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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.
http://hunter.unigame.me/

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Definition of vaccine

Vaccine refers to a vaccine-based preventive biological product for the prevention and control of the occurrence and prevalence of infectious diseases. The vaccine is also a virus or a bacterium that artificially reduces it, inactivates it, or breaks it. Through the treatment, the antigenic components must be retained. After injection into the human body, the human immune system recognizes, clears and produces antibodies. When the outside world is actually the original bacteria or virus, our body will remove it.

How is the vaccine developed?

The development of the vaccine can be divided into three periods, the first is the classical vaccine period, that is, the period of vaccine production based on repeated observation and exploration of the experience before the pathogen is discovered. The second is the traditional vaccine period, that is, the inactivated vaccine and the attenuated vaccine are prepared by using the diseased tissue, the chicken embryo or the cell proliferating virus; the inactivated vaccine and the attenuated vaccine are prepared by culturing the intact bacteria with the medium. The third is the engineering vaccine period, which uses DNA recombination technology to produce vaccines.

The development of vaccines dates back to ancient China. In China in the early 18th century, pus was inoculated to patients with smallpox - an ingenious method of inoculation of human pox to prevent smallpox. Although this kind of medical treatment for vaccinating human vaccination is not dangerous without any treatment, this method has pioneered the use of vaccines to prevent high-risk infectious diseases. In 1921, the vaccination method was introduced to the United Kingdom. British doctor Jenner also found that cows and cows vaccinated with vaccinia cattle did not suffer from smallpox. In 1976, Jenner took cytoplasm from a suckling woman infected with a milkmaid. Inoculated on the arm of an 8-year-old boy and then allowed to produce a smallpox pus, and the boy did not infect the smallpox, which proved that it was indeed immune to smallpox. This is also the first scientific experiment in which humans control infectious diseases through conscious vaccination. Vaccine, which means vaccines, vaccines, refers to all actively immunized biological products. A vaccine prepared from cattle used at the time of the largest scale - vaccinia vaccine.

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Vaccine development prospects

Vaccination is the most effective and economical means of preventing diseases. It is the only weapon that human beings can predict to eliminate a disease. With the advancement of treatment technology and disease prevention, vaccination has achieved remarkable results and is likely to expand to the goal of cancer prevention. There is also a vaccine that may prevent atherosclerosis. There is a significant potential for development in the vaccine market. Unmet needs remain, as many diseases still have lower immunization rates or no vaccine available. The World Health Organization expects the global market to soar to $10 billion by 2025, and 120 new products will be launched in the next 10 years. Increased awareness of infectious diseases: Governments of all countries are major customers, playing an infinite role in purchasing and enforcing safety regulations and affecting the spread of vaccines.

The global outbreak of vaccine-preventable diseases has boosted public awareness. The seasonal flu epidemic has caused thousands of deaths, placing a heavy burden on national health spending. The increase in consciousness prompted the government to adopt various programs to prevent the outbreak of future outbreaks. These programs have funded the popular immunization program, which has been instrumental in increasing vaccine use. The next generation of vaccine development depends on a platform strategy based on genomics, reverse vaccinology, high-throughput DNA sequencing, novel plant and insect-based expression and production systems, and new and more effective vaccine adjuvants. These developments are likely to rapidly produce new, optimal, cost-effective vaccine targets that have a greater chance of success in clinical development programs. New candidate vaccines with room for development (eg, meningococcal beta, GBS, methicillin-resistant Staphylococcus aureus, pneumococcal, and pathogenic E. coli) are already under development. These new platforms not only enhance the future of major infectious disease vaccines (eg, AIDS, tuberculosis, dengue and malaria), but also lay the foundation for vaccine development for the underlying treatment of emerging diseases.

 

Vaccine application:

With the advancement of molecular biology theory and technology, the theoretical basis and technical level of vaccine development have been continuously improved and improved. Some traditional classic vaccine varieties have been further transformed into new vaccines, while other vaccines that cannot be developed using classical techniques have been found. The way to solve the problem. Therefore, new vaccines such as subunit vaccines, recombinant vaccines, and nucleic acid vaccines for different infectious diseases and non-communicable diseases are constantly being introduced. Such as DNA vaccine, RNA vaccine, rBCG vaccine, Glycoconjugate Vaccines and Polysaccharide Vaccines.

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Generally, a cyclic peptide referred to in biology refers to a compound formed by amino acid peptide bonds. In phytochemistry, this concept is expanded to a class of compounds formed by amide bonds, and thus the range is expanded to include organic amines and macrocycles. Alkaloids are classified into cyclic peptides and linear peptides depending on whether they are cyclized or not. Cyclic peptide compounds have been reported to have a variety of biological activities, including anti-tumor, anti-HIV, antibacterial, anti-malarial, hypnosis, inhibition of platelet aggregation, hypotension, inhibition of tyrosinase, inhibition of cyclooxygenase, inhibition of lipids Biological activities such as peroxidase, estrogen-like, and immunosuppression.

 

Jame P. Tam et al. established a method for the preparation of non-protected cyclic peptides by intramolecular transfer of thiolactone and Ag+ ion assisted cyclization. For a linear polypeptide with a cysteine at the N-terminus and a thioester at the C-terminus, in a phosphate buffer at pH=7, a thiol group forms a covalent thiolactone with a thioester group. This thiolactone spontaneously passes through S. The atom to the N atom acyl group migrates to form a cyclic peptide.

 

The authors synthesized a series of Cyclo (Cys-Tyr-Gly-Xaa-Yaa-Leu) with N-terminal cysteine by the above method. In order to prevent the formation of disulfide bridge and accelerate the cyclization reaction, the reaction process was added. TCEP (tricarboxyethylphosphine), the reaction time is about 4 hours, the yield is between 78% and 92%, and no side reactions and oligomers are found by HPLC.
For the cyclization of a cysteine-free linear polypeptide, the N-terminal amino group of the linear polypeptide with a sulfophilic Ag+ ion-assisted coordination flexibility forms a cyclic intermediate with the C-terminal thioester, and promotes intramolecular activation by entropy activation. Loop. Similar to the principle of thiolactone cyclization, Ag+ ions promote intramolecular cyclization through a non-classical ring-chain structure.
A specific example of the synthesis of a cyclic peptide by the above method is the synthesis of Cyclo (Ala-Lys-Try-Gly-Gly-Phe-Leu). To the acetic acid buffer solution of pH 5.7, 10% DMSO was added as a co-solvent, and after 5 hours of reaction, a target of 67% yield was obtained.

 

Synthesis of cyclic dipeptides
The cyclic dipeptide (2,5-piperazinedione) is the smallest cyclic peptide, and many natural cyclic dipeptide compounds have clear biological activities, such as antibiotics, bittering agents, plant growth inhibitors, and hormone releasing inhibitors. The particularity of the structure of the cyclic dipeptide allows the synthesis of such compounds to be self-contained, and the target substance can be easily obtained by refluxing a linear peptide ester which is free at the N-terminus in a polar solvent. Although Fischer obtained a cyclic dipeptide by aminolysis of linear dipeptide methyl ester in methanol ammonia, it was found that this method easily caused racemization. Nitecki proposes that the N-terminal free linear dipeptide methyl ester can be refluxed to synthesize a cyclic dipeptide in a mixed solvent of butanol and toluene without causing racemization. Ueda uses methanol as a solvent for reflux, and a good yield of cyclic dipeptide is also obtained. Cook et al. used 1,2-ethanediol as a reaction solvent to obtain two diastereomeric cyclic dipeptides. The yield was 64.5% [95]. Recently, Wang Youchu reported that a series of cyclic dipeptides were synthesized with reference to Ueda and Cook. The yield ranged from 55% to 99%, and Cyclo (Phe-Pro), Cyclo (Ile-Ile) was discovered by biological activity experiments. And Cyclo (Met-Met) has a slight calcium antagonistic effect, and Cyclo (Ala-Ala) and Cyclo (Pro-Pro) show enhanced potassium-induced contraction effects.

 

Sum up
The method of synthesizing the end-to-end cyclic peptides to date has been described above. Since the number and type of amino acids contained in the precursor-linear peptide of the cyclic peptide vary widely, the method of synthesizing the cyclic peptide is diversified. Agents and methods that exhibit efficient, rapid condensation of certain linear peptides may become inefficient or ineffective for another peptide chain. Therefore, finding a corresponding cyclic peptide synthesis method based on the sequence of the target cyclic peptide must be carefully explored and worked hard.

 

 

About us

Creative Peptides is specialized in the process development and the manufacturing of bioactive peptides. We are dedicated to offering custom peptide synthesis, process development, GMP manufacturing as well as catalog products for customers in industry and research area. Here are some our products like: Peptide ModificationDe novo Peptide DesignLong Peptides SynthesisPeptide Drug Discovery, etc.

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What is CD molecule

CD molecule is the surface marker of white blood cells (including platelets and vascular endothelial cells, etc.) which appears or disappears in different lineages and stages of normal differentiation and maturation as well as during activation. Most of them are membrane-penetrating proteins or glycoproteins, including extracellular, membrane-penetrating and intracellular areas. A few CD molecules are carbohydrate hapten. Many CD molecules are functional molecules, some are enzymes, some are receptors, and some are signal transducers, ion channels or regulatory molecules.

The role of CD Molecule

CD molecule participates in some important physiological and pathological processes, such as: (1) mutual recognition of immune cells during immune response, recognition, activation, proliferation and differentiation of immune cell antigens, and exertion of immune function; (2) regulation of hematopoietic differentiation and hematopoietic process; (3) inflammation; and (4) migration of cells, such as metastasis of cancer cells.

CD antigen and its corresponding monoclonal antibodies have been widely used in basic and clinical immunology research.

In basic immunology, CD is mainly applied to:

(1) gene cloning of CD antigen, discovery of new CD antigen and new ligand; (2) relationship between structure and function of CD antigen; (3) transmission of cell activation pathway and membrane signal; (4) regulation of cell differentiation; and (5) function of cell subsets.

In clinical immunology research, CD monoclonal antibody can be used for: (1) detection of immune function of organism; (2) immunotyping of leukemia and lymphoma; (3) immunotoxin for cancer treatment, bone marrow transplantation and transplantation rejection prevention; and (4) immunomodulation therapy in vivo.

Some important CD molecules

  1. CD Molecules Related to Antigen Presentation

The CD molecule involved in antigen presentation mainly includes CD molecule constituting immunoglobulin Fc receptor and complement receptor and CD1. CD1 is the first human leukocyte surface differentiation antigen molecule detected by monoclonal antibody. CD1 is type I transmembrane glycoprotein. Its spatial structure is similar to that of MHC class I molecule and is linked to beta 2m, but its amino acid sequence is very different. According to the different amino acid sequences and glycosylation sites, CD1 molecules can be divided into CD1 (a, b, c) (category I) and CD1d (category II). CD1 molecule is mainly expressed on the surface of full-time APC, including most B cells, activated monocytes, Langerhans cells and dendritic cells. CD1 molecule is also expressed on immature double-negative thymocytes and CD8 T cells in the thymic cortex. CD1 molecule can present non-peptide antigens.

  1. The major CD molecules involved in T cell antigen recognition and activation include

TCR-CD3 complex: TCR binds to CD3 molecules on cell surface to form TCR-CD3 complex. TCR recognizes specific antigen peptide-MHC molecular complex. CD3 molecules transmit signals and promote T cell activation.

CD4: CD4 belongs to Ig superfamily and is a single-chain glycoprotein on the surface of cell membrane. Human CD4 molecule consists of 458 amino acid residues, including signal peptide 23 amino acid residues, extracellular domain 374 amino acid residues, transmembrane domain 21 amino acid residues, and intracytoplasmic domain 40 amino acid residues. There are four IgV-like functional areas in the extracellular area, including two glycosylation sites. CD4 is expressed on the surface of some T lymphocytes and thymocytes as well as some B lymphocytes, EBV-transformed B cells, mononuclear phagocytes and brain cells. The first and second functional regions of CD4 bind to the non-polymorphic parts of MHCII molecules, which can stabilize the interaction between MHCII-restricted T cells and APC cells with MHCII-antigen peptide complex. Therefore, CD4 molecules are also known as co-receptors involved in T cell activation. CD4 is also the main receptor of human immunodeficiency virus (HIV). The cytoplasmic region of CD4 molecule is linked to protein tyrosine kinase p56, which plays an important role in T cell signal transduction.

CD8: CD8 molecule is a membrane-penetrating glycoprotein composed of a and beta polypeptide chains. The molecular weight of a chain is 34 kDa and that of beta chain is 30 kDa. Each chain consists of an IgV-like functional area, a ligand, a membrane-penetrating area and a cytoplasmic area. CD8 molecules are distributed in some T lymphocytes and thymocytes.

The biological functions of CD8 molecule include: (1) as cell-cell adhesion molecule: MHCI class antigen is the ligand of CD8 molecule. CD8 binding with MHC class I can stabilize the binding of MHC class I restricted T cells (mainly CTL) to target cells with MHC class I molecule-antigen peptide complex. Therefore, CD8 is also known as a co-receptor involved in CTL cell activation. (2) After TCR binds to ligands, the intracellular domain of CD8 molecule is rapidly phosphorylated, leading to the activation of protein tyrosine kinase p56lck linked to CD8, which plays an important role in signal transduction of T cell proliferation and differentiation.

Some drugs about CD molecule

  1. Clenoliximab

Recombinant chimeric (primate/human) antibody expressed in CHO binding to human CD4/p55. Clenoliximab is a monoclonal antibody against CD4. It acts as an immunomodulator and has been investigated for the treatment of rheumatoid arthritis. The drug is a chimeric antibody from Macaca irus and Homo sapiens.

  1. Tregalizumab

Recombinant monoclonal antibody to CD4. Tregalizumab is an immunomodulator. It binds to CD4.

