers.

 

About Creative Biolabs

With more than ten-year corporate history, Creative Biolabs has accommodated many excellent researchers at home and abroad to develop high-quality and professional products and services belonging to the antibody field, which is now getting more and more popular and needed by clinical researches. The future for Creative Biolabs is getting more and more promising due to its professional scientists and research staff.

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Relapsing fever is transmitted via arthropod vectors. The human body louse (Pediculus humanus corporis) transmits Borrelia recurrentis, the causative organism of louse-borne relapsing fever (LBRF), while Ornithodoros ticks are vectors for the at least 15 different species of Borrelia that cause endemic or tick-borne relapsing fever (TBRF).    

While humans are the only known host for the LBRF spirochete, the TBRF-causing Borrelia species (with the exception of B. duttonii) have their reservoirs in small rodents and have been found in several locations worldwide. In North America, three species of Borrelia cause TBRF, and several outbreaks of the disease have been described. In East Africa, B. duttonii is the principal cause of TBRF.

Antibody responses against relapsing fever Borrelia species are primarily directed against outer surface lipoproteins. Two major protein groups have been identified, namely, variable small proteins (approximately 22 kDa) and variable large proteins (approximately 38 kDa). These proteins have been studied most thoroughly for B. hermsii and B. turicatae. It is probable that all of the relapsing fever Borrelia species share the same antigenic variation scheme as that described in detail for these two species.

When spirochetes are present in blood, they must evade the immune defense systems. Before the acquired immune responses lead to the production of antibodies, the alternative pathway of complement operates as a major innate immune defense system against the invading organisms. In the presence of antibodies, complement acts as an effector system, mainly via the classical pathway (CP). Both pathways lead to coating of the target surface with C3b. Together with their cleavage products, such as iC3b, the C3b molecules opsonize the target for phagocytosis. Further activation can lead to the formation of lytic membrane attack complexes. To avoid overconsumption of the components of the complement cascade and to protect self-cells from harmful attacks, complement activation must be tightly regulated. This is mediated by regulatory proteins in plasma and on cell surfaces.

The major fluid-phase regulators of complement are factor H (FH), for antibody-independent alternative pathway activation, and C4b-binding protein (C4BP), for antibody-dependent CP activation. These regulators accelerate decay of the C3 convertases (C3bBb and C4b2a, respectively) and act as cofactors for the irreversible inactivation of C3b and C4b, respectively. As a net effect, these functions prevent complement-mediated destruction of the target in both the absence and presence of antibodies. Acquisition of the host plasma complement regulator FH has been shown to be beneficial for complement evasion among other spirochetes, such as Borrelia burgdorferi sensu stricto and B. afzelii, which express at least two FH binding proteins. Also, the relapsing fever agents B. hermsii and B. parkeriihave been shown to bind FH, while no binding has been observed for B. turicataeB. hermsiiexpresses a unique 20kDa outer surface protein (FhbA) responsible for FH binding.

The bacteria that cause human Lyme disease belong to a group of at least 15 species, referred to as Borrelia burgdorferi sensu lato, or the Lyme disease agent bacterial group. Among these, B.burgdorferi sensu stricto causes Lyme disease in North America, while in Europe and eastern Asia Borrelia afzelii, Borrelia garinii, and Borrelia bavariensis sp. nov. are the best-known causes. However, more recently, Borrelia bissettii, Borrelia lusitaniae, Borrelia spielmanii, and Borrelia valaisiana have been isolated from Lyme disease patients. Other species in this bacterial group, such as Borrelia japonica and Borrelia sincia in Asia, have not been associated with human disease. To date, genome sequences have been reported for 14 B. burgdorferi isolates, two B. afzelii isolates, two B. garinii isolates, one B. bavariensis sp. nov. isolate and 1 isolate of unassigned species.

The complete genome sequences for three additional Borrelia species: B. valaisiana isolate VS116, B. bissettii isolate DN127 clone 9, and B. spielmanii isolate A14S. DNA samples from low-passage isolates were sequenced to minimize plasmid loss, and genomes are sequenced to about 8-fold coverage as previously described. Genome annotation is performed using the JCVI Prokaryotic Annotation Pipeline . The DN127 chromosome and 35 of 39 plasmid sequence contigs are closed, but in order to maximize the use of available funds, the sequences of a few replicons are not closed and some gaps remained in these sequences (two chromosomes and one cp9 and three cp32 plasmids, because they are much less variable than the other plasmids).

