Graphene family: the difference of graphane, graphyne, graphene ether

Graphene is a two-dimensional carbon nanomaterial with hexagonal honeycomb lattice composed of carbon atoms with sp² hybrid orbitals.

Graphene has excellent optical, electrical, and mechanical properties, and has important application prospects in materials science, micro / nano processing, energy, biomedicine, and drug delivery. It is considered to be a revolutionary material in the future.

Andrei Gem and Konstantin Novoselov, physicists at the University of Manchester, UK, successfully isolated graphene from graphite by micromechanical exfoliation, so they won the 2010 Nobel Prize in Physics . The common powder production methods of graphene are mechanical peeling method, redox method, SiC epitaxial growth method, and the thin film production method –chemical vapor deposition (CVD).

Recently, Professor Liu Chunsheng’s group at Nanjing University of Posts and Telecommunications has theoretically studied the effects of superconjugation on the energy bands, mechanical and electrical properties of two-dimensional materials. Drawing on the “bottom-up” method of assembling graphene, the authors assembled methyl ether molecules with a super-conjugation effect into a new two-dimensional oxycarbon compound, and named it “graphether”.

The research result was published in the journal Nanoscale, titled “Graphether: a two-dimensional oxocarbon as a direct wide-band-gap semiconductor with high mechanical and electrical performances” DOI: 10.1039 / C9NR08071F.

  1. Graphane

Simply put, it comes from the hydrogenation of graphene, and a hydrogen atom is introduced next to each carbon atom. Graphane is similar to graphene, a two-dimensional alkane. Its name is also based on the nomenclature of organic chemistry, which means saturated carbon Hydrogen compounds. Researchers said that although pure graphene is extremely stable in chemical properties, they found that hydrogen atoms can react with it, turning highly conductive graphene materials into new graphane materials with insulating properties. This experiment proved that the properties of graphene can be changed by chemical methods, which paved the way for the preparation of other graphene-based chemical derivatives. Similar to graphane, fluorine and nitrogen hybrids can also be introduced to produce other graphene derivatives.

In addition, corresponding to the complete hydrogenation of graphane, when the hydrogenation on graphene is incomplete, it is called hydrogenated graphene (including reduction of graphene oxide for hydrogenation). Hydrogenated graphene can exhibit a certain ferromagnetism and a band structure that can be adjusted according to the degree of hydrogenation. In addition, the material is also considered as a promising hydrogen storage material because reversible hydrogenation and dehydrogenation can occur.

  1. Graphyne

In 2010, researchers from the academician Li Yuliang of the Chinese Academy of Sciences Key Laboratory of Mechano-Solids have synthesized graphyne for the first time, opening up a new field of carbon materials. Graphyne is a full-carbon molecule with benzene rings conjugated by a 1,3-diyne bond to form a two-dimensional planar network structure. It has rich carbon chemical bonds, a large conjugate system, wide interplanar spacing, excellent chemical stability, and Semiconductor performance is expected to be widely used in electronics, semiconductors and new energy fields.

  1. Graphite ether(graphether)

Graphite ether has excellent dynamic and thermodynamic stability, is a direct band gap wide band gap semiconductor (energy gap 2.39 eV), and has good response in the ultraviolet region. In addition, it can maintain direct band gap characteristics under uniaxial or biaxial strain of -10% -10%. Due to the super-conjugation effect, the in-plane stiffness (459.8 N m-1) in the direction of the graphene armchair exceeds that of graphene (342 N m-1). Compared with the lower carrier mobility of hydrogenated and fluorinated graphene (101-2550px2V-1s-1), the electron mobility of graphene ether reached 2575px2V-1s-1 in both chair and zigzag directions. The above-mentioned superior properties make graphite ether materials expected to be used in nanoelectronic and photovoltaic devices.

Graphite ether is not only a direct band gap wide band gap semiconductor, but also has high in-plane stiffness and electron mobility. In addition, Pt (100) proved to be a potential substrate for the synthesis of graphene from the bottom up. These results are expected to provide new ideas for the design and preparation of graphene-like materials with superconjugation effects, and promote their innovative applications in next-generation electronic and optoelectronic devices.

 

How much do you know about interferon family

In terms of cytokine drugs, Interferon (IFN) may be familiar to you. At present, interferon is a very popular drug. At first, interferon is used to treat some small diseases such as flu, hepatitis, chickenpox, etc. Now it is used to deal with more complicated diseases, such as cancers and leukemia. And genetically engineered interferons have also been on the market for many years.

Interferon

Let’s take a look at these amazing cytokines:

Interferon (IFN) is a broad-spectrum antiviral agent that does not directly kill or inhibit the virus, but produces antiviral proteins through the action of cell surface receptors, thereby inhibiting the replication of the virus, and at the same time enhancing the vitality of natural killing cells (NK cells), macrophages and T lymphocytes, thereby playing an immunoregulatory role and enhancing antiviral capabilities. Interferon is a group of active proteins (mainly glycoproteins) with multiple functions. It is a cytokine produced by monocytes and lymphocytes. They have a wide range of biological activities such as anti-virus, affecting cell growth, differentiation, and regulating immune function on the same kind of cells. They are currently the most important anti-virus infection and anti-tumor biological products.

