Complement System and Targeted Therapy (II)

Homologous restriction of complement regulation

1. When both target cells and complement are from the same genus, the complement cytolytic effect is suppressed.

2. Regulatory proteins that cause homology restriction are called homologous restriction factors (HRF): CD46 (MCP), CD55 (DAF), CD59, CR1, etc.

Biological function of complement

(1) Bacteriolytic and cytolytic effects

After the complement system is activated through the classical pathway, the bypass pathway or the MBL pathway, it can form a membrane attack complex on the target cells, resulting in the lysis of the target cells;

This function of complement plays an important defense and immune surveillance role in the body’s immune system and can resist infection by pathogenic microorganisms;

When some patients have congenital or acquired complement defects, the most important manifestation is that they are susceptible to pathogenic microorganisms and recurrent infections.

(2) Conditioning effect

The opsonization effect is also called phagocytosis, and both complement and antibody have opsonization effects. There are a variety of complement receptors on the surface of phagocytic cells, such as CR1, CR2, CR3 and other complement fragments (C3b/C4b) that bind to target cells or antigens can specifically bind to complement receptors on the surface of phagocytic cells and promote contact between the two. Enhance phagocytosis and intracellular oxidation, and ultimately enhance the body’s ability to resist infection.

(3) Immune adhesion and removal of immune complexes

After bacteria or immune complexes activate complement and bind C3b/C4b, if they bind to RBC and platelets with corresponding complement receptors (CR1) on the surface, a larger polymer can be formed, which reaches the liver and spleen through blood circulation.

(4) Promote the effect of neutralizing and dissolving virus

Adding complement to the complex formed by the virus and the corresponding antibody can significantly enhance the neutralization effect of the antibody on the virus;

In the absence of antibodies, complement can also dissolve and inactivate viruses.

(5) Inflammation

C3a, C4a, C5a, with allergic toxins, can degranulate mast cells and basophils with corresponding receptors on the surface, release vasoactive substances such as histamine to cause vasodilation, enhanced permeability, smooth muscle contraction and Branch spasm, etc.;

C3a and C5a have a chemotactic effect on neutrophils, attracting neutrophils and monocyte phagocytic cells with corresponding receptors to migrate and aggregate to the inflammatory area activated by complement, enhancing the inflammatory response;

C2a has kinin-like effects: dilates small blood vessels, enhances permeability, and causes inflammatory hyperemia and edema.

(6) Immunomodulation

The active fragments produced during the activation of complement can interact with immune cells and regulate immune function. For example, C3d, Ba, Bb and other fragments can regulate the function of B cells; C5a can promote the production of various cytokines such as IL-1, IL-6, IL-8, and TNFa.

Complement system and diseases

1. Complement defects

Apart from the relatively common defects of C2 and C1 INH, defects of other complement components are very rare. The two major clinical manifestations of patients with congenital defects of complement are repeated infections and autoimmune diseases, which also confirms the importance of complement in anti-infection immunity and immune regulation from the negative side.

2. Complement and inflammatory diseases

The fragments produced by complement during activation have some new biological activities. Among them, C5a, C3a, and C4a have allergic toxin effects, and C5a has chemotactic activity. These fragments play an important role in promoting inflammatory reactions, and thus have some inflammation in related diseases, complement plays an important pathological role; Including autoimmune diseases, cardiovascular diseases, inflammatory tissue damage during infection, hyperacute transplant rejection, etc.;

By inhibiting complement, it is possible to suffer the effect of treating diseases.

3. Complement and virus infection

The relationship between complement receptors and complement membrane surface regulatory proteins and viral infections has received more and more attention,some viruses can infect host cells through complement receptors, etc.;

EB virus infects B cells through CR2, measles virus infects body cells through MCP, Coxsackie virus, Echovirus and enterovirus can infect cells through DAF, etc.;

In clinical practice, consider the use of complement receptor blockers to treat certain viral infections of a certain line.

  1. Complement and tumor

The body’s immunity to tumors includes cellular immunity and humoral immunity. In humoral immunity, antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are used as important anti-tumor mechanisms in humoral immunity;

Complement regulatory protein (CRP) plays a very important role in regulating complement attack on tumor cells and in evading immune attacks from tumors;

It is highly expressed complement regulatory proteins (CD46 (MCP), CD55 (DAF), CD59) on the surface of tumors such as gastric cancer, intestinal cancer, lung cancer, renal cancer, endometrial cancer, neurofibroma and breast cancer. Inhibits the cytolysis of complement on tumor cells, allowing tumors to escape immune attacks.