  1. CD38 Daratumumab

Recombinant monoclonal antibody to CD38. Daratumumab is an investigational anti-cancer drug. It binds to CD38.

  1. Keliximab

Recombinant monoclonal antibody to CD4. Keliximab is a monoclonal antibody for the treatment of severe chronic asthma. It suppresses the immune reaction by binding to white blood cells via the protein CD4. The drug is a chimeric antibody from Macaca irus and Homo sapiens.   

  1. Zanolimumab

Recombinant monoclonal antibody to CD4. Zanolimumab (expected trade name HuMax-CD4) is a human monoclonal antibody and an immunosuppressive drug. It is being developed for the treatment of rheumatoid arthritis, psoriasis, melanoma, cutaneous and peripheral T-cell lymphoma.

As the core component of hematopoietic immune tissue, human white blood cells have important biological functions. The study of differentiation antigens is not only helpful to understand the molecular structure, function, marker for cell subsets and differentiation stages, but also helpful to elucidate the differentiation, maturation, function and immunoregulation mechanism of immune cells. Therefore, it can deepen the understanding of immune response and immune diseases, and provide conditions for exploring new ways of clinical immunodiagnosis and treatment.    

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Bromelain is a pure natural plant protease obtained by extracting pineapple fruit stems, leaves and skins, purifying, purifying, concentrating, enzymatically immobilizing and freeze-drying. Its appearance is light gray powder, with a molecular weight of 33,000 and an isoelectric point of 9.55. The best quality bromelain is obtained by processing the middle stem of pineapple, filtering and concentrating by ultrafiltration method, and freezing and drying at low temperature. Products have been widely used in food, medicine and other industries.

 

Application
Application of bromelain in food processing industry
1. Baked food: Bromelain is added to the dough to degrade the gluten, and the dough is softened and easy to process. It can improve the taste and quality of biscuits and bread.
2. Cheese: used for the coagulation of casein.
3. Tenderization of meat: Bromelain hydrolyzes macromolecular proteins of meat proteins into easily absorbed small molecule amino acids and proteins. Can be widely used in the finishing of meat products.
4. Bromelain has been used in other food processing industries. Bromelain has been used to increase the PDI and NSI values of bean cake and soy flour to produce soluble protein products and breakfast, cereals and beverages containing soy flour. Others produce dehydrated beans, baby food and margarine; clarify apple juice; make soft candy; provide digestible food for patients; add flavor to everyday foods.

 

Application of bromelain in medicine and health care products
1. Inhibit the growth of tumor cells
According to relevant clinical research observations, bromelain can inhibit the growth of tumor cells.
2. Prevention and treatment of cardiovascular diseases
Bromelain is beneficial as a proteolytic enzyme for the prevention and treatment of cardiovascular diseases. It inhibits heart attacks and strokes caused by platelet aggregation, relieves symptoms of angina, relieves arterial contractions, and accelerates the breakdown of fibrinogen.
3. For burn dislocation
Bromelain selectively removes skin and allows new skin grafts to be performed as early as possible. Animal experiments have shown that bromelain has no adverse effects on adjacent normal skin. Topical use of antibiotics does not affect the effect of bromelain.
4. Anti-inflammatory effect
Bromelain is effective in treating inflammation and edema (including thrombophlebitis, skeletal muscle damage, hematoma, stomatitis, diabetic ulcers, and sports injuries) in various tissues. Bromelain has the potential to activate inflammatory responses. Bromelain can also treat diarrhea.
5. Improve drug absorption
Combining bromelain with various antibiotics (such as tetracycline, amoxicillin, etc.) can improve its efficacy. Related studies have shown that it can promote the transmission of antibiotics at the site of infection, thereby reducing the amount of antibiotics used. It is inferred that there is a similar effect on anticancer drugs. In addition, bromelain promotes the absorption of nutrients.

 

Application of bromelain in beauty and cosmetics industry
Bromelain has excellent effects on skin rejuvenation and whitening.
Basic principle: Bromelain acts on the aged stratum corneum on human skin, causing it to degenerate, decomposition, removal, promote skin metabolism, reduce skin color darkness caused by sun exposure. Make skin care a good white and tender state.

 

Application of bromelain preparation in feed
Adding bromelain to the feed formulation or directly mixing it in the feed can greatly increase protein utilization and conversion, and can develop a wider protein source, thereby reducing feed costs.
Action conditions:
Optimum temperature: 53±1°C
Deactivation temperature: ≥65°C
The best PH value: 5.0 ~ 8.0
Inactivated pH: less than 3.0 and large with 9.5
Recommended dosage: 0.05 to 0.1% for protein meter
Temperature range: 50 ~ 60 ° C
Function PH value: 6.0~9.0
package:
There are two types of packaging:
1) Aluminum foil bag packaging, net weight 1 kg / bag. 20 kg / carton.
2) Cardboard drum packaging, lined with transparent plastic bag, net weight 25 kg / barrel.
Storage and storage period:
It is recommended to store in a cool and dry place, the relative humidity is less than 60%, the temperature is lower than 25 °C; the storage period is 12 months, the storage period is 5 months at 5~15 °C in cool, low temperature and dry environment. It is best to keep away from odorous substances.

 

About us

Our products are used worldwide in academic, commercial, and government laboratories in diverse applications, including catalase, diamine oxidase, chitinase, and collagen. As a reliable supplier, Creative Enzymes supplies the products of high quality and competitive cost performance. We cooperate with a large number of satisfied customers in corresponding fields all over the world.

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The main biological function of GC is to combine and transport vitamin D and its metabolites. Each GC molecule contains a VD enzyme binding site, and normally only 1% to 2% of the sites participate in VD metabolism. GC participates in the transport of VD and its metabolites between blood and cell membranes, so most of the plasma is free 6C. GC can bind to actin in vivo or in vitro to form a 1:1 complex, which greatly increases the molecular weight and increases the electrophoretic migration string. The isoelectric point of the Gc actin complex is lower than that of free GC. Gc binding to actin facilitates the clearance of actin from tissues.

 

Vitamin D binding protein (also known as Gc protein), is an alpha globulin, a group of multi-gene super-including protein (ALB), prealbumin (PA), alpha-fetoprotein (AFP). The family, most of which is secreted by the liver parenchymal cells, has a relative molecular mass of about 55,000. It is abundant in serum and has many physiological functions. It not only binds vitamin D and clears actin, but also enhances C5 to neutral particles. The chemotactic activity of inflammatory cells such as cells, and activation of macrophages, regulation of osteoclast activity and transport of fatty acids and endotoxins, may also be involved in viral infection.

 

[What is the function of vitamin D binding protein?] 1 Structure of vitamin D receptor VDR is a member of the nuclear receptor superfamily such as thyroxine and corticosteroids. VDR has a large similarity in sequence and structure to subfamilies including retinal, thyroid hormone, and peroxisome proliferator-activated receptors. The study confirmed that the human VDR gene is located on chromosome 12, consisting of 11 exons and several introns, about 75 kb in length, and the protein consists of 427 amino acids. From the amino end to the carboxy end, it can be generally divided into six functional regions: A, B, C, D, E, and F.

 

The AB region is a ligand-independent, tissue-specific transcriptional activation self-regulating functional region AF1. The human VDR AB region consists of approximately 24 amino acid residues. No features were found in this domain. Region C: DNA binding domain DBD, involved in DNA sequence recognition, and is also partially involved in the formation of dimer interfaces. This region is highly conserved and the homology of human, rat and chicken VDR DBD is 98.15%. Zone E: This region is relatively large and functional: participating in binding ligands, thus referred to as the ligand binding domain LBD; forming a dimer with RXR; forming a ligand-dependent transcriptional activation or inhibition functional region AF2. In addition, the E region also has a synergistic effect on DNA recognition.

 

Zone D and Zone F: The functions of these two zones are unknown. Zone D may be a hinge region that primarily regulates the flexibility of the receptor and may be related to nuclear localization. 2 VDR regulates the calcium-binding protein gene VDR regulates many genes, most of which are genes in bone metabolism. Here we use the calcium-binding protein gene as an example to introduce the regulation of VDR genes. Calcium is second only to oxygen, carbon, hydrogen and nitrogen in the body, accounting for about 2% of the human body. Calcium absorption is a complex physiological and biochemical process, including active transport and passive diffusion. Active transport is the main factor when the body consumes less calcium. Active transport of calcium requires the assistance of vitamin D and calcium binding proteins.

 

Wasserman et al. first confirmed the formation of specific calcium-binding protein (CaBP) after treatment of rickets with vitamin D. This vitamin D-induced CaBP has been found in more than a dozen animals and is species-specific. The molecular weight of human intestinal calcium CaBP is 12000-21000, which has a high affinity with calcium, and the ability to bind calcium is about 2×10-5M-1. CaBP is highly concentrated on the absorption cells of the goblet cells and the brush border of the small intestine.
In different parts of the intestine, intestinal CaBP level and calcium absorption rate are consistent, with the highest in the duodenum, the second in the jejunum, and the lowest in the ileum. In addition, CaBP is also present in tissues such as the kidney, hypothalamus, cerebral cortex, adrenal gland, and parathyroid glands. CaBP plays an important role in the process of vitamin D-mediated intestinal calcium transport. Mammals can synthesize two CaBPs, namely CaBPD28K present in the kidney and central nervous system and CaBPD9K in the small intestine. Injection of 125OH2D3 into vitamin D-deficient rats and chicks resulted in rapid synthesis of CaBP mRNA and accumulation of CaBPD9K mRNA in intestinal mucosa and kidney cells.

 

 

About us

We provide a wide range of high quality normal human and animal cells, cell culture medium and reagents, FISH probes, tissue arrays, microorganisms and equipment. In addition, we also offer series of related services including cell services, biosample services and histology services for the researcher to make their project better and faster. Here are some products: FSTL5FTCDFTLFTO, etc.

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Chronic prostatitis includes chronic bacterial prostatitis and non-bacterial prostatitis. Among them, chronic bacterial prostatitis is mainly pathogen infection, mainly retrograde infection, the pathogen is mainly Staphylococcus, often repeated urinary tract infection attack history or prostate massage fluid in the persistent presence of pathogenic bacteria. Non-bacterial prostatitis is a complicated pathological change caused by many complicated causes and inducements, including inflammation, immunity and neuroendocrine. The main clinical manifestation is urethral irritation and chronic pelvic pain. And often associated with psychological symptoms of the disease, clinical manifestations are diverse. And the name of chronic prostatitis belongs to the old classfication system in which prostatitis is divided into acute bacterial prostatitis, chronic nonbacterial prostatiti and prostatodynia.

How much do you know chronic bacterial prostatitis?

The pathogenic factor of chronic bacterial prostatitis is mainly due to pathogen infection. When the body resistance is stronger or the pathogen virulence is weak, retrograde infection is the main form of infection. The main pathogens were Staphylococcus, followed by Escherichia coli, Corynebacterium and Enterococcus. Prostatic calculi and urinary reflux may be important causes of persistent pathogens and recurrence of infection.

How much do you know chronic non-bacterial prostatitis?

The nosogenesis of chronic non-bacterial prostatitis is still unkown. The nosogenesis of chronic non-bacterial prostatitis is very complicated with widespread controversy. Many hold a view that the reason that it may be caused by an initiator or may be multifactorial in the first place or more of which play a key role and interact with each other. Also, it may be many different diseases that are difficult to distinguish but with identical or similar clinical manifestations. What’s more, these diseases have been cured and the damage it causes continues to work independently with pathological changes. Most scholars believe that the main cause of the disease may be pathogen infection, inflammation and abnormal pelvic floor neuromuscular activity and immune abnormalities.

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How manyoncogenesis do you know?pathogen infection

 Although the pathogen can not be isolated by routine bacterial examination, it may still be related to some special pathogens, such as anaerobes, L-Proteus, nanobacteria, chlamydia trachomatis, mycoplasma and so on. Some studies have shown that the detection rate of local prokaryote DNA in this type of patients can be as high as 77%, and some clinical cases of "aseptic" prostatitis, which are mainly chronic inflammation and repeated attacks or aggravation, may be related to these pathogens. Other pathogens, such as parasites, fungi, viruses, trichomonas and Mycobacterium tuberculosis, may also be important pathogenic factors, but there is no reliable evidence, so far there is no unified opinion.

Urinary dysfunction

Some factors cause excessive contraction of the urethral sphincter, resulting in bladder outlet obstruction and residual urine formation, resulting in urine reverse flow into the prostate. Not only can the pathogen go into the prostate, but also directly stimulate the prostate to induce aseptic "chemical prostatitis", causing abnormal urination and pelvic pain.

Many prostatitis patients have a variety of urodynamic changes, such as decreased urinary flow rate, functional urinary tract obstruction, detrusor-urethral sphincter synergy, and so on. These abnormalities may only be a clinical phenomenon, and their nature may be related to underlying pathogenic factors.

Psychological factors 

More than half of the patients with chronic prostatitis have obvious psycho-psychological factors and personality changes, for example, anxiety, depression, hypochondria, hysteria, even suicidal tendency. The changes of these mental and psychological factors can cause autonomic nerve dysfunction to cause posterior urethral neuromuscular dysfunction, and lead to pelvic area pain and urination dysfunction or hypothalamus-pituitary-gonadal axis changes and affect sexual function. The further aggravate symptoms eliminate mental tension can make symptoms relief or recovery. However, it is not clear whether psycho-psychological change is its direct cause or secondary manifestation.