 

These three genome sequences include 3,914,891 bp in total (1,258,865, 1,403,466, and 1,252,560 bp for strains VS116, DN127, and A14S, respectively), with an average of 1,304,497 bp/genome. Like the sequences of other Borrelia species, they include numerous linear plasmids (6, 7, and 7, respectively) and circular plasmids (2, 2, and 2, respectively). Plasmid numbers in these three strains range from 11 in VS116 and 12 in A14S to 16 in DN127. Plasmids that are very similar to B. burgdorferi sensu stricto cp26, cp32 (7 in DN127, versus 3 in the other two strains analyzed), and lp54 plasmids are present in each of these isolates, and DN127 also contains an unusual fusion of four partial cp32 plasmids. Plasmids with predicted lp17 compatibility are also present in all three genomes, making it the only other plasmid type, in addition to cp26 and lp54, to be found in all 23 B. burgdorferi sensu lato sequenced genomes. However, the gene contents of the lp17s are much more variable than the other universally present plasmids.

The detailed analyses of these genome sequences will be a major step forward in attaining a complete understanding of B. burgdorferi sensu lato diversity. They will contribute to the development of species- and group-specific vaccines and diagnostic tools, as well as inform us whether these species are in genetic contact with the more-common Lyme disease-associated agents.

Reference

[1] Barbour A G, Hayes S F. Biology of Borrelia species. [J]. Microbiological Reviews, 1986, 50(4):381-400.

[2] Van H C, Comberbach M, De G D, et al. Evaluation of the safety, reactogenicity and immunogenicity of three recombinant outer surface protein (OspA) lyme vaccines in healthy adults [J]. Vaccine, 1996, 14(17–18):1620-1626.

[3] Kraiczy P. Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii. [J]. Journal of Bacteriology, 2012, 194(2):545-6.

[4] Cutler S J, Moss J, Fukunaga M, et al. Borrelia recurrentis characterization and comparison with relapsing-fever, Lyme-associated, and other Borrelia spp.[J]. International Journal of Systematic Bacteriology, 1997, 47(4):958-68.

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Antibody drugs are one of the fastest growing biopharmaceuticals, which bring huge benefits to the innovative drug market, with anti-tumor antibody drugs predominating. Since rituximab, the first antibody drug for cancer treatment, was approved by the US FDA in 1979, 17 antibody drugs have been approved for cancer treatment, but the gemtuzumab has withdrawn from the market. According to statistics from 2013, there are about 350 antibody drugs currently in clinical research, most of which are in the early evaluation stage. Therapeutic antibodies in Phase III clinical studies include 28 monoclonal antibodies and a mixture of monoclonal antibodies mainly used for cancer, inflammation or immune diseases, Alzheimer's disease and infectious diseases, 10 of which are cancer therapeutic antibodies.There are also many antibody drugs in preclinical studies, and their research trends are mainly focused on the following aspects, including: the discovery and confirmation of new antibody targets, new strategies to reduce antibody immunogenicity, conjugates of antibodies and drugs, and research on bispecific antibodies as well as other novel antibody drugs.

 

  1. 1.Discover new antibody target molecules

At present, the target molecules of antibody drugs are mainly divided into three categories: 1. Clinically validated target molecules, the efficacy of which has been confirmed, for example: CD20, HER2, EpCAM and CTLA-4; 2. Experimentally verified target molecules, the efficacy of this type of target molecule has been confirmed in cells and animals, such as IGF-1R, CD19, CD80, CXCR4 and ICAM-1; 3. New functional target molecules, newly discovered target molecules with certain efficacy, such as RAAG12, CD151, TSN-1, etc. To date, only a few target molecules for anti-tumor antibody drugs have been approved for marketing, and currently only nine. Many tumor-associated biomarker molecules have been reported, and thus so many target molecules for antibody therapy can be excavated. Even if the same target molecule has been developed, different molecular epitopes exist, and antibody drugs against different epitopes can be developed. For example, 5. Anti-CD20 antibody drugs have been marketed, and the 5 are now in clinical research.

In addition, many tumor cell transmembrane receptors and extracellular matrix-related genes can be spliced by differential splicing, which can generate new editing sequences and form new differential receptors and substrates as new antibody target molecules. The differentially spliced gene variants have been reported to be: FGFR variants, EGFR variants, EpCAM variants, L1CAM variants, Claudin18 and CD44 variants, and splicing of versican VN, fibronectin F and prion protein T Variants, etc. In recent years, many tumor-associated antibody target molecules have been studied, including EpCAM, PSMA and folate-binding proteins, as well as gangliosides, αVβ3, TRAILR2, FAP and toughlin. Recently, glypican proteoglycan has also been reported as a new target molecule for the treatment of liver cancer antibodies.