Interferon family classification

  1. Type I interferon: Type I interferon includes IFN-αand IFN-β. IFN β is produced by human fibroblasts; IFN-α is mainly produced by monocytes and macrophages; in addition, B cells and fibroblasts can also synthesize IFN-α; IFN-β is mainly produced by fibroblasts. Both IFN-α / β bind to the same receptor and are widely distributed, including monocytes-macrophages, polymorphonuclear leukocytes, B cells, T cells, platelets, epithelial cells, endothelial cells, and tumor cells.
  2. Type II interferon: Type II interferon, gamma interferon, is mainly produced by activated T cells (including Th0, TH1 cells and almost all CD8 + T cells) and NK cells. It is the so-called one type of lymphokine. IFN-γcan exist in the form of extracellular matrix connected, so the cell growth is controlled by a neighboring way, which can be distributed on the surface of almost all cells except mature red blood cells.

In the same type, according to the difference in amino acid sequence, it is divided into several subtypes. It is known that there are more than 23 subtypes of IFN α, which are represented by IFN-α1 and IFN-α2, respectively. There are only one or more subtypes of IFN β and IFN γ, and the physicochemical and biological properties of the three types of interferons are significantly different. Even among the subtypes of IFN α, their biological effects are not the same.

The discovery history

Speaking of the discovery of interferons, it goes back more than 80 years. In 1935, American scientists experimented with monkeys using yellow fever virus. Yellow fever is a malignant disease caused by a virus. There are several types of diseases that people and monkeys can get. They first infected the monkey with a weakly lethal virus. The monkey was safe and sound, and then they infected the same monkey with the highly pathogenic yellow fever virus, but the monkey did not respond. This phenomenon inspired American scientists. They thought the former virus may have produced something that would allow cells to defend themselves when attacked by a new virus. In 1937, a similar experiment was repeated, and it was confirmed that monkeys infected with Rift Valley Fever were injected with yellow fever virus, and the monkeys were fine. Repeated experimental evidence has led scientists to think that viruses in the biological world also have wonderful mutual interference phenomena.

In 1957, British virus biologist Alick Isaacs and Swiss researcher Jean Lindenmann learned that flu-infected cells can produce a factor, which affects other cells and interfere with virus replication, thus it is called interferon.

From 1966 to 1971, Friedman discovered the antiviral mechanism of interferon, which caused people’s attention to the antiviral effect of interferon, and then, the immune regulation of interferon and its antiviral, antiproliferative and antitumor effects were gradually recognized. .

In 1976, Greenberg et al. First reported that 4 cases of chronic active hepatitis B were treated with human interleukin, and 2 cases of HBeAg disappeared after treatment. However, because human leukocyte interferon has limited sources and is expensive, it has not been widely used in clinical practice.

In middle 1970s, the medical community found that patients with chronic hepatitis B have a low ability to produce interferons themselves. After the application of exogenous interferons, the patient not only showed the antiviral effects described above, but also the density of the human leukocyte tissue compatibility on the hepatocyte membrane increased, which  promoted the effectiveness of T cells in lysing infectious hepatocytes. After injection of (2 ~ 5) × 106 units of interferon in adults, the interferon activity in serum began to be measured at 3 hours, reached a high level at 6 hours, and disappeared at 48 hours. After more than ten years, IFN has been used to treat hepatitis B.

In early 1980s, Swiss scientists and American scientists succeeded in developing the first generation of genetically engineered IFN α almost simultaneously. From 1980 to 1982, scientists used genetic engineering methods to obtain interferon in E. coli and yeast cells, and from 20 to 40 milliliters of interferon per liter of cell culture.

In early 1981, Pestka et al. Synthesized and purified IFN α-2a and obtained FDA approval for clinical trials.

In the mid-1980s, after the first genetically engineered IFN α-2a was successfully developed and marketed, it was widely used in clinical practice. The second generation of genetically engineered IFN α-2b was introduced, and its molecular structure is almost the same as human IFN. It was approved by the FDA for the treatment of chronic hepatitis B in 1986. At the same time, Chinese Hou Yunde and other scholars are also studying the preparation of genetic engineering IFN. Since 1987, interferon produced by genetic engineering has entered industrial production and has been put on the market in large quantities.

In 2005, pegylated interferon alpha-2a was approved by the US FDA and officially used for hepatitis B treatment.

Until now, countries using genetic engineering techniques to obtain interferons include the United States, Japan, France, Belgium, Germany, the United Kingdom, and China. A variety of interferons were obtained in large quantities through methods such as DNA recombination and E. coli fermentation. Experiments have shown that the interferon thus prepared has certain effects on viruses such as hepatitis B, rabies, respiratory inflammation, and encephalitis. Interferon can slow the growth of cancer cells, is a promising anti-cancer drug, and has very attractive prospects.