Neuroendocrine factors

Patients with prostate pain are prone to fluctuations in heart rate and blood pressure, suggesting that autonomic nervous responses may be involved. The pain is characterized by visceral organ pain, local pathological stimulation of the prostate and urethra, triggering of spinal cord reflex through the afferent nerve of the prostate, activation of astrocytes in the lumbar and sacral spinal cord. Nerve impulses pass through the reproductive femoral nerve and ilioinguinal nerve efferent impulses. The sympathetic nerve endings release norepinephrine, prostaglandin, calcitonin gene-related peptide, substance P and so on, which cause bladder urethral dysfunction and lead to perineum. Abnormal pelvic floor muscle activity, persistent pain and traction in the corresponding area outside the prostate A wrench.

Immune response abnormality

Recent studies have shown that immune factors play a very important role in the development and progression of III prostatitis. Some cytokines may change in prostatic fluid, seminal plasma, tissue fluid or blood of the patient, such as IL- 12 FamilyIL- 7 Family, IL-8 Family, IL-10 Family, TNF- α and MCP-1. The level of IL-10 was positively correlated with the pain symptoms of patients with III prostatitis, and immunosuppressive therapy was effective.

Oxidative stress theory

Under normal conditions, the production, utilization and removal of oxygen free radicals are in dynamic equilibrium. The excessive production of oxygen free radicals and the decrease of scavenging effect of free radicals in patients with prostatitis may be one of the pathogenetic mechanisms, which can decrease the response ability of the body to antioxidant stress and increase the production and by-products of oxidative stress.

Pelvic disease factors

Some patients with prostatitis are often associated with prostatic peripheral venous plexus dilatation, hemorrhoids, varicocele and so on, suggesting that the symptoms of some patients with chronic prostatitis may be associated with pelvic venous congestion and blood stagnation. This may also be one of the reasons for the persistence of governance.

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Bispecific antibody is known as bsAb for short. The branch of technology in antibody drugs and bispecific antibody drugs over the past few years have been absolutely the hot spot. Anyone who makes drugs feels like it's hard to say not knowing this technology. Pharmaceutical companies feel like they might be inferior if they don't have a layout in this direction, and they're likely to miss out on big opportunities in the future. Is that the technology really important? Since the technical analysis is very professional, let me talk about it from the market.

The two bispecific antibodies which is currently available are Removab and Blincyto. Let's take a look at two stories about them.

Do you know First-in-Class is with complicated background and awkward status?

Removab, the first therapeutic antibody drug in the field of bispecific antibodies who was approved by the European Union on April 20, 2009, is certain to be First-in-Class consisting by antibody-peptide. Its original patent was granted in 1998 by Ascension GmbH, a smaller German biotechnology company. Shortly, after the deal was completed, TRION entered into an alliance with Fessenius, an established German company, and decided to jointly develop the later approved listing Removab. Ascension GmbH later sold all of its TRION holdings to Fessenius, a biotech subsidiary focused on developing biopharmaceuticals, which was later sold to Fesenius by Ascension GmbH, a subsidiary that specialized in the development of biological drugs. In 2011, Swedish Orphan Biovitum (Sobi) struck a marketing deal with Fresenius Biotech on which Removab was successfully approved for listing. Removab was approved for sale by EMA in June 2013. Also, in this month, Fessenius announced the sale of Fresenius Biotech would become as a whole to the Further family, the owner of Neopharm (the amount of the deal was not disclosed by the parties to the agreement). Fresenius Biotech GmbH was logically renamed Neovii Biotech GmbH.

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The indication for Removab approved in 2009 in the European Union is malignant ascites. If there is also a therapeutic effect on primary tumors, the market is worth imagining. But the market reaction was completely different. In 2009, the product was sold at $1.66 million, and in 2010-2012 it was 3.32 / 4.43 / 4.54 million dollars, respectively. The annual sales of the product have not been found in mainstream medical databases since 2013, followed by ovarian cancer, stomach cancer, non-small cell lung cancer, breast cancer, and other indications. In the II phase, dismal sales figures offer no hope for these new indications.

But Removab, has never been born. Its biological parents, Ascension GmnH, had no attachment to it from the start. Its two stepfathers, TRION and Fresenius, were just passing by in the process of his growth. And Neovii turned a blind eye to him in the end.

How is the market condition of Blincyto?

Let's take a look at the Blincyto of Amgen approved by FDA and EMA in December 2014, which is a double-specific antibody to CD3 on one end of CD19 that binds to the surface of white blood cell B cells, and on the other end of the surface of T cells. The basic idea is consistent with that of another popular cellular immunotherapy now. Its first indication is the second-line treatment of acute lymphoblastic leukemia.

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Blincyto has another American biology company, Micromet Inc. At the time, it was named MT103. In June 2003, Micromet signed an agreement with MedImmune to develop MT103. As everyone knows, in March 2009, after AstraZeneca acquired MedImmune2 in June 2007, Micromet bought back an interest in the original MedImmune agreement from AstraZeneca, so Micromet regained ownership. In January 2012, Amgen became Amgen's AMG103 by buying Micromet’s MT103 in full cash for $11 a share. Later, in May 2013, Amgen licensed the Japanese interest in the product to Astellas. In August 2013, it licensed its Indian interest to Dr. Reddy's in India.

Blincyto's U.S. sales were about $50 million in 2015, compared with $85 million in 2016. At the same time, Amgen is also carrying out clinical research on new indications such as diffuse large B-cell lymphoma and mantle cell lymphoma, which is expected to be further expanded. Some analysts believe the drug could reach annual sales of $178 million in 2017.

Amgen spent $1.16 billion in 2012 and certainly there would not spend small amount money on research and drug developing after the acquisition. Amgen's global sales figures for 2016 were $225 billion to $228 billion. However, Amgen will no doubt be a bit disappointed by the result of Blincyto alone in 2016.

Why the prospect of a variety on the market is not bright?

As for the next bispecific antibody drug most likely show in the market, Roche's ACE-910 (RG6013 Emicizumab) is considered to have no bright future. The drug is originally developed by Japanese and foreign pharmaceutical companies and they reached a global cooperative development agreement with Roche in August 2014.

Although severe adverse events with thromboembolism / thrombotic microvascular disease (Thrombotic microangiopathy, TMA) were reported in four participants in November 2016, the possibility that ACE-910 would eventually be approved for listing is high.

The indication for ACE910 is type A hemophilia with VIII inhibitors. We all know that hemophilia is a rare disease and the global market for hemophilia is estimated to be around $10 billion. But this huge market has been crowded into a number of heavyweights, including Shire,Baxalta, Northrop, Bayer, Baijian and CSL, of which Type A hemophilic drugs (such as Feiba,Advate,Adynovate) sold $2.84 billion in 2015. We can see that this market is large.

ACE-910 expects to hit the market around the end of 2019, which means we don't see a double antibody drug with annual sales of more than $500m over the next five years.

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Bispecifics, how many people are obsessed with you? How many people are manic for you? However, to me, it is really hard to say I love you.

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Abstract: In the long process of antibody research, three generations of different levels of antibody preparation techniques have been developed. A polyclonal antibody prepared by antigen-immunizing higher vertebrates is called a first-generation antibody; a monoclonal antibody produced by hybridoma technology only for a specific antigenic determinant is called a second-generation antibody; and recombinant DNA is applied; The technique or the method of gene mutation modifies the coding sequence of an antibody gene to produce an antibody protein molecule originally existing in nature called a genetically engineered antibody, that is, a third generation antibody. This genetic engineering is called antibody engineering. Human hybridoma cells have been tried in the early days to produce human monoclonal antibodies, but this technique has rarely been used due to the instability of human hybridoma cells, the low affinity of human monoclonal antibodies, and ethical controversy. In the mid-1980s, new technologies were introduced to combine the genetic structure and function of immunoglobulins with DNA recombination technology, and then the recombinant immunoglobulin genes were introduced into cells for expression. In the third generation of antibodies, mainly including humanized antibodies, small molecule antibodies, antibody fusion proteins and some specific types of antibodies, to some extent overcome the shortcomings of the first two generations of antibody technology. In addition, the construction of a phage antibody library, a ribosome display library, and the like allows specific antibodies to be obtained without antigen immunization. Recombinant antibody technology makes it possible to prepare humanized antibodies and human antibodies, which is not possible with other conventional polyclonal or monoclonal antibody preparation methods.

Keywords: recombinant antibody, monoclonal antibody, phage antibody technology

Chimeric antibody

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One method of reducing the immunogenicity of a murine monoclonal antibody is to link the variable region of the murine immunoglobulin to the constant region of the corresponding human immunoglobulin, thus producing a murine-human chimeric antibody, human region in 60%~70%. Using the recombinant DNA technology, the variable region gene of the murine monoclonal antibody is ligated to the human constant region gene, and the constructed chimeric gene is inserted into an appropriate expression plasmid, and then transfected into the corresponding cells for expression. The chimeric antibody produced has the function of binding antigen, and at the same time reduces the heterogeneity of the murine monoclonal antibody, such chimeric antibody is not much different in affinity from the corresponding murine monoclonal antibody, and humans have it The immune response will be reduced. However, since it still retains the heterogeneity of the murine immunoglobulin variable region, clinical experiments have shown that the chimeric antibody also produces an immunological response to the variable region of human anti-chimeric antibody (HACA) when applied. 

Small molecule antibody

In order to break through the limitations imposed by the oversize of monoclonal antibodies, techniques for the development of small molecule antibodies have been developed. The goal of these techniques is to obtain antigen-binding fragments (Fabs) of antibodies and variable regions (Fv) of antibodies. Such a fragment can be obtained by cleavage of an antibody, or by amplifying a gene associated with an immunoglobulin and cloning and expressing it in bacteria. Small molecule antibodies have small molecular weight, strong penetrability, low immunogenicity, and short half-life. At present, there are several studies and more practical prospects.

 Single chain antibody

The technology links a heavy chain variable region and a light chain variable region with a ligation peptide, and is expressed as a single-stranded polypeptide by a prokaryotic expression system and folded into a novel antibody consisting of a heavy chain and a light chain variable region.

 Multivalent antibody

The multiple antigen binding sites of the antibody have different specificities and are capable of binding different antigen molecules, which changes the deficiency that the traditional antibody can only bind to a single antigen molecule. At present, researchers are paying more attention to the application of bispecific monoclonal antibodies.

 Fab fragment

In the antibody, the Fab segment is mainly used to combine the role of the antigen. 

 

Phage antibody technology

In the early 1990s, based on the PCR technology, the expression of antibody Fab fragments in E. coli and the rapid development of phage display technology, antibody library technology based on molecular biological methods appeared. Among them, the phage antibody library technology is superior.

The basic procedure of phage antibody technology is to amplify a full set of antibody variable region genes or antibody fragments (Fab, Fv or seFv) genes by PCR and transfer them to the vector after phage capsid protein gene, which can be filamentous. The coat protein of the phage forms a fusion protein, and the antibody Fab fragment or single-chain antibody is expressed on the surface of the phage, and then the specific antibody and the gene encoding the same are obtained from a plurality of antibodies by affinity screening of the antigen. A phage population cloned and assembled using a full complement of antibody variable region genes of B lymphocytes is referred to as a phage antibody library. 

 

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The technology is simple to operate, does not require cell hybridization or complex PCR technology, has a short cycle and low cost; can simultaneously screen several antibodies using different antigens, and can be selected in millions to hundreds of millions of molecules; suitable for antibodies, hormones The preparation of proteins such as enzymes, drugs, and random polypeptides is more interesting because the technology can obtain antibodies that cannot be produced by immune animals due to the influence of immune tolerance mechanisms. Phage antibody technology has greatly promoted the development of genetically engineered antibodies, and their production has brought new hopes for the prevention, diagnosis and treatment of human and animal diseases.

References 

[1] Schillberg S, Fischer R, Emans N. Molecular farming of recombinant antibodies in plants [J]. Cell Mol Life Sci, 2003, 60(3): 433-445

[2]Bouquin T, Thomsen M, Nielsen L K, et al. Human anti-rhesus D IgGI antibody probuced in transgenic plants[J]. Transgenic Res, 2002, 11(2):15-22

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While buying a good quality pair of shoes may seem like something pretty obvious, it's not as easy as it sounds when you actually get into the store. Sure, style and looks have something to do with the right shoes, but there are a lot of other factors that need your consideration when you want to take care of your tootsies.

Before you make any important shoe decisions, take a moment to think about your feet. The comfort of your feet has a tremendous impact on your overall comfort. Just think about it - sore feet, cold feet, hot feet, itchy feet - all of these things are enough to drive you to distraction. If your feet are uncomfortable, the rest of you is uncomfortable. Keep this in mind the next time you walk into a shoe store and look at those brand-name, yet uncomfortable shoes.

You need to treat your feet right. This means that you not only have to buy a pair of shoes that look good, but you also need to find ones that fit your feet. This may sound obvious, but the majority of people wear shoes every day that aren't quite suited to their unique foot shape and size.

So how do you find a pair of shoes that are suited to your unique foot shape and size? It's not as easy as it sounds. Just follow these steps:

1. Have the salesperson measure your feet. He or she should measure both the length and width of each of your feet. Your feet will likely be slightly different in size, and it is important to know this when you're seeking the ideal comfortable shoe.

2. Use the larger foot's size. While you can use an insole to make a shoe that is a bit too big fit your smaller foot, you'll only hurt your larger foot by stuffing it into a shoe that is a bit too small.

3. Make the shoe fit your toes and your heel. There should be enough room to wiggle, but not enough to slip around.

4. Walk about the store to be sure that there isn't any pinching or slipping.

5. Don't assume that the shoe will break in. Have them fit when you buy them.

When it all comes down to it, shoes are your foot's best friend. Don't let your tootsies down!

Article Source: https://www.import-express.com/apa/shoes.html

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Ever wonder why dropship wholesalers for shoes are in much demand in today's market? The road to success for dropshipping shoes only takes sheer determination. Shoes are more than fashion items, it's a necessity. Who would want to go out to a supermarket or a club barefooted? With online selling at its peak, your chances of successfully selling shoes on the Internet is indeed high. However, finding your way to a trusted supplier can be an intimidating process. Here are insider secrets on how you can get your way to trusted dropship wholesalers.