Research progress of complement-related drugs

l SLE (lupus) (systemic lupus erythematosus)

l Rheumatoid arthritis

l Multiple Sclerosis

l Renal disease/nephritis

l Transplant rejection (organ transplantation)

l Cardiopulmonary bypass syndrome

l Myocardial infarction

l Septic shock

l Ischemia and reperfusion injury

l Stroke

The Principle of Single Cell Sequencing Technology and Its Development in the Biology Field (III)

3.2 Application of sc RNA-seq in the field of ophthalmology

The retina has a certain neuron diversity, which can be used to study neuronal differentiation and nervous system diversity. The retina is composed of various types of cells, such as ganglion cells, microglia cells, photoreceptor cells, etc. Due to its complex composition structure, conventional gene sequencing can no longer accurately reflect the specificity between retinal cells, but sc RNA-seq can overcome this difficulty and accurately reflect the gene expression of individual retinal cells. Transfected bipolar cells and Müller glial cells with GSP, collected GFP-positive cells by FACS, and applied large-scale Drop-seq to classify about 25,000 mouse retinal bipolar cells to obtain 15 bipolar cell subtypes. The matched molecular expression and cell morphology verified the accuracy of this classification method, and at the same time the method identified a variety of new genetic markers. The identification of genetic markers of individual retinal cell subtypes is conducive to the study of targets related to specific visual functions, enabling people to gain a deeper understanding of the source of cell function and cell heterogeneity.

Degeneration of the retina, such as age-related macular degeneration (AMD), is the result of damage to photoreceptor cells, which is also the main reason for the deterioration of vision in people in developed countries. The study found that the expression of transcription factors related to apoptosis is higher in patients with dry AMD, the expression of transcription factors related to angiogenesis is higher in patients with wet AMD, and all types of AMD have a cell-mediated immune response. Therefore, AMD may be a single disease with a common immune response.

 

3.3 Application of sc RNA-seq in the field of immunology research

The heterogeneity of the immune system helps to effectively defend against many different pathogens. In recent years, sc RNA-seq has been applied to the research of immune system. The response of immune cells to antigenic substances is characterized by complexity and heterogeneity. Lipopolysaccharide can activate Toll-like receptors in dendritic cells to trigger changes in the transcriptome. A new Th2 cell subpopulation can be identified by using sc RNA-seq to reveal the wide heterogeneity within the T helper 2 (Th2) cell population and marked with Cyp11a1 that regulates the steroid synthesis pathway. The technique of RNA transcription at the 5´-end can be used to construct a cDNA library and sequencing analysis of mouse bone marrow dendritic cells (BMDCs). The results showed that at the single cell level, dendritic cells response is highly heterogeneous. In some dendritic cells, many known genes that regulate the inflammatory response are in a fully activated state, while in other cells, they are only activated at a low level or not activated. The maturation state of protruding cells is related to the randomness of gene regulatory networks.

Although the application of SCS in the research of the immune system is relatively limited, SCS has shown strong potential in the immune cell subsets, detecting gene expression variability, differential splicing, and gene regulatory networks.

 

3.4 Application of sc RNA-seq in the field of animal breeding research

In juvenile in vitro Embryo Transfer (JIVET), scRNA-seq was used to screen out key genes that affect the maturation of lamb oocytes, and combined with real-time quantitative PCR technology to quantitatively verify the genes, namely, to study the regulation mechanism of oocyte maturation at the molecular level to improve the latent oocyte embryonic potential. And this experiment successfully found that MOS, RPS6KA1, CPEB1, ANAPC13 and CDK1 5 genes are extremely important for the maturation of lamb oocytes. JIVET uses oocytes on the ovary of young females as the source of eggs to cultivate embryos in vitro. The efficiency provides abundant genetic resources for livestock breeding.

 

Some studies have found that by analyzing and comparing transcriptomes, the ligand-receptor interaction signaling pathway for neural activity in the hypothalamus-pituitary-ovarian gonadal axis regulation of ovulation and follicle development plays an important role in regulating and screening out the production The related genes of lamb number enrich and supplement the information of sheep genome.