The latest progress

A recent study showed that boosting the body’s production of type I interferon can help clear viral infections. The results were published in the journal Cell.

In this study, the authors found that RIG-I-like receptor (RLR) -mediated production of interferon (IFN) plays a pivotal role in host immunity that enhances virus clearance and cancer immune surveillance. Previous studies have shown that glycolysis is the first step in breaking down glucose to extract cellular metabolic energy, and the authors found that during RLR activation, glycolysis is inhibited, and this inhibitory effect is the key to IFN-I production. Using pharmacological and genetic methods, scientists have shown that reducing lactic acid through inactivation of lactate dehydrogenase A (LDHA) can increase the production of type I IFN, thereby protecting mice from viral infections. The authors say that type I interferon (IFN) plays a vital role in host defenses against viral infections and cancer immune surveillance. In response, the authors plan to conduct additional studies in other animal models to prepare for potential clinical trials.

Principles and methods of food allergen detection

Allergy-related diseases affect nearly one quarter of the world’s population and are listed by the World Health Organization as one of the three major diseases to be controlled in the 21st century. Among them, adverse reactions to food have attracted much attention. About 30% of adults have experienced adverse reactions to food at least once in their lifetime, and 20% to 65% of patients have allergic bowel syndrome and indigestion to food allergies.

 

What is a food allergy?

An allergic reaction is an abnormal immune response that is inherent to a foreign substance, such as food. Any food that causes an allergic reaction is called an allergen.

With age and long-term consumption of a certain food, the allergenicity of that food will also change. When the immune system produces a large amount of antibodies to a certain food, it causes inflammation or allergies in different places.

Many unexplained chronic and recurrent symptoms are linked to food allergies.

Most people experience inexplicable allergic symptoms such as skin rash, diarrhea, headache, indigestion, flatulence, nasal congestion and nasal fluid. If you take into account some common minor discomfort symptoms: such as occasional anxiety, joint stiffness, edema, and dark circles, at least a quarter of the world’s population is affected by food allergies.

What factors will promote an allergic reaction?

Heavy metal poisoning, food additives, partial eclipse, stress, genetics, long-term infections, inflammation, drugs, environmental pollutants and toxins (such as toxins released from styrofoam food utensils) can promote an allergic reaction.

At present, the only effective treatment method is to avoid eating allergic foods, and it is particularly important to patients to detect which food is allergic to him/her. The study of food allergens is an important basis for the research and development of detection reagents and allergy mechanisms. The current detection methods of food allergens and the research progress of common allergens are summarized below.

First, the detection method of food allergens

(1) Double-blind food challenge test

(2) Skin prick test (SPT)

(3) In vitro 8IgE detection

  1. a. Radio allergosorbent test (RAST)
  2. b. Cap (CAP) Allergen Detection System
  3. Other reagents and methods
  4. Biochip

(4) Biological resonance technology

Second, research progress on common food allergens

Because all kinds of allergen detection reagents use recombinant or extracted allergens and allergens play an important role in the treatment of allergic diseases, it is of great significance to study allergens. Food allergens are generally proteins or glycoproteins with a relative molecular mass of 10,000 to 70,000. They can be divided into major allergens and minor allergens. Most allergic patients are sensitive to major allergens.

(1) Crustaceans and their products

As seafood has become more and more popular, reports of allergic reactions to such foods have gradually increased. Among them, shrimp allergens have attracted much attention. It is reported that 0.6% to 2.8% of patients with allergic diseases are allergic to shrimp. At least 13 IgE-binding proteins have been detected in shrimp, but tropomyosin has been identified as the only major allergen, with a relative molecular mass between 34,000 and 39,000. It is reported that tropomyosin is an important antigen of invertebrates such as shrimp, crab, oyster, and squid, and has a highly conserved amino acid sequence.

(2) Eggs and their products

Eggs are one of the most common causes of food allergic reactions in children, with a positive rate of 35% in food allergies in children and 12% in adults. The major allergens of the protein are oval mucin Galdl (relative molecular mass 28000), ovalbumin Gald2 (relative molecular mass 44000), oval transferrin Gald3 (relative molecular mass 77000), and lysozyme Gald4 (relative molecular mass) 14000); the main allergen for egg yolk is Ot yolk protein (relative molecular mass 70,000). According to reports, egg allergy is more likely to cause allergies than egg yolk, and ovalbumin is the main allergen.

(3) Milk and dairy products

Milk allergy rates among children under 2 years of age range from 1.6% to 2.8%, and 50% to 9% of children become tolerant before the age of 6 years. The main allergens of milk are casein (Bosd8, relative molecular mass is 20,000 to 30,000), Bosd5 (relative molecular mass is 18000), Ot. Lactalbumin (Bosd4, relative molecular weight is 14000), bovine serum albumin (Bosd6, relative molecular weight is 67000), and bovine immunoglobulins, among which casein is the most immunogenic and antigenic. Milk antigens are relatively stable and retain their immunogenicity after routine processing.