Visit a shoe manufacturer now. If you are lucky enough to be living in the same location of a shoe manufacturing company, you can get first hand information by visiting their office. You may not immediately get a deal, but you can get useful details, such as a list of the manufacturer's distributors and dropship wholesalers. This way you'll be able to get reliable suppliers for your online shoe store.

Visit offices of dropship wholesalers for shoes. Dropship wholesalers do not usually have websites and this is a fact most first time sellers do not know. So, once you get hold of wholesaler info, contact or visit their office immediately. Your competitor might be researching the same shoe wholesaler, so you must find way to that wholesaler your first.

Bring along with you relevant business documents and tax identification. Ask for samples, if available, but if not, you can always purchase a few pair of shoes as samples. While you have presented yourself as a legitimate seller, you also have the right to check the authenticity of the wholesale business by asking for business registration.

Contact dropship wholesale list providers. The world of dropshipping is a secret world. That's why sellers themselves do not usually disclose information about the wholesalers they deal with. But there are those who are in the business of selling wholesaler lists. These people are also dropship sellers themselves. For a fixed amount, you can get access to hundreds of dropship wholesalers for shoes that offer competitive prices, including those coming from China.

Trace the wholesaler by buying a product. You can get a little sneaky by buying a pair shoes from an online seller and inspecting the product details, which will more likely include the wholesaler info. With the wholesaler's name at hand, it will be easier for you to research the contact details of that supplier.

From visiting a shoe manufacturer to tracing the product details, all it takes for you to find trusted wholesalers for shoes is common sense and a few tricks. Remember, your goal is not get to the wholesaler fast. It is about getting a reliable dropshipper. So, if one tip doesn't work, you still have 3 other options towards getting reliable dropship wholesalers for shoes.

Article Source: https://www.import-express.com/apa/shoes.html

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High-quality reference and de novo genomes have been celebrated by geneticists, population biologists and conservationists alike, but it’s been a dream deferred for entomologists and others grappling with limited DNA samples, due to previous relatively high DNA input requirements (~5 μg for standard library protocol).

A new low-input protocol now makes it possible to create high-quality de novo genome assemblies from just 100 ng of starting genomic DNA, without the need for time-consuming inbreeding or pooling strategies. The targeted release date for the protocol is February 2019.

The protocol, developed as a collaboration by scientists at the Wellcome Sanger Institute and PacBio, was used to assemble the genome of an Anopheles coluzzii mosquito with unamplified DNA from a single individual female insect.  

As described in a bioXriv pre-print, Sarah B. Kingan, Haynes Heaton, et al. used a modified SMRTbell library construction protocol without DNA shearing and size selection to facilitate the use of lower input amounts, as shearing and clean up steps typically lead to loss of DNA material.

“This new low-input approach puts PacBio-based assemblies in reach for small and highly heterozygous organisms that comprise much of the diversity of life,” said co-corresponding author Jonas Korlach, our chief scientific officer.

The sample was run on the Sequel System with the latest v6.0 software, followed by de novo genome assembly with FALCON-Unzip, resulting in a highly continuous (contig N50 3.5 Mb) and complete (more than 98% of conserved genes were present and full-length) genome assembly.

About a third of the new de novo genome is haplotype-resolved and represented as two separate sequences for the two alleles, providing additional information about the extent and structure of heterozygosity that was not available in previous assemblies, all of which were constructed from many pooled individuals.

“The ability to generate high-quality genomes from single individuals greatly simplifies the assembly process and interpretation, and will allow far clearer lineage and evolutionary conclusions from the sequencing of members of different populations and species,” the authors state.

The first Anopheles gambiae genome, published in 2002, was created using BACs and Sanger sequencing. Further work over the years to order and orient contigs improved this reference and to date, AgamP4 remains the highest quality Anopheles genome among the 21 that have now been sequenced. However, AgamP4 still has 6,302 gaps of Ns in the primary chromosome scaffolds and a large bin of unplaced contigs known as the “UNKN” (unknown) chromosome.

The Sanger/PacBio single-insect assembly was able to place 667 (>90%) of the genes on the UNKN contigs into their appropriate chromosomal contexts.

The assembly’s “gap-less mega-base scale contiguity” will also provide insights into promoters, enhancers, repeat elements, large-scale structural variation relative to other species, and many other aspects relative to functional and comparative genomics questions, the authors state.  

The protocol’s potential could also extend to other areas with typically low DNA input regimes, such as metagenomic community characterizations of small biofilms, DNA isolated from needle biopsy samples, and minimization of amplification cycles for targeted or single-cell sequencing applications, the authors add.

Read more:https://www.cd-genomics.com/PacBio-SMRT-Sequencing.html

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What does coating nanoparticles mean?

As the name suggests, the process of applying a substance to the surface of a particle to modify it to form a composite particle is called coating of the nanoparticle. It has been found that this process can achieve uniform dispersion between different phase particles, and fully exert the excellent characteristics of different phase particles, and use it as a reinforcing particle. Because aluminum, iron-based, ceramic-based materials can have excellent properties such as high specific strength, wear resistance, small thermal expansion coefficient, and low cost, coating nanoparticles are widely used in aerospace, aviation, automotive, chemical, electronics, etc.

 

A typical representative of coated nanoparticles

Magnetic nanoparticles are nano-sized particles, generally composed of a magnetic core composed of a metal oxide such as iron, cobalt, or nickel, and a high molecular polymer/silicon/hydroxyapatite shell layer wrapped around a magnetic core. The most common core layer is made of Fe3O4 or γ-Fe2O3 with superparamagnetic or ferromagnetic properties. It has magnetic orientation (targeting). Under the action of external magnetic field, it can realize directional movement, convenient positioning and separation from medium. . Among them, the most common one is peg coated iron oxide nanoparticles.

 

 

Several satisfactory research results

Nanomaterials themselves exhibit different properties from conventional materials in terms of optical, thermal, electrical, magnetic, mechanical, physical, and chemical properties due to quantum size effects, surface effects, and macroscopic quantum tunneling effects that conventional materials do not possess. Moreover, the addition of nanoparticles can also improve the performance of traditional organic coatings. This topic has become a hot spot in the field of organic coatings research, and has obtained some satisfactory results:

 

Using the electromagnetic and optical properties of the nanoparticles to produce a multifunctional coating with stealth function;

The use of nanoparticles has a stronger flop effect, resulting in a more decorative coating for luxury car topcoats;

The addition of nanoparticles can also improve the aging resistance and protective properties of the coating;

Utilizing the absorption of ultraviolet rays by titania nanoparticles to form an ultraviolet shielding coating;

In the field of anti-corrosion coatings, in addition to improving the performance of the coating by adding nanoparticles, the nanoparticles can be further modified by the nanoparticles to improve the protection of the material.

 

The process of coating nanoparticles (taking SiC as an example)

Non-uniform nucleation

The principle is to use the coated particles as a nucleation matrix to control the concentration of the coating material between the critical value required for non-uniform nucleation and the critical value required for uniform nucleation, so that the nucleus of the coating material grows on the surface of the coated particles. The key to the process is to control the concentration of the appropriate coating material precipitation reaction and maintain the stable suspension characteristics of the coated particles.

 

 

Problems to be solved

At present, the research and application of SiC surface modification and coating at home and abroad are still in the initial stage. Due to various conditions, many related problems have not been solved, such as:

Surface modification of SiC nanoparticles, optimum process conditions for coating process, and modification and coating mechanism;

Development of low-phosphorus plating solution with low energy consumption, less pollution and renewable utilization;

Uniform dispersion of SiC nanoparticles in the plating solution and in the coating layer;

The behavior and mechanism of SiC nanoparticles in the coating layer;

At present, the processes used at home and abroad are relatively high in cost, and require high-pressure, high-temperature conditions and high-purity SiC nanoparticles, or complicated operation, many process flows, resulting in low yield or uneven coating, and is not strong. The quality of the product is not good.

In summary, reducing costs, simplifying steps, and ensuring quality will be an effort in the future.

 

Latest results: to improve electrochemical performance of electrode materials

At present, a Chinese R&D team has chosen to use nanoparticle surface coatings to improve the performance of lithium-rich electrodes.

 

 

The team constructed a carbon shell of NiCo nanoparticles on the prepared nanoparticles, and the resulting LLO@C&NiCo cathode exhibited excellent cycle and rate performance. Analysis shows that LLO@C&NiCo, as the cathode material of lithium battery, exhibits excellent electrochemical performance due to its protective C&NiCo shell. This research has been supported by China's national key research and development plan and national key basic research development plan.

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Do you understand these important concepts?

Nanoparticles refer to particles having a particle size between 1 and 100 nm, also known as ultrafine particles. They are in an over-extension between macroscopic objects and between micro-systems and macro-systems. They are a group of a small number of atoms or molecules, so they are neither a typical microscopic system nor an atypical macroscopic system.
The basic meaning of nanoscience and technology is to manipulate and arrange atomic and molecular innovations directly or indirectly in the nanometer category for making new substances.
Nanomedicine is an emerging edge interdisciplinary subject based on nanoscience or medicine. Its basic meaning refers to the use of nanotechnology theories and methods to carry out a new discipline of medical research and clinical treatment, which is mainly related to medicine in two aspects. Combination: First, the detection technology of disease; second, the treatment technology of disease. As an important tool in the field of nanomedicine, gold nanoparticles have become a research hotspot in the field of nanomedicine at home and abroad with its unique properties.
The emergence of nanoparticles has injected new impetus into the scientific development of the new century!
Preparation of nanoparticles
Ø Vapor Deposition: Synthesis of nanomaterials by chemical reaction of metal compound vapors. It is characterized by high purity and narrow particle size distribution.
Ø Precipitation method: After the precipitant is added to the salt solution for reaction, the precipitate is heat-treated to obtain a nano material. Its characteristics are simple and easy, but the purity is low and the particle radius is large.
Ø Hydrothermal synthesis method: synthesis in a fluid such as an aqueous solution or a vapor under high temperature and high pressure, and then separating and heat-treating to obtain nanoparticles. It is characterized by high purity, good dispersibility and easy control of particle size.
Ø Sol-gel method: a metal compound is solidified by a solution, a sol or a gel, and then subjected to a low heat treatment to form a nanoparticle. It is characterized by a large number of reaction species, uniform product particles, and easy process control.
 
Ø Microemulsion method: two mutually incompatible solvents form an emulsion under the action of a surfactant, and nucleation, coalescence, agglomeration and heat treatment in the microbubbles to obtain nanoparticles. It is characterized by monodisperse and good interface of particles. 
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Nanoparticles are widely used in biology, chemistry, medicine, etc.
Nanoparticle probe
Bioanalysis is mainly used in the frontier fields of biological macromolecule analysis, biopharmaceutical analysis, bioactive substance analysis and microbial analysis such as proteins and nucleic acids. Because the sensitivity of the existing methods to the specificity of the target molecule is not high, scientists have sought to find a more sensitive and specific method for bioanalysis. The emergence of nanoparticle probes provides a new way to solve this problem. It can accurately combine the target and has strong specificity. At the same time, the nanometer size of the probe can significantly improve the sensitivity, and the in-depth study of biomolecules. Convenience is provided.
Classification of nanoparticle probes:
² semiconductor nanoparticle probes
² metal nanoparticle probes
² composite nanoparticle probes
² magnetic nanoparticle probes
Applications of nanoparticle probes:
² DNA detection
² protein detection
² drug targeted delivery
Diagnosis of malignant tumors 
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As a new type of nanocarrier, mesoporous silica nanoparticles have a good application prospect in the field of biomedicine. It is different from the physicochemical properties of traditional inorganic materials and plays a key role in the diagnosis and treatment of malignant tumors. In particular, MSNs, as a drug-loading platform with high loading capacity, good biocompatibility, targeting and controllability for drug release, can be used to solve the problems encountered in the current clinical diagnosis and treatment of malignant tumors.
Nano pesticide
Nanotechnology is a highly attractive research area that is emerging to achieve these goals. It is a new approach to designing novel nano-sized active ingredients and their formulations and delivery systems, collectively referred to as "nano-pesticides." Nanopesticide is an emerging field called crop nanotechnology for crop protection. This field covers a wide range, including a basic understanding of the interaction between nanomaterials and insects, making existing pesticide active ingredients into nanoemulsions and dispersants, using nanomaterials as active pesticide ingredients or developing new nanopesticide formulations using nanomaterials as carriers. It is expected that research on nanopesticide will address the major limitations of existing pest management strategies, providing new and advanced nano-formulations that are stable and active in the target environment (less affected by sunlight, heat and rain) and can penetrate Target organisms (insects), which are resistant to pest defense mechanisms, are harmless to plants and mammals, are cost-effective to formulate and produce, and have new mechanisms of action.
Common carriers for pesticide delivery systems:
² Nano polymer particles
² inorganic nanoparticles
² other mineral nanoparticles
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Nanotechnology can reduce the use of crop protection chemicals and will make agriculture environmentally friendly and cost effective. Fertilizers, pesticides, and growth regulators are well-delivered, including nanosensors that detect soil conditions, crop growth, and pest and disease infestation in time, through the development of nanodevices and products. The application of nanotechnology in agriculture will have good prospects as well as in other fields, but progress will be relatively slow.
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Whole exome sequencing (WES), also named as exome sequencing, is a genomic technique to sequence all of the protein-coding genes in exome. It contains two parts: the first is to select only the subset of DNA that encodes proteins, known as exons. The second is to sequence the exonic DNA by any high-throughput DNA sequencing technology.