  1. Reduce antibody immunogenicity

Many antibody drugs are murine antibodies, which produce human anti-mouse antibody (HAMA) responses in clinical treatment. Therefore, for long-term treatment, an antibody drug that requires repeated administration is likely to produce a HAMA reaction, which requires humanization to reduce its immunogenicity. Humanized antibodies include chimeric antibodies, modified antibodies, and the like, which have biological properties such as reduced HAMA response and prolonged half-life in blood. In contrast to murine antibodies, the functional effector portion of the humanized antibody drug can be engineered as needed, and the humanized constant region of the antibody prevents the production of anti-isotype antibodies during treatment. The modified antibody further reduces the proportion of the murine fraction in the antibody compared to the chimeric antibody, and significantly reduces the HAMA response. In addition, the study of fully human antibodies has also received great attention and is one of the important development directions of antibody drug research. Fully human antibodies can be obtained by phage display technology, antibody library screening technology and transgenic animal technology. The anti-tumor antibody drugs produced by this technology have obvious inhibitory effects on cancer growth.

The immunogenicity of antibody drugs is one of its major toxic side effects, but there is no reliable prediction method for the frequency and timing of immunogenicity and antibody aggregation. If these problems are solved, new antibody detection techniques need to be developed. The results of studying the immunogenicity of human-derived antibodies, humanized antibodies and murine antibodies in humans indicate that: 1. More than 20% of mouse-derived antibodies induce tolerable immunogenicity, or negligible. These mouse antibodies do not need to be humanized; 2. Humanized antibodies can reduce immunogenicity, but their clinical therapeutic effects are also significantly reduced; 3. The immunogenicity of antibodies from different sources is not completely consistent with the law. A small number of humanized antibodies and fully human antibodies also produce significant immune responses; 4. Not only do we need to evaluate the utility of fully human and humanized antibodies item by item, but we also need to consider cost-effectiveness, while considering biochemical characteristics and targeted treatment indications. To date, there is no good way to completely eliminate the risk of antibodies binding to host cells. But classical research suggests that monomeric immunoglobulins are inherently tolerable if we are able to create methods to prevent antibody aggregation or immune complex formation.

To be continued in Section Two…

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Antibody Engineering comprises in vitro selection and modification of human antibodies, including humanization of mouse antibodies for therapy, diagnosis, and research. It holds the potential for creating antibodies with multiple specificities, greater affinity for their targets, and fewer side effects. In this article, the development history and types of antibody engineering drugs are talked.

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  1. Antibodies as therapeutic agents

An antibody is an immunoglobulin molecule with a specific amino acid sequence induced by an antigen, synthesized and secreted by lymphocytes (plasma cells). An antibody can bind to an antigen such as a bacterium, a virus or a toxin, and neutralize and remove the harmful substance by the following three or one of the following methods: a. directly inactivates the antigen; b. the immune effector cells phagocytose and destroy it; c. it is weakened by the surface of the antigen and thus be easily damaged by complement. The study of antibodies as therapeutic agents has been going on for a century. The lateral chain theory proposed by Ehrlich laid the foundation for immunology and immunotherapy. He believes that the surface of the cell has specific receptor molecules (or side chains) that bind only to specific groups in the toxin molecule; if the cells survive with the toxin, they will produce excess side chains. Some of the side chains are released into the blood, which is an anti-toxin. This is what is now called an antibody. Ehrlich uses organic arsenic compounds to treat syphilis and introduces the term "magic bullet" in the field of chemotherapy to strengthen the strategies and goals of specific therapies. As early as 1895, it was reported that Hericourt and Richet injected cancer cells into animals to produce antiserum for the treatment of cancer patients, and the symptoms of the patients were significantly improved. In the early 20th century, many researchers repeated the above tests, but failed to obtain a definite therapeutic effect, and sometimes even produced serious side effects. Later studies have found that antisera contain a large number of different kinds of antibodies, which are directed against different antigens on the surface of cancer cells, and these antigens are also present in normal tissue cells. Therefore, the cross-reactivity of antibodies with normal tissues may lead to serious consequences. Antiserum has long been effective in neutralizing exogenous toxins such as snake venom, but has not been effective in the treatment of tumors and other diseases.

  1. Monoclonal antibody

In 1975, Köhler and Milstein used B lymphocyte hybridoma technology to prepare monoclonal antibodies (MAb). Monoclonal antibodies have high specificity, uniform properties and are easy to mass produce. A variety of monoclonal antibodies can be prepared in vitro by cell engineering, which is an epoch-making advance in antibody production. It is especially important that monoclonal antibodies have high specificity for the corresponding antigens and homogeneity of the antibody molecules, which can greatly reduce cross-reactivity with normal tissues in vivo, and bring new hope for the treatment of diseases by using antibodies, especially for treating tumors. At that time, some people called it "magic bullet", and it is expected that monoclonal antibodies can specifically attack pathogenic cells or pathogens without causing toxic side effects. In the past 20 years, monoclonal antibodies have been widely used in the diagnosis of diseases, and make much breakthroughs have despite many obstacles. To distinguish, an antibody previously prepared by immunizing an animal with an antiserum pathway is referred to as a polyclonal antibody, or a conventional antibody.