 

The RNA-seq technology was used to perform transcriptomics sequencing and data comparative analysis of yak GV and MII oocytes, and 4 767 differentially expressed genes were screened out. After analyzing the differential genes through KEGG pathway interaction, it was found that metabolic pathways play a key role in regulating gene up- and down- pathways during the maturation of oocytes,. RNA-seq technology was used to perform transcriptome sequencing and comparative analysis on the ovaries in the estrus of yak and central plain yellow cattle, and found that the cell adhesion process is one of the important conditions for distinguishing the physiological activities of the ovary of yellow cattle and yak. Moreover, there is a close relationship between the complement and coagulation cascade pathways and the reproduction and ovarian development of yak, which may be related to the special natural environment of the yak.

 

Although single cell separation is difficult, many single cell separation techniques can be applied to the separation and extraction of different types of cells to the greatest extent. Obtain single cells in an ideal state; sc RNA-seq can obtain in-depth analysis of heterogeneity between different single cells, gene expression networks, etc. by obtaining the full transcriptome expression profile from the single cell level; High-throughput, high-efficiency and low-cost sequencing technology can directly sequence RNA and discover new transcripts. Direct sequencing of RNA makes the results more accurate. Based on this comprehensive advantage, the application field of sc RNA-seq has gradually expanded from the research of early embryonic development of mammals to the treatment of human diseases in immunology, ophthalmology, tumors, etc., and achieved good results.

 

References

[1] Moignard V, Macaulay I C, Swiers G, et al. Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis[J]. Nat Cell Biol, 2013, 15 (4) :363-372.

[2] Tang F, Barbacioru C, Bao S, et al. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis[J]. Cell Stem Cell, 2010, 6 (5) :468-478.

[3] Streets A M, Zhang X, Cao C, et al. Microfluidic single-cell whole-transcriptome sequencing[J]. Proc Natl Acad Sci USA2014, 111 (19) :7048-7053

[4] Wu A R, Kawahara T L, Rapicavoli N A, et al. High through put automated chromatin immuneprecipit at ion a s a platform for drug screening and antibody validation[J]. Lab Chip, 2012, 12 (12) :2190-2198.

[5] Rota L M, Lazzarino D A, Ziegler A N, et al. Determining mam-mosphere-forming potential: application of the limiting dilution analysis[J]. J Mammary Gland Biol, 2012, 17 (2) :119-123.

 

Introduction to TALEN

What is TALEN?

Transcription activator like effector nuclease (TALEN) is a powerful tool for genome editing. The chimeric nuclease consists of two parts: coding sequence-specific DNA binding modules and non-specific DNA cleavage domains. By inducing DNA double-strand breaks to stimulate error-prone non-homologous end connections or homologous directed repair at specific gene locations.

 

TAL effector (TALE) was originally discovered as an invasive strategy for bacterial infection of plants in a plant pathogen called Xanthomonas. These TALEs are injected into plant cells through the bacterial type III secretion system, and regulate transcription by targeting effector-specific gene promoters to promote bacterial colony formation. Due to the sequence-specific binding ability of TALE, researchers have formed a powerful class of tools with specific genome editing function, namely TALEN, by linking FokI nuclease with a segment of artificial TALE.

 

TALEN Typical Structure

A typical TALEN consists of an N-terminal domain containing a nuclear localization signal, NLS, a central domain containing a typical tandem TALE repeat sequence that recognizes a specific DNA sequence, and a C-terminal domain with FokI endonuclease function.Specific DNA_sequence lengths recognized by different types of TALEN elements differ greatly.In general, the length of specific DNA sequence recognized by natural TALEN elements is generally 17-18 bp, while that recognized by artificial TALEN elements is generally 14-20 bp.

 

TALEN Technical Principle

 

Figure 1. TALEN technical

The principle of TALEN technology is to target and combine TALEN elements to specific DNA sites by the DNA recognition module, then complete the shearing of specific sites under the action of FokI nuclease, and complete the insertion (or inversion), deletion and gene fusion of specific sequences by means of the inherent homologous directed repair (HDR) or non-homologous end joining pathway (NHEJ) repair process in cells. The core principle of TALEN is to achieve three different functions: directing into the nucleus, specific recognition of target site DNA, and cleavage of target site DNA in an orderly manner. The key to TALEN is to complete the specific recognition function of DNA, which is generally divided into two steps.

 

(1) Construct TAL target recognition module

The DNA-specific recognition unit of TAL is a double amino acid spaced by 32 constant amino acid residues. Binary amino acids correspond to the four nucleotide bases of AGCT: adenine (A) is recognized by NI, thymine (T) by NG, guanine (G) by N, and cytosine (C) by HD. In the experimental operation, we can deduce the duplex amino acid sequence that can specifically recognize this sequence through the DNA sequence of the target site, thus constructing the TAL target recognition module.