By using this technique, fixed-cost studies can sequence samples to much higher depth than that of whole genome sequencing. This additional depth makes exome sequencing well suited to several applications that need reliable variant calls, for example, rare variant mapping in complex disorders, discovery of Mendelian disorders, case studies, clinical diagnostics and direct-to-consumer exome sequencing.

In this article, we listed 20 frequently asked questions about whole exome sequencing.

1. Does the whole exome sequencing have a reference genome?

Yes, there should be a sequence of related species if there is no reference group, but the reliability of the capture results is not guaranteed. Since the capture probe is designed based on the provided reference sequence, it is not recommended if the target region is known to have a large discrepancy with the reference genome, such as a large fragment insertion.

2. Why does exon sequencing need to be compared with the whole genome in the analysis, rather than directly compared to the target area?

First, the target area is generally shorter than the whole genome, and may be discontinuous. If the target area is extracted separately, it will affect the sequence alignment effect at the edge of the area;

Second, the quality of the capture cannot be assessed, such as off-target rate, on target ratio, and the like.

3. How many coverage times are preferable for whole exome sequencing?

Generally, 100 × or 150 × is recommended. With a higher coverage, some rare mutations is easier to be found for the genetic deterioration of heterogeneity. In addition, the coverage of exon sequencing is random, so a higher average coverage is beneficial to ensure that most areas have sufficient coverage.

4. What is the significance of whole exome sequencing depth? How is the sequencing depth converted?

The sequencing depth represents the number of times of the sequence is covered by the probe set. The higher the number, the more accurate the sequencing result is, and the more accurate the subsequent statistical analysis. If you run tumor, low frequency mutation studies, it is recommended that the sequencing depth should be at least 150 ×. If you only look for classic SNPs, non-low frequency mutations, the sequencing depth should be at least 30×. Sequencing depth conversion method: The capture efficiency of the general target area is 60-70%, and the target area of the exon capture kits such as Agilent and Roche are about 60 Mb, that is, the sequencing depth = 10G*60%/60Mb=100×.

5. How many missing fragments can whole exome sequencing detect?

A 50 bp fragment deletion can be roughly measured. Because the coverage of exon sequencing is very uneven, if there is a large segment of deletion, it cannot be judged whether the hybridization is not captured or it is missing. What is currently detectable is a missing found in a read. The length of a read is about 150 bp, so fragment deletions below 50 bp can be detected from exon sequencing.

6. Can whole exome sequencing be used in CNV analysis? What are the methods for detecting CNV?

Whole exon sequencing has a hybrid capture efficiency problem because of a hybrid capture process. The hybridization efficiency of each exon is different, and the homologous competition is different, so the coverage difference of different exons is very large. Therefore, in general, exon sequencing cannot be used for CNV detection. However, in cancer research, CNV can be detected using cancer tissue and paracancerous tissue controls. There are two conventional methods for detecting CNV, one is whole genome resequencing and the other is Affymetrix SNP 6.0. Among them, Affymetrix SNP6.0 has a relatively low detection cost and is a relatively economical means.

7. Can whole exome sequencing be performed in the target region for methylation detection? Can RNA capture be performed?

Target region methylation assays can be directly used with methylation capture sequencing, such as SureSelect's MethylSeq or NimbleGen's SeqCap Epi. The principle is similar to capture sequencing, which is using specialized probes to capture and enrich target area. The target region is subjected to capture enrichment, sequencing and methylation detection after bisulfite treatment. RNA capture can be performed using SureSelect's RNA Capture or NimbleGen's SeqCapRNA System.

8. Can capture samples be mixed with multiple species?

Yes, as long as the captured target fragment has a corresponding reference genome, it can be captured by the probe. For example, viral sequences integrated in the host genome, mixed parasite sequences in blood, low abundance microbial sequences in environmental samples, and the like. Since the proportion of these sequences in the mixed sample is often very low, the efficiency of using the traditional resequencing method will be very low, and a very high amount of sequencing is required to obtain sufficient coverage depth, and a large amount of redundant data is generated. In such cases, capture sequencing has an absolute advantage over them.

9. Is there any indicator for the effect of the capture? What factors will reduce the capture effect?

Due to the limitation of sequencing technology, there are some repeating sequences, undetermined “N bases”, and the quality of the sample itself, which may cause the sequence to be uncovered. This is a problem that all sequencing technologies will encounter. Deep genome sequencing does not guarantee 100% complete coverage of the genome. Since each sample varies widely, it is not reliable to make a direct commitment to the capture effect. The best way is: client provides target area, and company designs and evaluates them to get detailed reports. In addition, there are ubiquitous repeat sequences in the species. Before designing, it is generally necessary to shield some difficult-to-cover repetitive masks and then design them, which will improve the quality of the capture, but at the same time reduce the overall coverage.

10. Can high GC content fragments be captured?

Yes, but there will be some impact on the capture efficiency of these fragments. Similarly, low complexity segments and containing fuzzy base segments have some difficulty in capturing. When designing the probe, company technicians appropriately increase the number of coverages and probe density according to the coverage, and send the expected capture effect to the customer for confirmation. In addition, if you are capturing full exons, UTR, etc., you can use preset probe sets. These probe designs are optimized, which improves the corresponding capture efficiency.

20 Frequently asked Questions (FAQs) about Whole Exome Sequencing (Part Two)

11. What is the efficiency of exon capture (whole exome sequencing)?

The hybridization process is used during exon sequencing. There are many parts of the human chromosome that are homologous to exons, and these homologous parts are likely to be captured during the hybridization process. Therefore, some of the sequences detected are not exon sequences. We refer to the ratio of the sequence of exons to the entire sequencing sequence as the capture efficiency. The efficiency of capture does not affect the quality of the data, but the effective proportion of the data.

12. When do you choose whole exon capture? When do you choose custom probe capture?

All exon captures of humans and some common species have pre-set probe products, of which human all-exon probes have been optimized for many times. If there are many target regions and all are exons (several tens of Mb or more), it is recommended to select whole exon capture directly, because the whole exon has a more mature probe set and lower cost, and the capture effect is better. Custom probes are recommended for smaller segments of interest, or for segments other than exons.

13. What information are needed for custom design exon capture probe?

Species, reference genomic information, target area information (coordinates, gene names or gene numbers, etc.) are needed. If only coordinate information is provided, it should be provided in the form of a table. If the gene name is provided, it is best to have the official name of NCBI and the corresponding gene number. Multiple target areas may overlap without affecting subsequent design and analysis. After the design is completed, the final regional information will be returned to the customer for confirmation. The target area supports a maximum of 200Mb. After providing the gene name, company will help customers find specific regions of the gene, such as exons, UTR, and so on. If you want to capture several or all of them, you need to indicate.

14. Can the conventional species or new species genomes, imperfect or much-error genome be used for exon capture sequencing?

Yes, the capture sequencing platform has no species restriction, as long as genomic sequence information and target region information are available. Capture sequencing design can be performed even if the reference genome of the species is new, inaccurate, or even very different. Correspondingly, the captured probes are designed according to the sequence provided by the customer, so the accuracy of the results cannot be fully guaranteed.

15. Can the degradation sample be used for post-library construction? What is the impact on subsequent data and analysis?

If the degradation of the sample is very serious, it is not recommended to build the database. The success rate of the database is low. If the sample is seriously degraded, the impact of constructing the exon library will be low, the effective data volume is low, and the properly mapped will be low.

16. What is the impact of viscosity sample, pore impurity contamination and RNA contamination on library construction? What is the success rate?

Slight protein contamination has little effect on the library construction, but if the protein or other impurities are seriously polluted, it will affect the quantification, and the enzyme efficiency in the database, which will reduce the success rate of the database; RNA pollution mainly affects the sample quantification and the sorting of DNA in the database. Therefore, if there is RNA contamination, RNA digestion is recommended when the total amount and quality of the sample are appropriate.

17. Can a tissue be co-extracted with DNA and RNA? What is the extraction method?

Co-extraction can be performed; there are generally two methods for co-extraction, one is to divide the tissue into two parts, each for DNA and RNA extraction; the other is to use the DNA/RNA co-extraction kit for extraction, but generally degradation of DNA and RNA is likely to happy after extraction.

18. What is the reason for the low DNA extraction yield of FFPE samples?

FFPE samples are generally stored for many years, and they have been seriously degraded by formaldehyde; and FFPE samples are generally precious, and the number of samples sent is less.

19. What is the difference between cfDNA and ctDNA?

cfDNA (cell free DNA) is a free DNA in the blood. It is released from the normal cells of the body or the rupture of white blood cells. It is harmless to the body and will soon be cleaned by itself. ctDNA (circulating tumor DNA) is the rupture of tumor cells. Free DNA released into the blood can be used as a highly specific tumor marker.

20. How to separate plasma samples when extracting cf/ctDNA?

Collect 5 mL of peripheral blood (usually in the morning), quickly transfer to the EDTA anticoagulant tube, carefully invert and mix (prevent hemolysis); run internal plasma separation within 1 hour (at room temperature) or 2 hours (at 4 °C): Centrifuge at 1000 rpm for 10 minutes at 4 °C, carefully pipette the plasma to a clean 1.5 mL EP tube (not to suck white blood cells during pipetting), then centrifuge at 12,000 rpm for 10 minutes at 4 °C to remove residual cells or debris, and carefully absorb the required supernatant volume into a new dispensing EP tube, mark with an oily marker, and store in an ultra-low refrigerator at -80 °C to avoid repeated freezing and thawing, and transport samples with dry ice.

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About Plasmid

What is a plasmid? A plasmid is a genetic unit capable of autonomous replication outside the chromosome, including organelles of eukaryotes and deoxyribonucleic acid (DNA) molecules except for chromosomes in bacterial cells. It is now customary to refer to DNA molecules in bacteria, yeasts, and actinomycetes other than chromosomes. In genetic engineering, plasmids are often used as vectors for genes. Many bacteria have a large number of small circular DNA molecules in addition to chromosomes. This is a plasmid (some plasmids are RNA). The plasmid often has antibiotic resistance genes, such as tetracycline resistance gene or kanamycin resistance gene. Some plasmids are called episomes, and these plasmids can be integrated into the chromosome of the bacteria. From the integration position, it becomes a DNA molecule that is free from extrachromosomes.

Plasmid categories

At present, hundreds of bacteria have been found to have plasmids. Most of the known bacterial plasmids are closed circular DNA molecules (cccDNA). The relative molecular mass of bacterial plasmids is generally small, about 0.5%~3% of bacterial chromosomes. According to the relative molecular mass, plasmid can be roughly divided into two types: the relative larger one is 40×106 or more, and the relative smaller one is 10×106 or less. (A few molecular masses of plasmids are somewhere in between.) The number of plasmids in each cell is mainly determined by the replication characteristics of the plasmid itself. According to the nature of replication, the plasmids can be divided into two categories: one is a tight-type plasmid. When the cell chromosome is replicated once, the plasmid is also replicated once, and there are only 1 or 2 plasmids in each cell; the other is a relaxed plasmid, which can continue to replicate when the chromosome replication is stopped. There are generally about 20 plasmids in each cell. The replication of these plasmids is under the control of the relaxation of the host cells, containing 10–200 copies per cell. If a certain drug treatment inhibits the synthesis of the host protein, the plasmid copy number is increased to several thousand. The earlier plasmid pBR322 belongs to the relaxation plasmid, which can be treated with chloramphenicol to reach higher copy number. Generally, the plasmid with larger molecular weight is tight-type. The plasmid with smaller molecular weight is relaxed-type. The replication of plasmids sometimes is related to their Host cell.

Plasmid application

In genetic engineering, artificially constructed plasmids are commonly used as vectors. Artificially constructed plasmids can integrate a variety of useful features, such as a variety of single enzyme cleavage sites, antibiotic resistance, etc. Commonly used artificial plasmid carriers have pBR322 and pSC101. pBR322 contains anti-tetracycline gene (Tcr) and ampicillin resistance gene (Apr), and contains a single point of 5 endonucleases. If the DNA fragment is inserted into the EcoRI cut point, it will not affect the two antibiotic genes expression. However, if the DNA fragment is inserted into the Hind III, Bam HI or Sal I point, the anti-tetracycline gene will be inactivated. At this time, the pBR322 containing the DNA insert will make the host bacterium resistant to ampicillin but sensitive to tetracycline. pBR322 without a DNA insert would make the host bacterium resistant to both ampicillin and tetracycline, whereas bacteria without the pBR322 plasmid would be sensitive to ampicillin and tetracycline. pSC101 is similar to pBR322 except that it has no anti-ampicillin gene and PstI nicking. The largest insert of the plasmid vector is approximately 10 kb (kb is expressed as kilobase pairs).

Plasmid sequencing

Before starting a plasmid sequencing project, one should consider:

The DNA quality and purity

Is there host DNA contamination?

Sole plasmid or genome and plasmid?

Plasmid sequencing Applications

Ÿ Mutation studies

Ÿ Vector verifications

Ÿ Characterization of production strains

CD Genomics provides complete plasmid DNA sequencing service, and our improved bioinformatics pipelines are available to perform de novo assembly with no reference required.

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CD Genomics, the USA-based genetic sequencing company, recently announces the launch of its proprietary GenSeqTM Technology to provide comprehensive Single-Cell Sequencing services.

We already knew that a typical human cell consists of around 2 x 3.3 billion base pairs of DNA and 600 million bases of mRNA. In normal condition, a mix of millions of cells are used during the course of sequencing the DNA or RNA with traditional methods (such as Sanger sequencing/ Illumina sequencing). With such deep sequencing of DNA and RNA from a single cell, the cellular functions can be investigated through-fully.