  1. Genetically engineered antibodies

Since the monoclonal antibodies prepared by the B lymphocyte hybridoma technique are mostly murine, the human anti-mouse antibody (HAMA) reaction can be induced in the human body, which limits the application of the monoclonal antibody as a therapeutic agent in humans. In order to overcome the heterologous reaction of murine monoclonal antibodies, in the mid-1980s, researchers explored the genetic engineering of mouse-derived monoclonal antibodies to prepare humanized antibodies. For example, they spliced the variable region of mouse Ig to the constant region of human Ig or modified the CDR region of the murine Ig to the human Ig. At the same time, considering the treatment of solid tumors with intact antibodies, the molecules are too large to penetrate the extracellular space into the deep tumor, so genetic engineering methods are used to miniaturize antibody molecules, such as single-chain antibodies (scFv), diabody, minibody, etc. The establishment and development of antibody library technology can directly clone the gene of specific antibody in the prokaryotic cell system by genetic engineering method, which greatly promotes the preparation and research and development of novel genetic engineering antibodies. Antibody engineering has become a key technology to promote the development of antibody drugs.

  1. Immunoconjugates and fusion proteins

The molecular structure and function of the antibody has two sides. One is the specific binding to the antigen and is completed by the variable region. The second is the effect function triggered by binding to the antigen, which is completed by the constant region. In order to enhance the effector function of antibody drugs, especially tumor antibody drugs, and to enhance their killing effect on tumor cells, an effector molecule or a "warhead" substance is commonly used to chemically connect with an antibody to prepare an immunoconjugate. The specificity of the antibody is utilized as a targeting vector; the "target" is used to kill tumor target cells. There are three main types of substances used as "warheads", namely radionuclides, chemotherapeutics and toxins. These "warhead" substances are linked to antibodies, which constitute radioimmunoconjugates, chemical immunoconjugates and immunotoxins, respectively. A fusion protein can be produced by a genetic engineering method, and the antibody portion of the fusion protein is generally a single-chain antibody scFv; the portion corresponding to the "warhead" is generally a toxin fragment, a cytokine or a peptide drug.

The monoclonal antibodies, antibody fragments, genetically engineered antibodies, and immunoconjugates described above can be collectively referred to as antibody-based drugs, also known as monoclonal antibody therapeutics. Such antibody drugs are prepared by a cell engineering method or a genetic engineering method or a combination of two methods, and may also be referred to as an antibody engineering drug.

  1. Antibody drugs for clinical application

The development of antibody drugs has a prominent position in the field of biotechnology drugs. The number of antibody drugs currently in preclinical and clinical research stages is among the highest in biotechnology drugs. The variety of antibody drugs in research and development is versatile, and the diseases that may be used for treatment include cancer, viruses and other infectious diseases, cardiovascular diseases and immune system diseases, among which cancer treatment is the first.

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  1. Bridging cells (in-transbinding)—bridging cell method

The most commonly used bridging cells are bridging effector T cells and tumor cells, also known as T cell-engaging bsAb (bsTCE). In addition, NK cells, stem cells, and tumor cells etc. are also used. bsTCE directly couples T cells and tumor cells to form immune synapses, activates in TCR, releases granzymes and perforin, and finally lyses tumor cells.

BsTCEs bind to CD3ε in the CD3ε-TCR complex and activate T cells without antigen presentation. Although this mechanism was proven to be highly effective in the laboratory by co-culture experiments, it caused rapid uncontrolled cytokine release syndrome (CRS), including the earliest marketed CD3×EpCAM bsTCE catumaxomab, via the Fc segment binding to FCγR expressing Kupffer cells in the liver and causes uncontrolled hepatotoxicity. Targeting bsTCEs by CD3 requires complete inhibition of Fc-mediated effector function to reduce non-target toxicity and maximize therapeutic effects.

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  1. Tumor target—Target expression and efficacy

The activity of some bsTCE is positively correlated with the expression level of tumor targets, such as CEA, CD33, HER2. There are also some no significant correlations with expression levels, such as EPHA2, PSMA. However, there is also a minimum required tumor target expression, such as RG6106 requires at least 50 bsTCE binds to the myeloma surface FCRL5, while Roche cibisatamab requires 10,000 CEA in combination with bsTCE to be activated.

  1. Structural design

Bivalent tumor targets can increase drug binding activity and enhance drug efficacy. Some tetravalent bsTCEs are under development, including Adaptir and TandAb, which have two CD3 and two TAA binding units. In addition, trivalent bsTCEs are under development, in which CD3 is monovalent and TAA is bivalent. For example, RG6026 (also known as RO7082859; Roche).