 

(2) Target recognition module recognition and expression of TAL

We need to connect a pair of TAL target recognition modules constructed according to the target DNA sequence in the previous step with the nuclear localization sequence at the N-terminal and FokI enzyme at the C-terminal to obtain a complete TALEN element. For one thing, we can adopt the eukaryotic expression vector system specially used to construct TALEN, clone a pair of specific TAL target recognition modules into the vector, and then introduce them into cells by transfection and other ways.

 

Application of TALEN

Since 2011, TALE and TALEN technologies have been rapidly applied in many species, of which zebrafish is the most prominent model animal. The application of TALEN technology has also been extended to more species and has been rapidly developed and tested. Some researchers used this technology to successfully obtain mutants expressing surface antigen H-2K (k) and hygromycin resistance protein, inserting or deleting mutants respectively, and then using magnetic separation and hygromycin treatment for cell separation, respectively. The results showed that the target cells were successfully isolated from cells greatly enriched with gene mutations, indicating that this technology can effectively enrich mutants. Therefore, they recommended that TALEN technology be more widely used in biomedical research, thereby greatly facilitating the efficiency of nucleic acid recombination. Researchers have also used this technology to genomically modify HEK293T cells and to mutate genes in model animals such as human induced pluripotent stem cells (iPS), Drosophila melanogaster, zebrafish, and Xenopus laevis, and it only takes 1 week to directly complete the evaluation in mammalian cells. Compared with the traditional yeast-based evaluation method, this method is more efficient, and the application of TALENS technology in various model organisms will provide a more convenient and effective method. In addition, TALEN was used to successfully knock out the proto-oncogenes of E6 and E7 proteins in human HPV-positive cell lines, thereby promoting cell apoptosis, inhibiting cell growth, reducing the possibility of tumorigenesis, and also enabling functional repair of p53 and RB genes. Thus, the TALEN-mediated genome editing of human cells will become a pillar technology in human biology and disease research. This new technology of gene editing can remove genes efficiently and quickly, and bring about the effect of efficient directional modification for the construction of model animals, gene therapy and modification, and cultivation of new varieties.

 

Expectation

TALEN has undoubtedly made breakthroughs in the field of gene editing since its application. In terms of precise gene modification, the application of TALEN makes it possible to study some complex gene functions, and TALEN-mediated gene editing is more effective and fast than traditional gene targeting schemes. At present, TALEN technology has been successful in many species, and its high efficiency has been recognized by the majority of scientific researchers. The only disadvantage of TALEN compared with ZFN is that its expression vector is larger, which may reduce the efficiency of plasmid transfection to some extent. Therefore, improving the introduction of TALEN is an important factor to improve the efficiency of TALEN cleavage.

 

Development History of Transgenic Technology

Transgenic technology refers to the use of DNA recombination, transformation and other technologies to transfer specific foreign target genes into recipient organisms, and make them produce predictable and targeted genetic changes. Transgenic technology is a core component of modern agricultural biotechnology.

A series of genetic research work has opened a new era for humans to transform organisms. This kind of technical system that uses modern biotechnological means to achieve the desired goals according to engineering design principles is called “genetic engineering” or “genetic engineering”. Plant genetic engineering is often referred to as “transgenic technology”, and the products obtained are called transgenic plants or transgenic crops, and sometimes names such as “genetically modified organisms” or “engineered crops”

The development history

In 1953, Watson JD and Crick FHC first proposed the double helix structure model of DNA and the semi-reserved replication hypothesis.

In 1966, the American scientist Nirenberg (MW) and others deciphered all the genetic code, proclaiming the birth of molecular biology. With the successive discovery of tool enzymes such as DNA restriction enzymes and DNA ligases, it provides a convenient tool for in vitro genetic manipulation.

In 1972, American scientists Boyer HW and Berg P successfully completed the work of splicing together two pieces of DNA from different sources, marking the birth of DNA recombination technology.

In 1974, Morrow JF et al. took the lead in expressing eukaryotic genes in E. coli.

In 1978, human brain hormone and human insulin genes were expressed in E. coli.

In 1983, scientists completed the genetic modification of plants (tobacco) for the first time.

The technical principles

Transgenic technology is the use of modern biological technology to artificially separate and recombine the desired gene into the genome of the organism, thereby improving the original traits of the organism or giving it new excellent traits.