Under the setting of rapid progress in the development of next-generation sequencing (NGS) technologies, this field has provided many valuable insights into complex biological systems and dramatically advanced cell biology, for example, from cancer genomics to diverse microbial identification. It has been treated as a promising tool for approaching a set of seemingly inaccessible problems. For example, heterogeneous samples, rare cell types, cell lineage relationships, mosaicism of somatic tissues, analyses of microbes that cannot be cultured, and disease evolution can all be elucidated through single cell sequencing. With such progress, researchers are now increasingly moving their focus from whole to the characterization of individual cells-based technologies for genomics, transcriptomics, and epigenomics.

“These single-cell analyses will allow researchers to uncover new and potentially unexpected biological discoveries relative to traditional profiling methods that assess bulk populations. Single-cell RNA sequencing (scRNA-seq), for example, can reveal complex and rare cell populations, uncover regulatory relationships between genes, and track the trajectories of distinct cell lineages in development”, as said by Byungjin Hwang.

Single-cell sequencing could be applied in:

  • Profiling scarce clinical samples
  • Measuring intra-tumor heterogeneity and guiding chemotherapy
  • Cancer cells evolution analysis during tumor progression
  • Pre-implantation genetic diagnosis (PGD)

Similar to NGS experiments, single cell sequencing protocols generally cover the following four steps: isolation of a single cell, nucleic acid extraction and amplification, sequencing library preparation, sequencing and bioinformatic data analysis.

About CD Genomics Single-Cell Sequencing services

CD Genomics’ Single-Cell kit produces amplified DNA fragments suitable for Copy Number Variation (CNV) analysis using oligonucleotide aCGH or qPCR; SNP genotyping, mutation detection and sequencing.

Advantages of CD Genomics Single-Cell Sequencing

  • Complete: End-to-end workflow for whole transcriptome analysis of individual cells.
  • Highest throughput: Unprecedented parallel processing of up to 96 single cells per run.
  • Easiest to use: Less than three hours total hands-on time, working directly from single cells, with no RNA fragmentation and purification step.
  • Affordable: One-eighth the cost of other library preparation system.
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The signal pathway means that when a certain reaction occurs in a cell, the signal transmits a message from outside the cell to the cell, and the cell responds according to the information. The signal pathway was first traced back to 1972, but it was called signal transmission at that time. In 1980, M. Rodbell referred to signal transduction in a review, and the concept has since been widely used. A signaling pathway is a series of enzymatic reaction pathways that can exert extracellular molecular signals through the cell membrane and into cells. These extracellular molecular signals (called ligands) include hormones, growth factors, cytokines, neurotransmitters, and other small molecule compounds.

Definition

How does the signal in the cell pass when the ligand specifically binds to the receptor in the cell membrane or cell?

The various biochemical pathways in the cell are composed of a series of different proteins that perform different physiological and biochemical functions. The regulation of downstream protein activity (including activation or inhibition) by upstream proteins in each signaling pathway is primarily accomplished by the addition or removal of phosphate groups, thereby altering the stereoconfiguration of downstream proteins. Therefore, the major members of the signaling pathway are protein kinases and phosphatases, which are capable of rapidly altering and restoring the conformation of downstream proteins. Receiving an external signal from a cell receptor to finally making a comprehensive response is not only a signal transduction process, but more importantly, a process of gradually amplifying the external signal. Receptor proteins convert extracellular signals into intracellular signals, which are amplified, dispersed, and regulated by signal cascades, resulting in a comprehensive array of cellular responses, including regulation of downstream gene expression, changes in intracellular enzyme activity, and cellular bone architecture. Type and changes in DNA synthesis, etc. These changes are not all caused by one type of signal, but different reactions can be produced by different combinations of several signals.

Classification

First, when the signal molecules are lipids such as cholesterol, they can easily cross the cell membrane and bind to the target receptor in the cytoplasm;

Second, when the signal molecules are polypeptides, they can only bind to receptors such as proteins on the cell membrane. These receptors are mostly transmembrane proteins. Through conformational changes, signals are transmitted from the extracellular domain to the domain within the membrane, and then Acting with the next level of receptors, the next level of pathway is activated by modification such as phosphorylation.

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Common signal path

NF-κB signal

NF-kB (nuclear factor-kappa B) is a transcription factor found in the nuclear extract of B lymphocytes in 1986. It binds specifically to the enhancer B sequence GGGACTTTCC of the immunoglobulin kappa light chain gene and promotes κ. Light chain gene expression, hence the name. It is a member of the Rel family of eukaryotic transcription factors and is widely found in various mammalian cells [1]. To date, five NF-kB/Rel family members have been found in mammalian cells, which are RelA (ie, p65), RelB, C-Rel, p50/NF-kB1 (ie, p50/RelA) and p52/NF. -kB2. These members all have a Rel homology domain (RHD) of approximately 300 amino acids. This highly conserved domain mediates the formation of a homologous or heterodimer of the Rel protein, which is also a region of specific binding of NF-kB to the DNA sequence of the target gene.

The activation process of NF-kB in cells is finely regulated. Normally, NF-kB in the cytoplasm is inactivated and binds to the inhibitory protein of NF-kB to form a trimer complex. When TNF-a signaling, inflammatory factors, and external stimuli such as LPS and ultraviolet rays are present, cytokines bind to TNF receptors on the surface of cell membranes, and TNF receptors multimerize and interact with TRADD molecules in the cytoplasm. TRADD recruits TRAF (TNFR-associated factor) and kinase RIP (receptor interacting protein), and signals are transmitted by RIP to IKK (IkB kinase). IKK plays a very important role in the NF-kB signaling pathway, and although it is different in the upstream signal path, it eventually aggregates into IKK. IKK consists of three subunits a, b and g. IKK as a kinase phosphorylates the Ser32 and Ser36 residues of the a subunit of IkB and the Ser19 and Ser23 residues of the b subunit. IkB is then dissociated from the p50/p65/IkB heterotrimer and degraded by proteasome after ubiquitination. Thus, NF-kB, which is inhibited by IkB, is exposed to its nuclear localization signals (NLS), rapidly entering the nucleus from the cytoplasm, and binding to specific sequences on the DNA in the nucleus to initiate or enhance transcription of related genes.

2. JAK-STAT signaling pathway

1) JAK and STAT proteins

The JAK-STAT signaling pathway is a signal transduction pathway stimulated by cytokines discovered in recent years, and is involved in many important biological processes such as cell proliferation, differentiation, apoptosis and immune regulation. Compared with other signaling pathways, this signaling pathway is relatively simple to transfer. It consists of three components, namely tyrosine kinase-related receptor, tyrosine kinase JAK and transcription factor STAT.

2) JAK-STAT signaling pathway

The transfer of the JAK-STAT signaling pathway is relatively simple compared to other signaling pathways. The signaling process is as follows: Binding of cytokines to the corresponding receptor causes dimerization of the receptor molecule, which allows the JAK kinases coupled to the receptor to be close to each other and activated by interactive tyrosine phosphorylation. After JAK activation, the tyrosine residues on the catalytic receptor are phosphorylated, and then these phosphorylated tyrosine sites form a "docking site" with the surrounding amino acid sequence, and contain the SH2 domain. STAT protein was recruited to this "parking site." Finally, the kinase JAK catalyzes the phosphorylation of the STAT protein bound to the receptor, and the activated STAT protein enters the nucleus in the form of a dimer to bind to the target gene and regulate gene transcription. It is worth mentioning that a JAK kinase can be involved in the signal transduction of many cytokines. A cytokine signaling pathway can also activate multiple JAK kinases, but cytokines have certain choices for activated STAT molecules. Sex. For example, IL-4 activates STAT6, while IL-12 specifically activates STAT4.

3.Ras, PI (3)K and mTOR signals

With the completion of the sequencing of the human genome, hundreds of protein kinases have been discovered. Based on their structural similarities, these kinases can be divided into multiple protein families that play important biological functions in cell proliferation, growth, differentiation, and apoptosis. Ras, PI(3)K and mTOR are a class of protein kinases closely related to cell proliferation. The normal growth of eukaryotic cells is limited by the nutrients provided by the surrounding environment. Ras and PI (3)K signaling play a key role in regulating cell growth by regulating the downstream molecule mTOR.

In most human tumor cells, key mutations in the Ras and PI (3)K signaling pathways have undergone significant mutations. The reason is that if this signal pathway is mutated, it will lead to cell survival and growth no longer limited by environmental conditions such as nutrients, and then induce cell cancer. It is worth noting that some tumor suppressors, such as TSC1, TSC2 and LKB1, attenuate the intensity of the mTOR signaling pathway under nutrient-deficient conditions. Correspondingly, inactivation mutations in TSC1, TSC2 or LKB1 result in similar cancer symptoms with a common clinical manifestation. Therefore, this signal pathway that ensures that cells proliferate under environmentally appropriate conditions can be used by cancer cells to survive and grow under conditions of lack of nutrients. In the process of screening for kinase inhibitors, a series of drugs targeting kinases such as mTOR, PI (3)K, RTKs and Raf have been designed. In the molecular mechanism of cancer research, although this signaling pathway is the most thoroughly studied, the physiological functions of these kinases in cells and organisms are far more complicated than we think.

4.Wnt signal

The Wnt signaling pathway is widely distributed in invertebrates and vertebrates and is a highly conserved signaling pathway during species evolution. Wnt signaling plays a crucial role in the early development of animal embryos, organ formation, tissue regeneration and other physiological processes. If a key protein in this signaling pathway is mutated, resulting in abnormal activation of the signal, it may induce cancer. In 1982, R. Nusse and H.E. Varmus cloned the first Wnt gene in mouse breast cancer cells, initially named Int1 (integration 1). Later studies found that the mouse Int gene and the wingless gene wg (wingless) of Drosophila are homologous genes, and thus the two are collectively referred to as Wnt. H.E. Varmus I also won the 1989 Nobel Prize in Physiology and Medicine for his outstanding contribution to cancer research.

Wnt is a type of secreted glycoprotein that acts through autocrine or paracrine. The main components of the Wnt signaling pathway include: the secreted protein Wnt family, the transmembrane receptor Frizzled family, CK1, Deskerled, GSK3, APC, Axin, β-Catenin, and the transcription factor TCF/LEF family. The Wnt signaling pathway is a complex regulatory network that is currently thought to include three branches: the canonical Wnt signaling pathway, which activates gene transcription via β-Catenin; the Wnt/PCP pathway (planner cell polarity pathway), which activates JNK via small G proteins (c -Jun N-terminal kinase) regulates cytoskeletal rearrangement; the Wnt/Ca2+ pathway affects cell adhesion and related gene expression by releasing intracellular Ca2+. It is generally mentioned that the Wnt signaling pathway mainly refers to the classical Wnt signaling pathway mediated by β-Catenin.

5. BMP signaling pathway

BMP (bone morphogenetic protein) is an important member of the TGF-β (transforming growth factor-β) superfamily [6]. By regulating the activity of a range of downstream genes, many important biological processes such as mesoderm formation, nervous system differentiation, tooth and bone development, and cancer development are controlled. The BMP signal is specifically transmitted by the ligand BMP to specifically bind to the serine/threonine kinase receptor (BMPR) on the cell membrane to form a ligand receptor binary complex. At the same time, the type II receptor (BMPR2) is able to activate the type I receptor (BMPR1) and further transmit signals to the Smad molecules in the cell. The Smad protein plays a key role in the transmission of BMP and TGF-β signals from the cell membrane to the nucleus. Activated type I receptor (BMPR1) further phosphorylates Smad proteins (Smad1, Smad5, and Smad8), causing Smad molecules to detach from cell membrane receptors and bind to Smad4 molecules (common Smad, Co-Smad) in the cytoplasm Enter the nucleus. In the nucleus, the Smad multi-component complex acts on a specific target gene with the participation of other DNA-binding proteins to regulate the transcription of the target gene.

6. Ras2MAPK signal transduction pathway

1) Ras upstream channel

Ras can be activated by complex networks. First, phosphorylation-activated receptors, such as PDGFR, EGFR directly bind to growth factor receptor binding protein (Grb2), which also bind indirectly and phosphorylate proteins containing the src homology region 2 (SH2) domain (For example, Shc, Syp), then activate Grb2. In addition, the src homology 3 (SH3) domain of Grb2 binds to target proteins such as mSos1, mSos2, C3G and dynamin. The SH3 structure of C3G and connexin Crk Domain binding after coupling tyrosine phosphorylation to activate Ras. Crk can also bind mSos1 to activate Ras.Grb2 to bind to activated receptors to promote the localization of guanylate exchange factor (Sos) protein on the cell membrane adjacent to Ras. Sos forms a complex with Ras. After GTP replaces GDP and Ras, Ras is activated. When GTP is hydrolyzed into GDP, Ras is inactivated. Ras has intrinsic GTPase activity, and its activity can be regulated by RasGAPs, thus RasGAPs act as Ras activity regulation. The role of the agent. In addition, Ras inactivation is also highly regulated. Currently, there are three proteins that can hydrolyze GTP to inactivate Ras, which are P120GAP, neurofibromin and GAP1m, collectively referred to as RasGAPs.

2) Ras downstream path

At present, the Ras/Raf pathway is the clearest signal transduction pathway. When GTP replaces GDP and Ras binds, Ras is activated, then activates the silrethine kinase cascade amplification effect, recruiting intracellular Raf1 serine kinase to the cell membrane, Raf kinase phosphorylates MAPK kinase (MAPKK), and MAPKK activates MAPK. MAPK is activated and then transferred to the nucleus to directly activate transcription factors. In addition, MAPK stimulates Fos, Jun transcription factor forms transcription factor AP1, which is related to the specific DNA sequence next to the myc gene binds to initiate transcription. The myc gene product is also a transcription factor that activates other genes. Ultimately, these signals are concentrated to induce the expression and activity of D-type Cyclin. D-type Cyclin and Cyclin-dependent kinases (such as CDK4 and CDK6) form a complex that forms the cell from the G1 phase to the S phase. Therefore, the Ras/Raf pathway plays a key role between the receptor signal and G1 phase progression, however, Ras/Raf Pathway is not the only pathway regulating G1 progression. Ras and Raf alone do not promote Raf kinase activity, while Raf can be activated by Ras-independent mechanisms (eg, activated by Src tyrosine kinase and PKC), MAPK Can be independent of Ra the s mechanism (eg, by modulating the activity of integrins) is activated. This indicates that each signaling protein in the cascade reaction can be activated by multiple upstream proteins, and they may have additional target proteins.