  1. Immune checkpoint

Clinically administration of monoclonal antibody against CTLA-4, PD1/PD-L1 immunological checkpoints have been quite successful. More than 10 corresponding bispecific antibodies have entered the early clinical stage worldwide, involving PD1/PD-L, CTLA4, lymphocyte activation Gene 3 (LAG3) or T cell immunoglobulin mucin protein 3 (TIM3; also known as HAVCR2)

Previously, two checkpoint antibodies were combined to improve efficacy. For example, treatment of melanoma patients with ipilimumab (anti-CTLA4) + nivolumab (anti-PD1) improves survival compared with ipilimumab alone. However, clinical immune-related side effects have also multiplied.

In order to improve the safety of the combined targeting of PD-1 and CTLA 4, an Fc-silencing bsAb, a high affinity inhibition of PD-1, and a low affinity binding inhibition of CTLA 4 were designed to inhibit the PD-1 pathway. This design is beneficial for inhibiting PD-1-CTLA 4 double positive tumor infiltrating lymphocytes, and at the same time reducing the binding of CTLA 4 expressing peripheral T cells, thereby providing better safety.

Currently, the safety and early efficacy of the four PD-1×CTLA4 bsAbs are being evaluated in early clinical trials. The concept of blocking two immunological checkpoint inhibitors is also clinically used for other target combinations, such as PD-1 x LAG 3, PD-1 x TIM3, and PD-L1 x CTLA 4.

  1. Bridging receptors (in-cisbinding)—bridge acceptor inhibitory activity

Targeting inhibition of tumor receptor tyrosine kinases (RTKs) such as EGFR and HER 2 is a successful anticancer approach, but the development of drug resistance is one of the major limiting factors for such treatment. Drug resistance typically involves up-regulation of other RTKs that bypass specific receptor inhibition to activate parallel signaling pathways. For example, in the tyrosine protein kinase MET pathway, non-small cell lung cancer (NSCLC) tumors are resistant to EGFR tyrosine kinase inhibitors.

JNJ-61186372 (Janssen Pharmaceuticals) is an EGFR x MET bsA that blocks EGFR and MET signaling by inhibiting ligand-induced activation and receptor degradation by cFAE. JNJ-61186372 has antibody-dependent cytotoxic activity (ADCC) which is produced by Fc containing low fucose. The glycotypic analysis in the quality control of such bispecific antibodies should be noted.

  1. Bridging receptors (in-cisbinding)—bridge acceptor agonist receptor activity

In contrast to blocking pathogenic signals with inhibitory antibodies, some therapeutic concepts require receptor signals to be activated by agonistic antibodies. Specific bsAbs are also particularly suitable for activating multi-component receptor complexes because they require simultaneous binding of the receptor and the co-receptor to activate. Activation of the fibroblast growth factor 21 (FGF 21) pathway has been reported to improve obesity and diabetes. However, the pharmacokinetic properties of recombinant FGF21 are poor, and chronic treatment has a risk of adverse reactions. Thus, the agonist bFKB8488A (Roche) was designed to activate this metabolic pathway by selectively targeting the fibroblast growth factor receptor 1C (FGFR1C)-β-Klotho (KLB) receptor complex. KLB is selectively expressed in liver, fat, and pancreatic tissues, while FGFR1C is widely distributed in liver, fat, and pancreatic tissues. Thus, co-targeting of these receptors may limit signal activation to only tissues that co-express KLB and FGFR1C and limit the adverse consequences of extensive FGFR activation, such as induction of cell proliferation. Preliminary results from an ongoing human first trial showed an improvement in cardiac metabolism in obese patients with insulin resistance.

  1. Cofactor mimetics

Sampi and his colleagues began producing a bsAb to replace FVIII as a potential treatment for hemophilia A to prevent bleeding from FVIII dysfunction. The role of bsAb is to mimic the activated form of FVIII, FVIIIa, which binds FX and FIXa to enhance the catalytic activity of FIXa. In a wide range of screening efforts, a dual-characteristic antibody called AE 910 or emicizumab was finally selected and finally approved by the FDA for routine prevention to reduce bleeding in patients with hemophilia A using FVIII inhibitors. Further approval in October 2018 also included the use of emicizumab for prevention in patients without FVIII inhibitors. Emicizumab was first approved in Europe in March 2018.