In addition to the transfer of new foreign genes, the genetic characteristics of organisms can also be modified, such as processing, knocking out, and masking of organism genes through transgenic technology, to obtain the desired traits. The main processes of this technology include cloning of foreign genes, construction of expression vectors, establishment of genetic transformation systems, screening of genetic transformants, analysis of genetic stability, and backcrossing.

The technical classification

  1.  Plant transgenic technology

Plant transgene technology adopts cloning and other methods to insert foreign DNA into recipient cells. Typical methods of use include vector-mediated method and direct DNA uptake method. At present, the most widely used and the most ideal effect is the Agrobacterium-mediated method. This method uses Agrobacterium rhizogenes as a carrier to implant Agrobacterium in plant cells to achieve the purpose of cell transformation.

  1.  Animal transgenic technology

Microinjection method is to use a glass needle to inject DNA into the animal embryo cell nucleus, and then transplant the DNA into the animal body to make it develop normally. Somatic cell nuclear transfer method is to first cultivate cells in vitro, select high-quality genes, and then transplant them to egg cells, and then transplanted into the mother.

Technical advantages and disadvantages

Advantage

1. Food quality is improved: genetically modified products have a certain degree of stress resistance, and some biological properties are strengthened, which improves the taste quality and nutritional value of foods, and some insect-resistant plants not only reduce the use of pesticides, but also It can ensure that the food surface is non-toxic and pollution-free, and will not cause pesticide accumulation in the human body. There have been no cases of diseases caused by the consumption of genetically modified products in the world. The approved genetically modified rice in China has fully met the food safety standards in the safety evaluation for more than ten years, and the food safety evaluation indicators of some genetically modified crops are even high In accordance with international standards.

2. Significant achievements in environmental protection: the GM planted in China has insect resistance, drought resistance, and salinity resistance, which reduces the use of pesticides, and the negative impact of planting GM rice may be far less than planting non-GM crop. In the future, the advantages of “biological agriculture” will far exceed that of “chemical agriculture”. This trend is inevitable and irreversible.

3. Has inestimable social benefits: In the social economy, genetically modified technology can increase material productivity and become a new driving force for economic growth and quality improvement. At the same time, GM technology also has certain advantages in the field of medicine. The health industry dominated by GM technology will gradually become a pillar industry in the world economy. In terms of social culture, genetically modified culture is also an important part of advanced social culture. It also reflects to a certain extent the economic strength of a country and the international status of a country.

Disadvantages

1. Food safety issues: While genetically modified products bring huge economic and social benefits to people, the threat to human health cannot be ignored. First, changes in crop genes may cause undesirable effects. Newly introduced proteins may have toxicity or allergic problems; antibiotic marker genes in genetically modified crops may cause antibiotic treatment to fail. Taken together, genetically modified products are still unknown and uncertain.

2. The impact of genetically modified products on the ecological environment: the genes of genetically modified organisms will flow to the natural biological community, for example, the genes for the resistance to herbicides may escape to the weeds, making the weeds super resistant to weeds. The difficulty of removing this weed, and the mutual transfer of genes between these organisms, may affect the fair competition between species, destroy the original ecological balance, and thus turn some of the original dominant species into disadvantages or even extinction. .

3. Impact on biodiversity: Genetically modified organisms bring convenience to humans while destroying the laws of natural genetic evolution. While inhibiting the normal growth of harmful organisms, they may also affect or exterminate beneficial organisms; second, the genes of genetically modified organisms may also promote the evolution of insects, viruses, and bacteria. Studies have shown that cotton bollworm has developed resistance to transgenic insect-resistant cotton.

ABOUT US

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

Our plant breeding and culture services support increasingly stringent safety and quality standards in the agricultural industry. Relying on our revolutionary techniques, various molecular breeding services and molecular diagnostic methods are offered for a wider range of agriculture-related sectors. Meanwhile, extensive analytical solutions reach out to a wider community of researchers in environmental and energy fields. Holding a strong tie between biological innovation and transformation in the ecosystem, Lifeasible now leverages the expertise and strengths of each to create its unique platform that is accessible to all leaders working in agriculture, botany, biology, ecology and environmental science. Having taken on step forward, we also provide lab-to-field solutions that advance the breeding process for farmers and breeders. Diverse analytical tools are available for breeding or quality control purposes in identification and production of plants. Here are some our products: plant hormone productsaspergillus vitricolaamino acids cell culturebyproducts of passive solar energy, etc.