(4) Peanuts and Products

Due to the severity and high incidence of peanut allergic reactions, this allergen has caused widespread concern in medical institutions. Peanut allergies account for 10% to 47% of food-induced allergic reactions. The main peanut allergens are Arah1 (relative molecular mass is 63500), Arah2 (relative molecular mass is 17000), and other related antigens are Arah3 (relative molecular mass is 60,000) and Arah4 (relative molecular mass is 14000). The secondary antigens are Arah6, Arah7 and actin. ELISA inhibition test confirmed that Arah2 was the main antigen that caused cross-reaction of peanut with hazelnut and almond. Different food processing methods have different effects on peanut antigens. It is reported that baking will increase Amhl content and make IgE antibody-binding epitopes more easily bind to antibodies. Although peanut and legumes have cross-reactive proteins, clinical cross-reactivity is rare. Peanut allergy is more common in individuals who are allergic to eggs, milk, walnuts, etc., but it has not been found to have cross-reactive proteins with walnuts. In most cases, peanut allergy persists and turn worsen. In recent years, various technologies such as immunoelectrophoresis, ELISA, PCR, and biosensors have been applied to the study of food allergens, which has greatly improved people’s understanding of food allergens. However, the cross-reactivity of food allergens and different treatments on its allergenicity and the allergenicity of genetically modified foods need to be further explored.

The summary

In summary, in recent years, the incidence of food allergies has been increasing, and the detection of allergens is of great significance for the prevention and treatment of diseases. With the development of immunology, food allergen detection technology has made great progress, but there are also shortcomings: (1) because food allergens are (glyco) proteins, some of these (glyco) proteins are stable, and some are not stable, which limits the sensitivity of the test; (2) Some nut foods and seafood have cross-reactions, so the food allergen detection is not specific and further improvements are needed in the future. There are also methods for detecting food allergens such as blood IGG test, histamine release test, and basophil granulocyte activation test. No method can detect all food allergens, so combination should be used in clinical practice when necessary.

The development of molecular biology and immunology has made people have a better understanding of the structure and immunogenicity of food allergens. The development of bioinformatics has provided a strong support for the study of allergens. The recognition of T-lymphocyte and B-lymphocyte epitopes and the assessment of allergenicity and cross-reactivity are complementary to traditional laboratory techniques. Now the cross-reactivity of food proteins is available, and the allergenicity of genetically modified foods based on their genetic sequences is evaluated. It is believed that in the near future, human beings will have a deeper understanding of food allergens, which will provide a good platform for future research on food allergy mechanisms and research and development of allergen detection reagents.

Recent advances in peptide cancer vaccines

In the field of cancer treatment, a number of achievements in immunotherapy have emerged. Cancer vaccines, as one of them, have emerged as a research hotspot. Compared to conventional vaccines, cancer vaccines, such as cervical cancer vaccines, not only have the preventive effects, but also can be used as therapeutic vaccines to activate the human immune system and kill tumor cells.

There are many types of cancer vaccines, including cell vaccines, nucleic acid vaccines, protein peptide vaccines, and genetically engineered vaccines. No matter what kind of vaccine, tumor antigens are presented to immune cells to activate the immune response, thereby achieving anti-cancer effects. Here is a list of recent advances in cancer vaccines.

Peptide vaccine

Polypeptide vaccine is a vaccine prepared by chemical synthesis technology according to the amino acid sequence of a certain antigen epitope in a pathogen antigen gene. If a specific antigen in a tumor cell can be found and a cancer vaccine can be developed based on this amino acid sequence, the relevant immune cells can be activated to kill tumor cells of the same specific antigen.

ASCO has two researches on peptide cancer vaccines: the first is the application of multiple peptide vaccines in aggressive tumors; the second is the clinical efficacy of peptide vaccines based on new antigens.

In the first study, there was not much research on tumor and lymph node microenvironment in patients with aggressive cancer, and there was no effective way to regulate it. However, multiple peptide vaccine treatments may be able to solve this problem, and can also be guaranteed in clinical safety.

The study recruited a total of 11 patients and identified numerous targets through various methods. Patients were vaccinated subcutaneously at the lymph nodes every other week for a total of 8 times. The vaccine was then injected subcutaneously in the active site of the tumor by CT or PET for a total of 10 times.

The results of the study showed that 100% of patients responded to treatment regardless of disease progression, 80% of them reached CR, and 20% of them with pseudo-progression later developed CR.

Granzyme B levels were increased in 100% of patients targeting Bcl-2 (p = 0.001), VCP (p = 0.0001), Ape-1 (p = 0.005) and RCAS1 (P = 0.0001), and scan data were similar after treatment result.