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Technical advances in RNA-seq

Sanger sequencing and microarrays. Sanger sequencing technology was first used for transcriptomics, which enabled methods such as SAGE (serial analysis of gene expression). SAGE was one of the first attempts to quantify gene expression on a global basis. Almost instantaneously, microarrays utilizing complementary probe hybridization, quickly emerged and come to dominate the field of transcriptomics profiling for the next decade.

NGS. The advent of next-generation technologies has enabled the sequencing approach to surpass microarray approach. In 2006, the first RNA-seq paper was published by utilizing454/Roche technology. The era of RNA-seq dominance began in 2008 with the maturity of Illumina/Solexa technology. The most popular technical platforms for RNA-Seq has been the Illumina Genome Analyzer and Hi-Seq. While the Illumina/Solexa technology can generate gigabases of data per run (initially 1GB per run for the Genome Analyzer in 2006 and 600 GB per run for the HiSeq in 2012), Roche/454 technology generates reads long enough for RNA-seq but are hampered by the relatively low throughput and high cost.

Third generation sequencing. Despite the popularization of the NGS technologies, the application of third generation sequencing in RNA-seq is on its way. For examples, Heliscope sequencing and single-molecule real-time (SMRT) sequencing have already been applied in some RNA-seq studies. PacBio SMRT long reads sequencing technology can easily cover complete transcript from the 5′ end to the 3′-poly A tail without the need of fragmentation to obtain full-length cDNA sequences, which is useful to identify new transcripts and new introns, thereby accurately identifying isoforms, alternative splicing sites, fusion gene expression, and allelic expression.

Table 1. The advantages of RNA-seq compared with other transcriptomics approaches (Wang et al. 2009).

Technology

Tiling microarray

cDNA or EST sequencing

RNA-seq

Technology specifications

Principle

hybridization

Sanger sequencing

High-throughput sequencing

Resolution

From several to 100 bp

Single base

Single base

Throughput

High

Low

High

Reliance on genomic sequence

Yes

No

In some cases

Background noise

High

Low

Low

Application

Simultaneously map transcribed regions and gene expression

Yes

Limited for gene expression

Yes

Dynamic range to quantify gene expression level

Up to a few-hundredfold

Not practical

>8,000-fold

Ability to distinguish different isoforms

Limited

Yes

Yes

Ability to distinguish allelic expression

Limited

Yes

Yes

Practical issues

Required amount of RNA

High

High

Low

Cost for mapping transcriptomes of large genomes

High

High

Relatively low

Challenges of RNA-seq

  • Short-read. Illumina sequencing technology has steadily increased read length and throughput since its introduction in 2007. Long paired-end strand-specific reads are commonly used for higher levels of mappability and de novoassembly of transcriptomes. Furthermore, the third generation sequencing technology (such as PacBio and Ion-Torrent) enables full-length transcripts sequencing.
  • PCR biases. Another concern is the impact of PCR amplification on the accuracy of gene expression quantitation via RNA-seq. Helicos and some of the third sequencer used an amplification-free technology. There are also PCR-free methods for Illumina sequencing.

Workflow of RNA-seq based on NGS

The workflow of RNA-seq by utilizing high-throughput sequencing technology is illustrated in Figure 1. Briefly, long RNAs are first converted into a library of cDNA fragments through RNA or DNA fragmentation. Sequencing adaptors are then attached to each cDNA fragment and sequence data are generated in a high-throughput manner from both ends (paired-end sequencing). The resulting sequence reads are subsequently aligned with the reference genome or transcriptome, and are classifies into three types: exonic reads, junction reads and poly(A) end-reads. A base-resolution expression profile can be generated by using these three types of sequence reads.

149921824?profile=original
Figure 1. A typical workflow of RNA-seq (Wang et al. 2009).

  • Library construction

149926785?profile=original
Figure 2. A typical library construction pipeline of RNA-seq.

Following sample collection, total RNA is usually isolated via organic extraction and/or silica-membranes of spin columns. Total RNA sample is subsequently processed either by direct selection of poly(A) RNA or by selective removal of rRNA because the abundant rRNA is usually not the research focus and greatly reduces the coverage of the useful transcript. Oligo(dT)-based mRNA purification procedure is widely used in eukaryotes. However, some RNA transcripts that lack the poly(A) tails are missed. Compared to the poly(A) RNA selection, ribo-depletion approach is preferred because it enriches all nonribosomal RNA species, including tRNA, ncRNAs, nonpoly(A) mRNA, and preprocessed RNA. The two most popular rRNA depletion methods are: (i) hybridization of rRNA with biotin-labeled anti-rRNA probes, followed by removal with streptavidin-caoted magnetic beads; and (ii) selective degradation of rRNA by a 5’-3’ exonuclease that specifically recognizes rRNA with a 5’ phosphate.

Fragmentation is subsequently conducted to reach the desired length for different NGS technologies. Some small RNAs, such as microRNAs, piwi-interacting RNAs, and short interfering RNAs, can be directly sequenced without fragmentation. Larger RNA molecules need to be fragmented into smaller pieces (200-500 bp) before deep-sequencing technologies. cDNA fragmentation (DNase I treatment or sonication) and RNA hydrolysis or nebulization. However, each of these methods can create a different bias in the outcome. For example, cDNA fragmentation is usually strongly biased towards the identification of sequences from the 3’ ends of transcripts, while RNA fragmentation has little bias over the transcript but is depleted for transcript ends. Therefore, cDNA fragmentation provides valuable information about the precise identity of these ends and RNA fragmentation provides access to precisely identity of the transcript body.

In the classic NGS protocols, adapters are ligated onto shared double-stranded DNA fragments. However, a major drawback of this approach is the loss of information on transcriptional direction. Pre-treat the RNA samples with sodium bisulphate can convert the cytidine into uridine. Widespread C-T transition thereby marks the coding stand of each transcript. Some other methods that maintain strand-specificity have been proposed, such as direct ligation of RNA adaptors to the RNA sample before reverse transcription.

  • Sequencing

The RNA-seq is currently dominated by three different platforms: Illumina (Genome Analyzer and HiSeq), Applied Biosystems SOLID, and Roche 454 Life Science systems. Read lengths range from 30-100 bp for Illumina and SOLiD, and 200-500 bp for 454 pyrosequencing system. 454-based RNA-seq is particularly attractive for non-model organisms without reference genomes or transcriptomes. Longer reads or paired-end short reads can reveal connectivity between multiple exons. RNA-seq is a powerful method to study complex transcriptomes and reveal sequence variations in the transcribed regions.

  • Bioinformatics

149929857?profile=original
Figure 3. A typical analysis pipeline of RNA-seq data.

Quality assessment is the first step for the bioinformatics analysis of RNA-seq, which ensures a coherent final result by removal of low-quality sequences, over-represented sequences, and adapter sequences. Once all reads have been filtered and mapped or assembled, gene expression levels can thus be inferred, leading to a genome-scale transcriptome map in terms of quality and quantity. RNA-seq also allows detecting differential expression (DE) across treatments of conditions. Normalization has to be conducted to adjust the differences between samples such as library size and gene-specific features. Furthermore, RNA-seq enables us to identify SNPs, fusion genes, and post-transcriptional gene regulation, such as RNA editing, degradation, and translation.

If you want more information about the applications of RNA-seq or bioinformatics workflow of RNA-seq, you can refer to the article.

References:

  1. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nature reviews genetics, 2009, 10(1): 57.
  2. Qian X, Ba Y, Zhuang Q, et al. RNA-Seq technology and its application in fish transcriptomics. Omics: a journal of integrative biology, 2014, 18(2): 98-110.
  3. Marguerat S, Bähler J. RNA-seq: from technology to biology. Cellular and molecular life sciences, 2010, 67(4): 569-579.
  4. Wilhelm B T, Landry J R. RNA-Seq—quantitative measurement of expression through massively parallel RNA-sequencing. Methods, 2009, 48(3): 249-257.
  5. McGettigan P A. Transcriptomics in the RNA-seq era. Current opinion in chemical biology, 2013, 17(1): 4-11.
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Introduction to ChIP-Seq

ChIP-sequencing (also known as ChIP-seq), which combines chromatin immunoprecipitation (ChIP) assays with DNA sequencing, is a powerful technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. ChIP is a type of immunoprecipitation (IP) experimental method used to isolate specific DNA sites in direct physical interaction with transcription factors and other proteins. In ChIP, specific antibodies are used to enrich DNA fragments bound by particular proteins or nucleosomes.

ChIP-seq was one of the early applications of NGS (next-generation sequencing), and the first study of large-scale profiling of the genome-wide histone methylations using ChIP-seq was published in 2007  (Barski et al., 2007). The sequencing of this study was performed on the platform of Solexa 1G genome analyzer. At the same year, Johnson et al. (2007) used ChIP-seq to generate the genome-wide mapping of transcription factor binding sites. Robertson et al. (2007) developed ChIP-seq to identify mammalian DNA sequences bound by transcription factors in vivo. These two papers also demonstrated the increased sensitivity and specificity of ChIP-seq. Owing to the rapid progress of NGS technology and the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for characterization of epigenomes and gene regulation study (Park, 2009).

Comparison of ChIP-chip and ChIP-seq

ChIP-chip, ChIP coupled with microarrays, and ChIP-seq are two standard techniques for identification of the genome wide DNA-proteins binding interactions. Take advantages of the sequencing technology, ChIP-seq offers many advantages over ChIP-chip, as summarized in Table 1 (Park, 2009; Schones and Zhao, 2008).

Table 1. Comparison of ChIP-chip and ChIP-seq.

 

ChIP-chip

ChIP-seq

Maximum resolution

Array-specific, generally 30-100 bp

Single nucleotide

Coverage

Limited by sequences on the array; repetitive regions are usually masked out

Limited only by alignability of reads to the genome; increases with read length; many repetitive regions can be covered

Flexibility

Dependent on available products; multiple arrays may be needed for large genomes

Genome-wide assay of any sequenced organism

Source of platform noise

Cross-hybridization between probes and nonspecific targets

Some GC bias can be present

Experimental design

Single- or double-channel, depending on the platform

Single channel

Cost-effective cases

Profiling of selected regions; when a large fraction of the genome is enriched for the modification or protein of interest (broad binding)

Large genomes; when a small fraction of the genome is enriched for the modification or protein of interest (sharp binding)

Required amount of ChIP DNA

High (a few micrograms)

Low (10-50 ng)

Dynamic range

Lower detection limit; saturation at high signal

Not limited

Amplification

More required

Less required; single-molecule sequencing without amplification is available

Multiplexing

Not possible

Possible

Workflow of ChIP-seq

The workflow of ChIP-seq used to profile the specific DNA binding sites for transcription factors, DNA-binding enzymes or other DNA-associated proteins (non-histone ChIP) and DNA sites correspond to modified nucleosomes (histone ChIP) is illustrated in Figure 1 (Park, 2009). Following ChIP protocols, the chromatin is fragmentated and crosslinked proteins or modified nucleosomes immunoprecipitated using an antibody specific to the protein or the histone modification. After DNA purification and library construction, DNA fragments can be sequenced simultaneously on any of the sequencing platforms, such as Illumina Solexa Genome Analyzer, Roche 454 and Applied Biosystems (ABI) SOLiD platforms, and HeliScope by Helicos, as illustrated in Figure 1. With the tremendous progress of NGS technology, the Illumina platform, such as Hiseq, has been the most widely used platform for sequencing.

 296967103b39b46df443872ba5325030.png

Figure 1. Overview of a ChIP-seq experiment (Park, 2009).

Experimental Design of ChIP-seq

The Encyclopedia of DNA Elements (ENCODE) and model organism ENCODE (modENCODE) consortia have developed a set of working standards and guidelines for ChIP-seq experiments based on experience of hundreds of ChIP-seq experiments (Landt et al., 2012). To obtain high-quality ChIP-seq data, there are several technical aspects should be considered in the ChIP-seq experimental design, including antibodies, cell number, controls, replicates, chromatin fragmentation, library construction and sequencing (Kidder et al., 2011).

  • Antibodies
    • The quality of antibodies used for ChIP is one of the most important factors that contribute to the quality of ChIP-seq data.
    • A sensitive and specific antibody will give a high level of enrichment. Limited efficiency of antibody is the main reason for failed ChIP-seq experiments.
    • Antibody validation and characterization should be done before the ChIP begin.
  • Cell number
    • As the signal-to-noise ratio (SNR) is directly correlated with the cell number, using the correct number of cells can help to diminish the background noise.
    • The abundance of the protein or histone modification to be investigated and the quality of the antibody should be considered when determining the number of cells.
  • Controls
    • An important part of ChIP-seq experimental design is determining which controls to use. A ChIP-seq peak should be compared with the same region in a matched control.
    • There are several different control types but no consensus on which is the most appropriate:
      • Input DNA.
      • Mock IP: DNA obtained from IP without antibody.
      • Nonspecific IP: using an antibody against a protein that is not known to be involved in DNA binding.
    • Replicates
      • High-quality ChIP-seq data sets are valuable resources for the community. Many factors, including cell-culture conditions, ChIP and library construction, may contribute to variability between data sets.
      • To ensure reliability of the data, biological replicate experiments are necessary.
    • Chromatin fragmentation
      • Before ChIP, chromatin must be fragmented into a manageable size.
      • ChIP-seq for DNA-binding proteins uses endonuclease digestion or sonication to fragment DNA.
      • ChIP-seq for histone modifications uses micrococcal nuclease (MNase) digestion to fragment DNA.
    • Library construction and sequencing
      • Libraries may be constructed from ChIP DNA by standard protocols specific to the sequencing platform.
      • Process in library construction and sequencing, including size selection, gel purification, PCR, single-end or paired-end sequencing strategy and sequencing depth, would affect the ChIP-seq data quality.