A Brief Look of Biosynthesis of Isoprenoid

Isoprene like compounds are widely distributed in nature. The lower terpenoids are mainly found in higher plants, algae, mosses and lichens, and also in insects and microorganisms. However, steroids (Fig. 1) mainly exist in animal kingdom, plant kingdom and microorganism. At present, there are more than 23000 isoprene like compounds, and they are increasing at the rate of dozens every week.

Isoprenoids (ISOs) compounds, also known as terpenoids (Terpenoids) compounds, as organic compounds are more or less present in various types of animals and plants on the earth, in the form of huge and diverse natural lipids, It has become the world’s largest natural product extracted from animals and plants, widely used in cosmetics, flavoring, coloring, food additives, antibacterial, anti-tumor and other pharmaceutical applications. Among them, aromatic lipids extracted from plants are the most popular, and the world market size in 2013 reached US$1 billion. However, in general, the ISOs compounds have slower growth and lower yields. The more difficult the collection, the richer the content of animals and plants, which seriously restricts the sustainable development of related industries.

Isoprenoid derivatives of plants have different functions. In addition to some common physiological, metabolic and structural functions, many special terpenoid complexes have communication and defense functions.

Isoprene like is a kind of natural compound widely existing in nature, which has important economic value. At present, great progress has been made in the research of isoprene like synthesis at the level of molecular biology. More and more scientists have successfully cloned enzyme genes and transferred these genes into other organisms.

Isoprenoid biosynthesis

Wallach put forward the isopentene rule as early as 1887, and speculated that the carbon framework of terpenes was formed by polymerization of isoprene. This hypothesis was verified until lynen experiment proved the existence of isopentene pyrophosphate (IPP). In 1956, folkers also proved that the key precursor of iPP synthesis was mevalonate (MVA).

In isoprene like biosynthesis pathway , IPP is an important precursor in this pathway. It is synthesized by MVA pathway in cytoplasm and glyceraldehyde-3-phosphate pathway in plastid. A series of C10 (GPP), C15 (FPP), C20 (GGPP) and more high molecular weight c5n (PPP) compounds were formed by increasing IPP one by one, starting from dmapp, a carbon 5 (C5) molecule isomerized with IPP. With GPP, FPP and GGPP as the parent materials, various isoprene like compounds with different functions were formed under the catalysis of various enzymes.

The study of isoprene like compounds is gradually showing the details of the biosynthesis pathway and regulation of this complex compound. In this paper, the main enzymes and their regulation in isoprene like biosynthesis pathway are mainly introduced.

The transgenic work of isoprene like biosynthesis can improve the color and taste of food, enhance the commercial value of industrial products, and also enable biological defense against diseases and pests. The benefits of this work, like the form and function of isoprene like biosynthesis, seem unlimited. Therefore, The study of enzymes and regulation in isoprene – like biosynthesis is of great significance to make full use of nature.

Detection Methods of 2019 Novel Coronavirus (2019-nCoV) (II)

The nucleic acid extraction is followed by an amplification step, which is achieved by several tubes of reagents provided by the manufacturer. The detection process is called RT-PCR, which is reverse transcription-polymerase chain reaction. This is an operation that uses the nucleic acid replication mechanism that exists in nature to achieve the in vitro replication of DNA molecules. It has been invented for nearly 40 years. It is a basic technology for molecular diagnosis and an important foundational technology for modern biology and medical progress.

In addition, the selection of the probe sequence, that is, the primer sequence, is very particular. In the sequence of about 30,000 bases in length, it takes a lot of effort to detect segment.

A group of Korean scientists recently published a comparison result, which made a relatively objective comparison of the effectiveness of the detection sites of the nucleic acid tests. The sites identified by scientists from various countries for diagnosis are different.

Can nucleic acid testing be improved? First look at the speed, a standard nucleic acid detection process, sampling is fast, and then store it in the transport medium, when the sample is stored to a certain amount, concentrated inactivation, 56 degrees, 30 minutes, and then nucleic acid extraction. The nucleic acid extraction is about 30-90 minutes, and the PCR reaction is 40-60 minutes. This can be done with a batch of samples. After they come out, you can quickly determine the results. The whole experiment is about half a day. Can this process be accelerated? If it is a single sample, do it directly after sample collection, you can skip many steps, such as putting it directly in the lysate, and then doing rapid nucleic acid extraction, or even doing rapid PCR without extraction, which may take about 30 minutes. It can be seen that PCR detection itself can be done quickly, especially when the sample volume is small, but it is better to operate batch by batch when the samples are more efficient.