  1. Piggyback approaches

The first target-specific transport of a second specific target using the bsAb is referred to as a "piggyback transport" or "hijacking" method for accessing a restricted cell chamber. Raso and colleagues described the first case of the use of hijacking concept-specific transport proteins, which linked bispecific receptors to ricin A, mediating toxin internalization and toxicity. A recent example is the hijacking of the transferrin transport pathway to cross the blood-brain barrier and enter the immune-immunized brain region. The researchers targeted the transferrin receptor (Tfr) by using a binding arm of the bsAb, and the second binding arm carries β-secretase 1 (BACE 1), which transports BACE1 into the brain, and TfR×BACE 1 bsAb can reduce amyloid (Aβ, Aβ) peptide levels in brain tissue and cerebrospinal fluid.

Psl×pcrvbsAb MEDI3902 is a full-length IgG 1 antibody inserted between Fabs and fc to form a symmetrical 2+2 format. This antibody enhances P. aeruginosa killing effect by neutrophils using a similar piggyback mechanism.

Reference

  1. Oberst, .mAbs 6, 1571–1584 (2014). Laszlo, GS et al. Blood 123, 554–561 (2014). Lopez-Albaitero, A. et al. . Oncoimmunology 6, e1267891 (2017)
  2. Wolchok, J. D. et al. N.Engl. J. Med. 377, 1345–1356 (2017). Dovedi, S. J. et al. Cancer Res. 78,2776(2018).
  3. Hedvat, M. et al.Presented at the 2018 Society for ImmunotherapyofCancer (SITC) (2018).
  4. Xu, J. et al. . Am. J. Physiol. Endocrinol. Metab. 297,E1105–E1114(2009).Wu, A. L. et al.Sci. Transl Med. 3, 113ra126
  5. Xu, J. et al. . Am. J. Physiol. Endocrinol. Metab. 297, E1105–E1114 (2009). Wu, A. L. et al. Sci. Transl Med. 3, 113ra126
  6. Sampei, Z. et al. PLOS ONE 8, e57479 (2013)
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Anti-drug conjugate (ADC) is an antibody that binds to cytotoxic drugs and targets cytotoxic drugs to tumors through the targeting of antibodies, thereby reducing the non-specific systemic toxicity of drugs commonly found in chemotherapy. The study of antibody-drug conjugate (ADC) can date back to 1980s.

A successful antibody drug conjugate drug includes four main parts: a suitable target (tumor antigen), a highly specific antibody, an ideal linker, and a highly cytotoxic drug.

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Firgure. The model of ADC action

  1. The basis for target selection

Nowadays, ADC drugs are mainly used for anti-tumor. When selecting a target, the ideal target antigen should be overexpressed on the surface of tumor cells, but no expression or very low expression in normal tissues. Moreover, when the antibody and the target are aggregated in the ADC drug, it can be effectively internalized, and the drug is released into the cell to kill the target cell.

  1. Antibody specificity, affinity, and pharmacokinetics

The high affinity of the antibody and the target antigen is the core of the effective targeting of the ADC. It is generally believed that the affinity index KD 10 nM is a basic requirement for the antibody. On the basis of this, antibodies that are low in immunogenicity, long in half-life, and stable in blood are screened.

  1. Selection and intracellular drug release

The ideal linker can maintain stability in the blood and effectively release the drug in the target cells. The commonly used Linkers can be divided into two categories: cleavable linkers and non-cleavable linkers. The current study found that seven B cell receptors (CD19, CD20, CD21, CD22, CD79b, and CD180) have effective effects by cleavable linkers. In contrast, when non-cleavable linkers are used, only CD22 and CD79b antigens can bind to the antibody, effectively transport ADC to lysosomes, and release the drug to kill target cells. Therefore, when choosing which linker to be uses, the nature of the target should be considered.

  1. Selection of cytotoxic drugs

Since the antibody enters the body and can effectively enter the tumor site by about 0.003–0.08% of the total amount, it is necessary to have a highly effective and highly sensitive killing effect on the target cells (free drug IC50: 10-1 1–10-9M). ). There are two main types of commonly used drugs at present - microtubule inhibitors and DNA-damaging agents.

ADC development trend

  1. Site-specific conjugation

At present, the most advanced ADC drugs are using traditional no-specific conjugation. The biggest disadvantage is that the product obtained is a mixture of different drug molecules per antibody. Site-specific conjugated drugs, and more importantly, uniform data (eg, PK) for clinical evaluation is difficult to obtain. In oder to solve these shortcomings, site specific conjugation technology has become a hot spot for major companies. Using site specific conjugation techniques, the same number of drug molecules can be carried on each antibody to obtain a uniform ADC drug. It is conducive to pharmacodynamic research and evaluation. And in the clinical can get more stable and effective results. Among them, A mbrx's Unatural Amino acid (pAcPhe) technology has more applications and promotion prospects.

  1. Multivalent ADC drugs

The development of antibody drugs and vaccines has progressed from monovalent drugs to multivalent drugs. ADC might also follow this development process, that is, to link several small molecules that are synergistic with each other in the same antibody to improve the drug's efficacy. This requires a more sophisticated conjugation technology, to integrate two or more technologies. But now, the site-specific technology, excessively pursuits the coupling of a specific molecules at a specific site and neglects the diversity of coupling.