Patients showed more CD8 T cell infiltration at tumor sites (p = 0.002) than non-tumor sites (p = 0.01). The number of disappearance of lung (p = 0.004) and liver (p = 0.001) metastases in patients was significantly related to the increase of IL-12.

This study shows that injection of multiple peptide vaccines into the lymph nodes and tumor sites of multiple solid tumors is not only safe and reliable, but also can improve the clinical benefit of advanced patients.

In the second study, neoantigen vaccines can activate T cells and kill tumor cells. However, most peptide vaccines either choose HLA-binding epitopes or HLA-presenting epitopes, and neither can induce a personalized T cell response. This study suggests that personalized epitopes that bind to multiple autologous HLA alleles can induce T-cell responses.

The researchers injected patients with synthetic long-chain polypeptide (SLP) vaccines in two clinical trials, identified epitopes of the single HLA and 3 HLA alleles, and validated them with CD8 and CD4 T cell responses. Ultimately, they developed a personalized vaccine that binds 14 HLA alleles from 12 tumor antigen sources.

The results showed that there was no correlation between single HLA-binding epitopes and HPV-specific T cell responses. In contrast, the consistency of the first-type HLA epitope and CD8 T cell response was 90% (p <0.001), and the response of the second-type HLA personalized epitope and CD4 T cell response was 69% (p = 0.005).

The value of the individualized epitope can predict the T cell response rate in clinical trials of SLP vaccines. It is predicted that 91% and 100% of peptide vaccines can induce CD8 and CD4 T cell responses, respective.

 

Innovative Technologies for Rapid HIV Detection in Recent Years

This article describes the development of new technologies in the field of HIV testing in recent years.

 

[1]Science: Imaging technology to monitor the spread of HIV in vivo

How the transcriptional viruses (such as HIV) spread in the host, scientists are not clear, recently, researchers from Yale University designed a method to observe the HIV diffusion process in living organisms. Related research was published in the international journal Science, in which researchers successfully observed how HIV reaches and spreads in mouse lymph nodes.

 

Researcher Professor Walther Mothes said that the way we observed the spread of the virus is not the same as people imagined. In the experiment, we tracked the fluorescently labeled virus in the mouse body and used complex imaging techniques to capture the combination of viral particles. The process of phagocytosis, which is accomplished by viscous proteins located on the surface of the lymph nodes.

 

The researchers said that the captured virus particles can be opened to a rare type of B cells, and then the virus particles will adsorb themselves to the tail of these B cells and be dragged into the lymph nodes. These B cells will be the same within one to two days. The organization establishes a stable connection to promote the complete transmission of the virus. The video taken by the researchers describes a potential way to help suppress the tissues surrounding the HIV-infected area. If researchers can develop a way to block HIV’s use of sticky proteins to bind macrophages, then the virus Diffusion propagation is suppressed.

 

[2] Scientists develop the world’s first next-generation sequencing technology to measure HIV drug tolerance mutations

At the ACCC Annual Meeting and Clinical Lab Expo, researchers from Singapore’s genetic sequencing company, Vela Diagnostics, launched the world’s first HIV drug resistance mutation. A new generation of sequencing technology that plays an important role in helping clinicians optimize HIV treatment systems, while also helping scientists to take the initiative to minimize the global antiretroviral drug tolerant epidemic.

 

The use of antiretroviral therapy for HIV infection has grown dramatically over the past decade and is part of the current global adoption of the AIDS public health threat program; according to data from the World Health Organization, HIV drug tolerance Concurrent growth will counteract the efforts of scientists over the years by offsetting the effects of antiretroviral drugs on HIV and AIDS progression; therefore, testing patients’ tolerance to HIV drugs is key to ensuring patients receive effective treatment. At the same time, it is also very important to effectively manage the tolerance of antiretroviral drugs.

 

[3]Analy Chem: A new mobile DNA detection technology for monitoring HIV

Recently, in a research report published in the international magazine Analytical Chemistry, scientists from the University of Rice in the United States have developed a simple and high-accuracy detection technology to detect the signs of HIV and the degree of disease development in patients’ bodies.

 

Researcher Rebecca Richards-Kortum said that the current gold standard for diagnosing HIV status in infants relies on laboratory equipment and currently available detection techniques to monitor viral load in their bodies, and researchers in this paper have developed New HIV-based detection technology based on nucleic acids.

 

The new detection technology is based on a PCR-based method for amplification of recombinant polymerases (RPA antibody), which replaces complex laboratory testing steps that allow rapid multi-amplification of genetic markers in the blood, resulting in genetic markers of the virus. The object can be easily detected; the qRPA test can be used to perform targeted labeling of fluorescent probes on specific sequences in HIV DNA, thereby using a machine for quantitative detection, and finally by software analysis of fluorescent DNA, doctors can master Dynamic information about HIV in the patient’s body.

 

[4] Brushing saliva for 15 minutes to test AIDS

The most common method of AIDS testing is to use the kit to detect and pump blood tests. Kit testing typically requires 50 or more people to work together, and the results typically take several days. The rapid blood test can get the test results within 30 minutes, but it requires professional doctors to operate around. So, is there a simpler and more practical detection method?