In addition to NEAR, there are many isothermal amplification reactions that can be used for nucleic acid detection. For example, a relatively well-known LAMP loop mediates isothermal amplification, which was invented by Japanese scientist. This method also uses four or six primer sequences to add to the substance to be tested.

There are other methods, one of which is RPA, which is relatively easy. It uses a combination of two enzymes, recombinase and polymerase, put together, through a very complex cycle, to achieve the amplification of nucleic acid molecules, although complex but very efficient and very fast. Based on RPA, , a Chinese scientist in Boston, United States, combined it with gene editing and grafted it to achieve RNA detection. They named it SHERLOCK.

Comparing these two methods, they can achieve 30 to 50 minutes of detection, which are relatively fast. These two methods can read the results in the form of test strips, and are easily understood by everyone, without the need for complicated instruments.

In addition to shortening the time, for large-scale testing, it is very important to increase the throughput of the test. The kit can be used to carry out multiple tests, but the samples must be tested one by one. There is also the work of nucleic acid extraction, which is particularly physically intensive and requires experience, skills, and training. It is best to leave it to a machine that is tireless and not easy to make mistakes. Therefore, we see a large number of automated nucleic acid extraction instruments appearing on the testing site. Such instruments are found in designated hospitals and disease control centers at all levels.

As the epidemic progresses, more and more testing needs emerge. As home isolation becomes the norm, can self-testing and self-sampling be feasible?

Less than a month ago, scientists at Yale University in the United States published a paper comparing the effects of PCR nucleic acid detection on saliva and nasopharyngeal swabs. Experimental results show that, compared with nasopharyngeal swabs, the stability of saliva sampling is quite good, even better. What’s more interesting is that the results seen on most patients have more viruses detected in saliva. The results may be more accurate and reduce the occurrence of false negatives. This is a good starting point, so a few days ago, Rutgers University in New Jersey and their affiliated testing agencies obtained approval from the U.S. Food and Drug Administration to start using samples collected from saliva for the detection of new coronavirus. They used an automated machine with large-scale pipeline processing, which can test 10,000 samples per day. Saliva sampling detection is a very encouraging new method, and if it is promoted, it is expected to well solve the problems we faced in the past sampling.

Antibody detection

First of all, the samples are very different, they are all tested with blood or serum. Antibody detection methods are diverse; the most common are enzyme-linked immunosorbent assay and immunochemiluminescence. The principle of these two methods is very similar, and it is also the most common detection method in laboratory research. Taking the principle of enzyme-linked immunosorbent detection as an example, we first lay a layer of artificially prepared antigen on the detection substrate, and then add the serum sample. After incubation for a period of time, it specifically binds to the antibody; then we elute it, leaving specifically bound antibody; at this time, add enzyme-labeled secondary antibody, the secondary antibody can recognize immune antibody, and it also has catalytic enzyme on it; then incubate for a period of time, leaving specific secondary antibody after elution, Adding a substrate will enzymatically catalyze, which will lead to the development of color reaction, so that the color of the solution will change. Finally, we use the concentration of the substance that produces the color reaction to derive the concentration of the antibody you want to detect. This method has a simple principle and specificity is ensured by antibody recognition. It does not require particularly complicated operations. It can be automated or semi-automated, and the experimental throughput is not low.

Another method is to use colloidal gold color test paper detection method.

The reason why the colloidal gold color test is done is because this experiment can be applied to large-scale screening. Many places have to investigate the infection rate. If the infection rate is high and most people have antibodies, the virus will not spread easily. If the infection rate is very low and most people do not have antibodies, the virus will spread easily. This is to better prevent the next epidemic.

3D Printing and Oncolytic Virus Contribute to Precise Medical Solutions

Abstract: Many drugs have been developed or are under research for cancer treatment. With the advancement of technology, precise medical solutions for individuals may provide better effect with cheaper cost.

On the morning of May 19, 2014, at the Innovator Conference held in the lobby of the New York Historical Society, an Autodesk Genetic Engineer, Andrew Hessel, told all the representatives of the large pharmaceutical factories how he will treat cancer: first to test the virus that can kill tumor cells, and then use a 3D printer to cultivate these viruses in the laboratory.

In an interview with Le Monde, Hessel introduced, “These viruses are called oncolytic viruses, and scientists have studied them decades ago. These viruses are weakly infectious pathogens, but can infect tumor cells without damaging healthy body cells. Some companies have used these viruses to conduct clinical trials and have achieved success. But now, no one can use computers to design these viruses and cultivate them according to the needs of each patient.”