Practical and traditional techniques for multivalent coupling of drugs require simultaneous coupling of multiple drugs on one antibody. In this case, the singularity of the antibody itself to modify the linking group will result in a mixed product, and there is no guarantee that each antibody carries a different drug at the same time.

This problem can be solved by Site-specific techniques. When performing Site-specific modification, a variety of different coupling groups can be designed, which can use a group to carry out drug couples for the linker with the corresponding group.

Monoclonal antibodies and their conjugates are macromolecular substances. Large drug molecules are difficult to penetrate deep into the solid tumor through the capillary endothelium and through the extracellular space of the tumor. The use of antibody fragments, such as Fab, to prepare conjugates with lower molecular weight, may increase the permeability to the extracellular space and increase the amount of drug reaching deep tumor cells. "Small size or moderate miniaturization is an important way to develop ADC drugs."

Reference

  1. Beck, Alain, et al. "Strategies and challenges for the next generation of antibody–drug conjugates." Nature reviews Drug discovery 16.5 (2017): 315.
  2. Okeley NM, et al. (2010) Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res 16:888–897.
  3. Wu AM, Senter PD (2005) Arming antibodies: Prospects and challenges for immunoconjugates. Nat Biotechnol 23:1137–1146.
  4. Nagayama, Aiko, et al. "Antibody–Drug Conjugates for the Treatment of Solid Tumors: Clinical Experience and Latest Developments." Targeted oncology 12.6 (2017): 719-739.
  5. Polakis P (2005) Arming antibodies for cancer therapy. Curr Opin Pharmacol 5:382–387.
  6. Dan, Nirnoy, et al. "Antibody-drug conjugates for cancer therapy: chemistry to clinical implications." Pharmaceuticals 11.2 (2018): 32.
  7. Lambert JM (2005) Drug-conjugated monoclonal antibodies for the treatment of cancer. Curr Opin Pharmacol 5:543–549.
  8. Sau, Samaresh, et al. "Advances in antibody–drug conjugates: a new era of targeted cancer therapy." Drug discovery today (2017).
  9. Vater CA, Goldmacher VS (2010) Antibody-cytotoxic compound conjugates for oncology. Macromolecular Anticancer Therapeutics, Part 4:331–369.
  10. Zhou, Qun. "Site-specific antibody conjugation for ADC and beyond." Biomedicines 5.4 (2017): 64.
  11. Kovtun YV, Goldmacher VS (2007) Cell killing by antibody-drug conjugates. Cancer Lett 255:232–240.
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What is metabolomics?

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We know that metabolism is the general term for various chemical reactions in living organisms. It is one of the most important life activities of animals and plants. Individuals adapt to changes in internal and external environment through various metabolic adjustments, which is one of the basic characteristics of life activities. A "metabolome" is a collection of all metabolites in a biological sample. There are many kinds of metabolites (may be more than one million species) with various structures, and there are large differences in distribution and concentration in different tissues and body fluids. The main types include lipids, amino acids, organic acids, carbohydrates, nucleic acids, and so on.

Metabolomics is an independent discipline, systematically studying metabolomes that provides unique biochemical fingerprints of all cellular processes. It can be used to identify metabolic biomarkers elucidating underlying disease mechanisms or predicting environmental changes or external interventions reaction.

What is lipidomics?

Lipolipomics is an independent discipline that systematically studies lipid groups. As a method for large-scale qualitative and quantitative study of lipid compounds and understanding their functions and changes under different physiological and pathological conditions, it can accurately and comprehensively provide a full-fat information spectrum of biological samples under different physiological conditions.

As can be seen from the above definition, "lipolipomics" is actually a branch of "metabolomics." However, because lipid metabolism is the most major category of animal and plant metabolism (about 70% of metabolites in plasma is lipid), it is the hot spot of animal and plant metabolism research. Lipid metabolism is involved in energy transportation and intercellular information communication and network regulation in the process of growth and development. As a major component of cell membranes and lipid droplets, various structural lipids play an important role in a wide range of biological processes, such as signal transduction, transport, and biomacromolecule sorting with different biochemical properties. With the rapid development of lipidomics in recent years, scientists have gradually divided the lipid group research from mebabolism to “lipidomics”. Currently, when we refer to “metabolomics”, the lipid group analysis is no longer included.

Clinical application of lipidomics studies

Comprehensive analysis of lipids, i.e. lipidomics is a prerequisite for understanding the subtle dynamic changes and pathogenesis of lipids during cell changes. Lipid metabolism disorders and reproductive development defects are closely related to a variety of major diseases such as diabetes, cardiovascular disease, fatty liver, obesity, cancer, and Alzheimer's disease. The changes of lipid composition can directly reflect the physiological and pathological state of the organism.