 

In the Technology Incubation Park in the High-tech Zone of Suzhou, China, Suzhou Wanmuchun Biotechnology Co., Ltd. revealed that they independently developed the first integrated HIV saliva detector in China and successfully obtained the highest level of production license for the national medical device. . “Efficient, it can be read in 10 to 15 minutes; it is convenient to brush saliva; protect privacy, patients can self-test at home.” The company’s R&D personnel took a small test stick from the box and showed it to reporters, as long as Stretch the gums in the entrance cavity, and after the test stick is taken out of the mouth for a while, the “display window” will show colored lines, and the test results will be clear at a glance.

 

[5] British scientists have developed a fast and cheap HIV test method

Scientists at Imperial College of Science and Technology have developed a new HIV test that is 10 times more sensitive than current tests, but at a relatively low cost, which is a diagnosis for HIV-infected people in developing countries. Treatment brings a new gospel.

 

At present, the use of human saliva to detect HIV has been quite simple and fast, but its shortcoming is that it can only be detected when the viral load reaches a certain concentration. In some cases, the viral load of the test sample is too low, and it is difficult to obtain accurate results using some of the original detection methods (such as saliva detection), and a “false negative” test result usually occurs. The new method can accurately detect the situation where the viral load is too low.

 

The new detection method uses nanotechnology, which can display the detection result of the sample to be tested as red or blue, which can be distinguished by ordinary naked eyes. The method is to test the serum and detect in the disposable vessel whether there is an HIV biomarker called p24. If p24 is present, it will cause tiny gold nanoparticles to condense in an irregular manner, which in turn turns the solution blue; a negative result can separate the gold nanoparticles into a spherical shape. The solution is shown in red.

 

[6] American scientists developed molecular microscopy for in situ detection of HIV

The HIV in situ analysis technology has once again made a breakthrough. At the International AIDS Conference held last week, US scientists demonstrated their new detection technology and test results. This probe called “Molecular Microscope” can accurately detect the hidden places of HIV inside and outside the cell.

 

Ricardo Pul, deputy director of the Vaccine Research Center of the American Institute of Allergy and Infectious Diseases, said that the new technology of this molecular microscope is magical, and its super powers can fully understand the traces of HIV in any cell, and ultimately Help clear the mystery of the long-term survival of HIV and completely remove it from the body.

Basic information about amplicon sequencing

Overview

Amplicon sequencing is a highly targeted method for analyzing genetic variations in specific genomic regions. Ultra-deep sequencing of PCR products (amplicons) can effectively identify mutations and characterize them. The general idea is to capture the target area in a targeted manner, and then perform next-generation sequencing (NGS), analyze the sequencing results, and obtain corresponding information.

Amplicon sequencing mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing and target region amplicon sequencing. The sequence of a high-variation region of 16S/18S/ITS determined by a second-generation high-throughput sequencing platform to reflect the differences between species in the classification of bacteria, fungi, and archaea for the study of oceans, soils, and intestines, and the composition of microorganisms in the environment such as feces has an important guiding role, and is also a widely used method in phylogeny and taxonomic studies, especially in different metagenomic samples.

The overall route of amplicon sequencing

The principle of amplicon sequencing

Taking QIAGEN’s amplicon sequencing kit as an example, we briefly introduce the principle. PCR amplification is the main method of amplicon sequencing because the target DNA fragment can be enriched by PCR amplification. However, there may be errors in the PCR amplification and sequencing process that ultimately lead to false positives in the sequencing results. In order to overcome the human error introduced in PCR amplification, UMIs (unique molecular indices) are incorporated into the starting DNA material before any amplification, thereby preserving the uniqueness of the starting DNA molecule and overcoming the PCR amplification, as well as the bias introduced in the library construction process.

Each original DNA molecule carries unique UMIs. When analyzing data, the same UMIs are derived from the same DNA. All the same UMIs have the same diversity site in the DNA. If only one or several of the UMIs contain diversity sites, this diversity can be judged as a false positive. This method has good accuracy, specificity, sensitivity and versatility.

The applications of amplicon sequencing

(1) Cancer gene sequencing: Targeted cancer sequencing focuses on a set of genes, gene regions or amplicons that have known associations with cancer, pre-designed collections and custom collections, both can be used to study target-targeted genes;

(2) Disease screening: NGS can be used to simultaneously detect multiple genes to discover the pathogenic variation of genetic diseases. Compared with traditional methods, using NGS can reduce costs because traditional methods usually cost a lot and the results are uncertain, and require extensive testing;

(3) Plant and animal sequencing: Pre-set genomic region-targeted resequencing can reveal genetic variations in animals and plants that may represent beneficial mutations that can help making breeding decisions and reveal mutations associated with disease susceptibility.