As we all know, chemotherapy is one of the common methods of cancer treatment. While chemotherapy kills tumor cells, it also kills normal cells in the body. Therefore, its side effects can cause other diseases. This is equivalent to dropping a nuclear bomb into New York City in order to eliminate a criminal gang.

Putting a new drug on the market is a very tortuous process, which requires decades of clinical trials and government approval. Therefore, Hessel does not want to produce and sell medicines, but uses continuously developing genetic engineering and powerful computers to tailor the treatment plan for each cancer patient.

Hessel heads the Nanotechnology Department of Autodesk in California. Their most successful product is AutoCAD (Automatic Computer Aided Design Software). At that time, genetic technology began to achieve some initial achievements, which Hessel found could provide a good opportunity for the digitization of medicine.

“A friend of mine mentioned the oncolytic virus to me, and I think that if we cultivate it for each patient, we can improve the efficacy and efficiency. Our cells are like a small computer, DNA is its operating system. Genetic technology enables us to digitize the information of this operating system, just like digitizing a text, a song.” He said.

Hassell’s cancer treatment plan is very different from those of large pharmaceutical factories. Their goal is to obtain approval from the Food and Drug Administration to place drugs with few side effects on the market. But Hessel thought if individual cancer patients could receive tailor-made treatment plans, the treatment will be more effective.

Some scientists in the scientific community questioned his treatment plan and warned him that cancer was not as simple as it seemed. Hessel just thought his plan was the beginning of a revolution, just like the advent of the first personal computers.

Today, about 600 investors support his plan. He guarantees that the cost of designing and nurturing a cancer patient will only cost about 730 euros, and with the enhancement of computer functions, treatment will become much cheaper and simpler. But at present, their treatment can be applied to humans.

According to Hessel, although success is still unreal for therapies like oncolytic viruses, it is not just a scene in science fiction. The cure for an individual’s cancer could be a small-market drug or a virus that’s tailor-made just for them. And the future fighting against cancer is looking brighter by the day.

Alfa Chemistry Offers PEG linkers for the Science Community in Bioconjugation and Pegylation

Earlier this month in May, 2020, the world’s leading chemical supplier Alfa Chemistry announced that a total of close to 800 PEG linker products are now available for its customers worldwide, including acid PEG linkers, alkyl PEG linkers, amino PEG linkers, azido PEG linkers, benzyl PEG linkers, biotin PEG linkers, fmoc PEG linkers, NHBoc PEG linkers, NHS ester PEG linkers, t-butyl ester PEG linkers, THP PEG linkers, and other PEG Linkers.

 

Polyethylene glycol (PEG) linkers are necessary for bioconjugation and pegylation. Due to their characteristics of water solubility, lack of toxicity, low immunogenicity, well defined chain lengths and molecular weights, PEG linkers are widely used in pharmaceutical and biotechnology applications.

 

PEGylation is considered one of the most successful techniques to enhance the therapeutic and biotechnological potential of molecules, such as therapeutic peptides, proteins, small molecules, oligonucleotides, and antibodies. The PEG based products differs from one another because they have different linker lengths and various reactive groups. Usually, PEG linkers contain a methoxy group and another functional group. And it is said that all common target moieties can be specifically addressed with the appropriate functionalized PEG linker.

 

The comprehensive portfolio of PEG linkers at Alfa Chemistry can be further divided into the following subtypes:

 

Acid PEG linkers

l Alkyl PEG linkers

l Amino PEG linkers

l Azido PEG linkers

l Benzyl PEG linkers

Biotin PEG linkers

l Fmoc PEG linkers

l NHBoc PEG linkers

l NHS ester PEG linkers

l t-butyl ester PEG linkers

l THP PEG linkers

l Other PEG Linkers

 

“The high purity PEGylation products we offered are kept in stock, with a hope to empower our clients’ research on bioconjugation, PEGylation, crosslinking, as well as ADC drug development and biolabeling in the pharmaceutical and biotech area,” says a senior scientist from Alfa Chemistry.

 

For more information about Alfa Chemistry’s PEG linkers, please visit https://www.alfa-chemistry.com/ to learn more.

 

About Alfa Chemistry

As a professional and reliable supplier of building blocks, reagents, catalysts and reference materials, Alfa Chemistry is a preferred choice of partner for many universities, research institutes as well as other organizations. Its broad range of product offerings cover heterocyclic organic compounds, boronic compounds, optoelectronic materials, organic building blocks, fluorinated building blocks, steroidal compounds, precious metal catalyst, material & chemicals, metal organics, nanomaterials, and more.