Therefore, it is of great significance to analyze the changes of lipid spectrometry during the growth and development of tissues and organs and to analyze the mechanism of related metabolic regulation network.

With an integrated set of separation, characterization, identification and quantification system, featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective targeted lipidomics, untargeted lipidomics, Exosome Lipidomics and MALDI Imaging Lipidomics Service.

Advantages of Creative Proteomics Services

  • The compounds we test are widely covered, ranging from small water-soluble molecules to large lipids.
  • We can analyze any biological materials, including but not limited to biofluids and tissues from animals, cell cultures and humans.
  • A comprehensive platform contains advanced instruments, including MS, GC-MS, LC-MS, NMR, and so on.
  • A complete analysis report is offered, including method interpretation, data, and result files.
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Lipids are the general term for fats, cholesterol, phospholipids, lipoproteins and glycolipids. It is one of the important nutrients needed by the human body. It provides the energy needed by the body and the essential fatty acids needed by the body. Lipid is one of the three nutrients that can produce energy, together with proteins and carbohydrates, and plays an important role in providing energy for the human body. It is the constituent of human cell tissue, among which phospholipids and cholesterol are the main components of all biofilms. The human body needs to absorb a certain amount of lipid substances every day, but excessive intake can lead to the occurrence and development of hyperlipidemia, atherosclerosis and other diseases. It is a compound insoluble in water but soluble in organic solvents, including fats and lipids. Fat accounts for about 15% of total human body weight, with a minimum of 13% and a maximum of 50%. Animal oils provide mainly saturated fatty acids while vegetable oils mainly unsaturated fatty acids.

The main functions of lipids are: 1. Main components of cell membrane and biofilm, fixing body tissues and organs, and fat is the isolation layer of organs and joints, filling and avoiding friction. 2. Supply and store energy. Fat intake should be 20% to 25% of total energy. One gram of fat provides 37.6 kilojoules of heat. You also need 70 to 80 grams of fat per day. 3. Promote digestion and absorption of fat-soluble vitamins.4. Maintain body temperature. Here we will introduce some important lipids below:

1. Phospholipids

Phospholipids are lipids containing phosphoric acid, which are composed of glycerophospholipids and sphingolipids. Glycerophospholipids are formed by condensation of phosphoric acid and glycerol, while sphingolipids are formed by condensation of sphingosine-1-phosphate and phosphoric acid. Phospholipids mainly act as emulsifiers, meaning that a molecule of phospholipids has both a hydrophilic part bound to water and a hydrophobic part bound to oil. So, it's an intermediate medium of water and oil that doesn't blend together, and makes it work very well. Most of the phospholipids in foods are in the form of soft phospholipids. As a plant extract, phospholipids are often found in egg yolks, animal livers and legumes. Egg yolk, soybean, fish head, sesame, mushroom, yam and black fungus also contain a certain number of phospholipids.

2. Sphingolipids

Sphingolipids consist of a molecule of long-chain fatty acid, a molecule of sphingosine or its derivatives and a molecule of polar head alcohol. Sphingosine is the parent compound of many long chain amino alcohols in sphingolipids and is abundant in mammals. The polar head group of sphingolipids binds to the hydroxy group of sphingosines, while the fatty acid part forms amide bonds with the amino group. Therefore, sphingolipids, with a polar head group and two non-polar tails (long hydrocarbon chains of fatty acids and sphingolipids), are polar lipids, the second largest group of membrane lipids after phospholipids. Sphingolipids can be divided into sphingolipids, sphingolipids and gangliosides.

3. Steroids

Steroids are a broad class of cyclopentane derivatives of total hydrogenation phenanthrene widely distributed in the biological world. Steroid compounds contain no bound fatty acids and are no saponifiable lipids; These compounds are isoprene like substances, which are generated by triterpene cyclization and chemical modification. Steroids include sterols (such as cholesterol, lanosterol, glusterol, soy sterol, ergosterol), bile acids and bile alcohols, steroid hormones (such as adrenocorticosteroids, androgen, estrogen), insect ecdysis hormones, cardiac glycosides, saponin ligands and bufotoxin. In addition, there are synthetic steroids such as anti-inflammatory agents (hydro prednisone, dexamethasone), steroids that promote protein synthesis and oral contraceptives. Anabolic steroids are similar to synthetic male hormones. They are a kind of synthetic chemical derivatives similar to human testosterone in structure and activity. On October 27, 2017, the world health organization's international agency for research on cancer released a preliminary list of carcinogens for reference, androgen (anabolic) steroids in the 2A list of carcinogens.

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