Advances in plant CRISPR genome editing technology (Part Three)

2.2 Optimizing gRNA expression

CRISPR / Cas9’s simple and powerful multi-site editing capability is one of its significant advantages. Realizing multi-site editing requires simultaneous expression of multiple gRNA molecules. At present, there are mainly three strategies to use multiple vectors to simultaneously express multiple gRNAs. The first is to clone multiple expression units in a plasmid vector, each unit containing a Pol III promoter and gRNA. The second is to use Csy4 nuclease to cleave transcripts containing multiple gRNAs into a single gRNA, where the gRNAs are linked by Csy4’s RNA substrate. Third, the endogenous transfer RNA (tRNA) of the organism is fused with gRNA, and multiple tRNA-gRNA structures are arranged in series into one polycistronic. The tRNA processing enzyme in the organism is used to convert this polycistronic. The daughter cuts into mature gRNA for multi-site editing. In addition, self-cleaving ribozyme can also be used for the expression of multiple gRNAs. These different methods provide mature multi-site CRISPR / Cas9 genome editing technologies for plant genetic manipulation.

2.3 Limitations of Reducing PAM

DNA target sites must be present PAM is the main factor limiting CRISPR / Cas9 for genome editing. According to the sequence analysis of the reference genomes of several model plants, the average genome has an average of 6−11 PAM (5′-NGG-3 ′) per 100 bp. Although this frequency has basically met the needs of conventional gene function research, it has greatly limited the scope of CRISPR / Cas9 applications.

In order to eliminate or reduce the PAM-dependent restriction of CRISPR / Cas9, it is necessary to find or create CRISPR / Cas9 that recognizes different PAM sequences for genome editing. In the past years, several CRISPR / Cas9 systems have been discovered, greatly expanding the space for CRISPR / Cas targeted gene editing. In addition, the researchers modified Cas9, the most commonly used Streptococcus pyogenes, to modify the amino acid sites related to PAM recognition in Cas9 protein, and obtained recognition 5′-NGAN-3 ′ and 5′-NGNG-3 ′. Cas9 mutants with different PAM sequences and have been used for plant genome editing. To synthesize these new developments, the existing CRISPR / Cas9 tools basically have the ability to target the whole genome.

3.Beyond CRISPR / Cas9

3.1 Targeted single base editing

In genome editing technology, the most common application is to introduce indel at the target site through CRISPR / Cas9, thereby destroying the function of the target gene. However, in the practice of genetic improvement of crops, it is difficult to obtain materials of breeding or application value by completely destroying the function of the gene. More often, it is necessary to modify the function of the gene by directional modification of a single or several bases, so as to improve the crop Agronomic traits. Therefore, efficient and targeted transformation of the genome sequence is the key to CRISPR / Cas9 technology for genetic improvement practices, and single base editing technology is meeting this need.

Figure. The principle of targeted editing of a single base based on CRISPR / Cas9

The principle of targeted editing of a single base based on CRISPR / Cas9 is shown in Figure above. Cas9n (or dCas9) is fused with Cytidine deaminase. When gRNA guides the fusion protein to the target site, the target site Nearby cytosine C is converted to uracil U by deaminase, and U has the same base pairing rules as thymine T. U will be converted to T when DNA is copied or repaired, thus completing the C → T base Conversion. In order to improve the efficiency of C → U → T, related components will be added to the editing tool to suppress the recovery of U → C. At present, there are two successful single-base editing tools: one is the BE (Baseeditor) system constructed by David Liu’s laboratory at the Massachusetts Institute of Technology, which is obtained by fusing dCas9 or Cas9n with mammalian deaminase APOBEC1. The second is the Target-AID, CRISPR-X and TAM systems obtained by fusing dCas9 or Cas9n with AID (Activation-induced deaminase). These different single base editing tools are slightly different in specific design, and the window of target bases that can be edited are also slightly different, but high efficiency C → T single base replacement can be achieved in animal cells.

Although Cas9-mediated knock-in can also be used to achieve single-base substitution, the efficiency and ease of implementation of HDR are incomparable with single-base editing tools such as BE and Target-AID / TAM / CRISPR-X. When editing a single base on both APOE4 and p53 genes in a mammalian cell line, Cas9-mediated HDR has a base conversion efficiency of up to 0.3%, while the efficiency of the BE3 system reaches a surprising 58% −75 % (APOE4) and 3% −8% (p53). The latest results also indicate that the single-base editing tools engineered with CRISPR / Cas9 are very specific and no off-target editing was detected. These CRISPR single-base editing tools have taken gene editing technology to a new level, laying an important foundation for CRISPR / Cas9 technology for clinical treatment, basic research and crop genetic improvement.

Because single-base editing has great application potential in crop genetics and breeding, these editing techniques have been rapidly applied to crops. For example, if BE (Cas9n-APOBEC1) is used for rice, wheat, and corn, the exact replacement of C → T can be realized in the 3-9 bp edit window of protospacer, with a maximum efficiency of 43.48%. Target-AID was used in rice, and single-base substitution of the ALS (Acetolactate Synthase) gene yielded herbicide-resistant rice material. These successful examples initially demonstrate the great potential of CRISPR / Cas9 single base editing.

3.2 Genome editing technology based on CRISPR / Cpf1 system

The huge success of CRISPR / Cas9 from S. pyogenes for genome editing has prompted researchers to find and modify other CRISPR / Cas9 systems to expand the gene editing toolbox. The most potential currently is the CRISPR / Cpf1 system, which is considered to be a new generation of the best in genome editing tools. In 2015, the Zhang Feng laboratory at the Massachusetts Institute of Technology in the United States first discovered the Cpf1 endonuclease, a member of the 5th subfamily of the class II CRISPR system. The three highly homologous proteins AsCpf1, LbCpf1 and FnCpf1 of the genus Franisella novicida have RNA-guided endonuclease activity, and the efficiency of AsCpf1 and LbCpf1 for animal genome editing is close to the original CRISPR / Cas9 system.

To be continued in Part Four…

 

Application of Metabolomics in Food Testing

The most striking feature of metabolomics is its overall analytical ability to better reflect the impact of the environment on food composition. However, metabolomics does not appear to be rapidly hotter as proteomics, but rather shows a slowly rising trend.

 

In order to ensure the interests of consumers, the identification of food quality, especially traceability of the origin, has become the focus of food analysis technology. From the new products launched by many testing companies, the application of metabolomics in the analysis of food authenticity is generally optimistic. With the further study of metabolomics, the authenticity of food is expected to be known.

 

Authenticity identification has become a hot area

Driven by huge economic interests, the falsification of the origin of food industry and the incorporation of cheap raw materials in food production have repeatedly been banned. For this reason, more and more analytical techniques are beginning to be applied to the identification of food quality and origin.

 

At the 5th International Forum on China’s Food and Agricultural Products Safety Testing Technology and Quality Control held recently, experts in the field of food safety testing have proposed many effective methods for various endangered food fraud (adhesion) events.

 

For example, for the identification of the species characteristics of meat products, Shi Xiju, a researcher at the Beijing Entry-Exit Inspection and Quarantine Bureau, said that microscopic methods for the observation of microstructures, PCR and restriction enzymes for detecting nucleic acids, and fluorescent PCR methods are commonly used, and the future tends to be high-throughput and fast. Simple methods, such as LC/MS technology and test strips. In the fields of grain fats, meat and dairy products, fermentation and nutrition, low-field NMR technology has a wide range of applications such as non-destructive, rapid, and more intuitive imaging, such as grain quality classification, grease quality testing, non-destructive testing of fruits and vegetables, Microbial fermentation monitoring, meat moisture migration, etc. In addition, infrared spectroscopy can simultaneously measure multiple component data of a substance by simply measuring the primary infrared spectrum of the sample in tens of seconds or even seconds. It is now also used for the identification of various food adulterations. .

 

Metabolomics is expected to be an effective means

As an important branch of systems biology, metabolomics is widely used in drug research, disease diagnosis, plant breeding, environmental science and other fields. In a general sense, metabolomics uses statistical analysis to compare differences between two or more sets of samples, to find and identify biomarkers, and to analyze metabolic pathways to reveal biological significance.

 

Metabolomics analyzes metabolites on a large scale, focusing on the widest range of small molecules rather than just focusing on a certain set of metabolites. Metabolomics is the observation of changes in small molecule metabolites with relative molecular weights of less than 1200 Daltons or their changes over time by examining the stimulation or perturbation of biological systems. Metabolomics analysis techniques can be divided into targeted metabolomics and untargeted metabolomics, depending on the subject and purpose of the study. Known and unknown chemicals can be analyzed. It can be said that when traditional sensory index evaluation and routine quality index detection can not effectively distinguish adulterated and counterfeit food, metabolomics technology can quickly and accurately identify adulterated food.

 

Taking the identification of the most common red wine producing areas as an example, the mass spectrometry and spectral data generated by metabolic fingerprinting and metabolic profile can identify the type, source and even vintage of the wine. The traditional method for judging the quality of grapes used for winemaking is generally to determine their sugar, acidity, pH and total phenolic content, respectively. By establishing metabolite fingerprints of mature grapes from different wine producing regions, they can be identified and some are temporarily unrecognizable. Metabolite fingerprinting of metabolite components is not possible with traditional methods.

 

A great advance in metabolomics is expected, using new mass spectrometry interfaces that require little sample preparation, and the ability to analyze protein and metabolites at the tissue and single cell levels using MALDI imaging mass spectrometry to obtain information on the spatial distribution of specific molecules. Sample preparation methods and improvements in analytical platforms will enhance the relevance of food metabolomics research. In addition, capillary electrophoresis and capillary mass spectrometry are ideal tools for metabolomics research because they do not require extensive sample preparation, have a wide range of applications, high efficiency, high resolution, and low sample consumption.