Considering above technical aspects, a ChIP-seq experimental design that would obtain high-quality data is illustrated in Figure 2 (Kidder et al., 2011). At first, the appropriate controls for antibody specificity should be determined before ChIP. Chromatin is sheared into an ideal size range by sonication or enzymatic means after isolation of the ideal number of cells. Next, high-quality antibodies are used for ChIP. After purification of ChIP-enriched DNA, a library is constructed to allow sequencing on NGS platforms.

 5d15170e997b2290d31620095e8e7f0d.png

Figure 2. ChIP-seq experimental design (Kidder et al., 2011).

At CD Genomics, we provide you with high-quality sequencing and integrated bioinformatics analysis for your ChIP-Seq project, enabling accurately screen and determine the protein binding sites in the whole genome. If you have additional requirements or questions, please feel free to contact us.

Additional reading:

Pipeline and Tools for ChIP-seq Analysis

References:

  1. Barski, A., Cuddapah, S., Cui, K., Roh, T.Y., Schones, D.E., Wang, Z., Wei, G., Chepelev, I., and Zhao, K. (2007). High-resolution profiling of histone methylations in the human genome. Cell129, 823-837.
  2. Johnson, D.S., Mortazavi, A., Myers, R.M., and Wold, B. (2007). Genome-wide mapping of in vivo protein-DNA interactions. Science316, 1497-1502.
  3. Kidder, B.L., Hu, G., and Zhao, K. (2011). ChIP-Seq: technical considerations for obtaining high-quality data. Nature immunology12, 918-922.
  4. Landt, S.G., Marinov, G.K., Kundaje, A., Kheradpour, P., Pauli, F., Batzoglou, S., Bernstein, B.E., Bickel, P., Brown, J.B., Cayting, P., et al. (2012). ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome research22, 1813-1831.
  5. Park, P.J. (2009). ChIP-seq: advantages and challenges of a maturing technology. Nature reviews Genetics10, 669-680.
  6. Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao, Y., Zeng, T., Euskirchen, G., Bernier, B., Varhol, R., Delaney, A., et al. (2007). Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature methods4, 651-657.
  7. Schones, D.E., and Zhao, K. (2008). Genome-wide approaches to studying chromatin modifications. Nature reviews Genetics9, 179-191.
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The challenges of Chip-seq

ChIP-seq is a powerful method to identify genome-wide DNA binding sites for a protein of interest. Mapping the chromosomal locations of transcription factors (TFs), nucleosomes, histone modifications, chromatin remodeling enzymes, chaperones, and polymerases is one of the key tasks of modern biology. To this end, ChIP-seq is the standard methodology (Bailey et al., 2013). Multiple challenges presented in ChIP-seq are not only in sample preparation and sequencing but also in computational analysis.

Unlike other types of massively parallel sequencing data, the ChIP-seq data have several characteristics:

  • Histone modifications cover broader regions of DNA than TFs.
  • Reads are trimmed to within a smaller number of bases.
  • Fragments are quite large relative to binding sites of TFs.
  • Measurements of histone modification often undulate following well-positioned nucleosomes.

To extract meaningful data from the raw sequence reads, the ChIP-seq data analysis should:

  • Identify genomic regions – ‘peaks’ – where TF binds or histones are modified.
  • Quantify and compare levels of binding or histone modification between samples.
  • Characterize the relationships among chromatin state and gene expression or splicing.

Bioinformatics analysis workflow for ChIP-seq data

Bioinformatics analysis workflow for ChIP-seq data and the considerations for each step is illustrated in Figure 1 (Nakato and Shirahige, 2017). The procedure of sample preparation, sequencing and mapping (Figure 1A) is common in both experiments with single or a few samples (Figure 1B) and experiments with many samples (Figure 1C). Initially, sequencing reads of ChIP-seq are analyzed to assess the quality of the reads. After quality metrics, reads are mapped to the reference genome. Compared with input reads, genomic regions that are significantly enriched for ChIP reads are detected as peaks. Other genomic regions are regarded as non-specific background. Read densities can be visualized along the genome. Adjusting peak-calling strategy and parameters to each sample’s property is possible in sample-scale analysis (Figure 1B). But one-by-one adjusting is difficult for large-scale analysis (Figure 1C), in which objective quality metrics for multilateral quantitative assessment is necessary to filter poor-quality data automatically. The called peaks represent candidates of histone modification and targeted protein or DNA-binding sites, which can be used to identify associated functional annotations, such as binding motifs.

 137237160?profile=original

Figure 1. ChIP-seq analysis workflow. Adapted from (Nakato and Shirahige, 2017)

A comprehensive comparison of tools for differential ChIP-seq data analysis

There has been a large effort to improve analytical tools that are used in analysis of ChIP-seq data, and each step has led to the development of specialized software tools. A subset of software tools available for mapping and peak calling are briefly listed in Table 1 (Furey, 2012).

Table 1. A subset of software tools available for mapping and peak calling in the analysis of ChIP-seq data.

Tool

Notes

Web address

Short-read aligners

BWA (Burrows-Wheeler Aligner)

Fast and efficient; based on the Burrows-Wheeler transform

http://bio-bwa.sourceforge.net

Bowtie

Similar to BWA, part of suite of tools that includes TopHat and CuffLinks for RNA-seq processing

http://bowtie-bio.sourceforge.net

GSNAP (Genomic Short-read Nucleotide Alignment Program)

Considers a set of variant allele inputs to better align to heterozygous sites

http://research-pub.gene.com/gmap

Wikipedia list of aligners

A comprehensive list of available short-read aligners, with descriptions and links to download the software

http://en.wikipedia.org/wiki/List_of_sequence_alignment_software#Short- Read_Sequence_Alignment

Peak callers

MACS (Model-based Analysis for ChIP-seq)

Fits data to a dynamic Poisson distribution; works with and without control data

http://liulab.dfci.harvard.edu/MACS

PeakSeq

Takes into account differences in mappability of genomic regions; enrichment based on FDR (false-discovery rate) calculation

http://info.gersteinlab.org/PeakSeq

ZINBA (Zero-Inflated Negative Binomial Algorithm)

Can incorporate multiple genomic factors, such as mappability and GC content; can work with point-source and broad-source peak data

http://code.google.com/p/zinba

Besides detection of enriched or bound regions in ChIP-seq data analysis, an important question is to determine differences between conditions. Owing to the complexity of ChIP-seq data in terms of noisiness and variability, the question is particularly challenging for ChIP-seq. Many different computational tools have been developed and published in recent years for differential ChIP-seq analysis. These tools show important differences in their algorithmic setups, in the number and size of detected differential regions (DR), and in the range of applicability. Description of 14 different tools for differential ChIP-seq data analysis is listed in Table 2 (Steinhauser et al., 2016).

Table 2. Description of different tools for differential ChIP-seq data analysis.

Tool

Language

Peak Calling

Web address

SICER

Bash/Python

Window based approach, merging of eligible clusters in proximity closer than the defined gap size

https://home.gwu.edu/~wpeng/
Software.htm

MACS2

Python

Not required

https://github.com/taoliu/MACS/

ODIN

Python

Not required

http://costalab.org/wp/
odin

RSEG

C++

Not required

http://smithlabresearch.org/software
/rseg/

MAnorm

R

Requires peak calling e.g. with MACS

http://bcb.dfci.harvard.edu/~gcyuan
/MAnorm/MAnorm.htm

HOMER

Perl & C++

Window based approach Peak calling done by HOMER

http://homer.salk.edu/homer
/index.html

QChIPat

R, Perl & C++

Peak calling possible with BELT, MACS, SISSRs or FindPeaks

http://motif.bmi.ohio-state.edu/
QChIPat/

diffReps

Perl

Sliding window approach

https://github.com/shenlab
-sinai/diffreps

DBChip

R

Requires peak calling e.g. with MACS

http://pages.cs.wisc.edu/
~kliang/DBChIP/

ChIPComp

R

Requires peak calling e.g. with MACS

http://web1.sph.emory.edu/users
/hwu30/software/ChIPComp.html

MultiGPS

Java

Expectation maximization learning

http://mahonylab.org/software
/multigps/

MMDiff

R

Requires peak calling e.g. with MACS

https://bioconductor.riken.jp/
packages/3.1/bioc/html/MMDiff.html

DiffBind

R

Requires peak calling e.g. with MACS

http://bioconductor.org/packages
/release/bioc/html/DiffBind.html

PePr

Python

Window based approach

https://github.com/shawnzhangyx
/PePr

Decision tree indicating the proper choice of tool is illustrated in Figure 2. The choice of tool depends on several factors: shape of the signal (sharp peaks or broad ChIP enrichments), presence of replicates and presence of an external set of regions of interest. The tools indicated in black give good results using default settings, and the tools in gray would require more extensive fine-tuning of parameters to achieve optimal results.

 137237280?profile=original

Figure 2. Decision tree indicating the proper choice of tool. Adapted from (Steinhauser et al., 2016).

Technical guidelines for the comprehensive analysis of ChIP-seq data

Recent advances in sequencing technologies and analyses enable us to handle hundreds of ChIP samples simultaneously. But there are still some issues in analysis of ChIP-seq data, such as the false positive peaks, the multiple mapped reads and the poor overlap between peak-finding algorithm results. To obtain high-quality results from the computational analysis of ChIP-seq data, some technical aspects should be considered, which have been listed below (Bailey et al., 2013):

1) Sequencing Depth

  • Effective analysis of ChIP-seq data requires enough coverage by sequence reads (sequencing depth). The required sequencing depth mainly depends on the size of the genome and the number and size of the binding sites of the protein.
  • 20 million reads may be adequate for mammalian TFs and chromatin modifications which are typically localized at specific, narrow sites, such as enhancer-associated histone marks (Landt et al., 2012).
  • Proteins with broader factors, including most histone marks, or more binding sites, such as RNA Pol II, will require up to 60 million reads for mammalian ChIP-seq (Chen et al., 2012).
  • Control samples should be sequenced significantly deeper than the ChIP ones.

2) Read Mapping and Quality Metrics

  • Before mapping to the reference genome, the reads should be filtered by applying a quality cutoff.
  • It is important to consider the percentage of uniquely mapped reads reported by the mapping tools.

3) Peak Calling

  • The analysis for ChIP-seq data is to predict the regions of the genome where the ChIPed protein is bound by finding regions with peaks.
  • A fine balance between sensitivity and specificity depends on choosing an appropriate peak-calling algorithm and normalization method based on the type of protein ChIPed.

4) Assessment of Reproducibility

  • To ensure the reproducibility of the experimental results, at least two biological replicates of each ChIP-seq experiment are recommended to be performed.
  • The reproducibility of both reads and identified peaks should be examined.

5) Differential Binding Analysis

  • Comparative ChIP-seq analysis of an increasing number of protein-bound regions across conditions or tissues is expected with the steady raise of NGS (next-generation sequencing) projects.
  • The direct calculation of differentially bound regions between treatment samples without controls is not recommended.

6) Peak Annotation

The aim of the annotation is to associate the ChIP-seq peaks with functionally relevant genomic regions, such as gene promoters, transcription start sites, intergenic regions, etc.

7) Motif Analysis

  • Motif analysis is useful for much more than just identifying the causal DNA-binding motif in TF ChIP-seq peaks.
  • When the motif of the ChIPed protein is already known, motif analysis provides validation of the success of the experiment.

Additional reading:

The Advantages and Workflow of ChIP-Seq

References:

  1. Bailey, T., Krajewski, P., Ladunga, I., Lefebvre, C., Li, Q., Liu, T., Madrigal, P., Taslim, C., and Zhang, J. (2013). Practical guidelines for the comprehensive analysis of ChIP-seq data. PLoS computational biology 9, e1003326.
  2. Chen, Y., Negre, N., Li, Q., Mieczkowska, J.O., Slattery, M., Liu, T., Zhang, Y., Kim, T.K., He, H.H., Zieba, J., et al.(2012). Systematic evaluation of factors influencing ChIP-seq fidelity. Nature methods 9, 609-614.
  3. Furey, T.S. (2012). ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nature reviews Genetics 13, 840-852.
  4. Landt, S.G., Marinov, G.K., Kundaje, A., Kheradpour, P., Pauli, F., Batzoglou, S., Bernstein, B.E., Bickel, P., Brown, J.B., Cayting, P., et al.(2012). ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome research 22, 1813-1831.
  5. Machanick, P., and Bailey, T.L. (2011). MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696-1697.
  6. McLean, C.Y., Bristor, D., Hiller, M., Clarke, S.L., Schaar, B.T., Lowe, C.B., Wenger, A.M., and Bejerano, G. (2010). GREAT improves functional interpretation of cis-regulatory regions. Nature biotechnology 28, 495-501.
  7. Nakato, R., and Shirahige, K. (2017). Recent advances in ChIP-seq analysis: from quality management to whole-genome annotation. Briefings in bioinformatics 18, 279-290.
  8. Steinhauser, S., Kurzawa, N., Eils, R., and Herrmann, C. (2016). A comprehensive comparison of tools for differential ChIP-seq analysis. Briefings in bioinformatics 17, 953-966.
  9. Thomas-Chollier, M., Herrmann, C., Defrance, M., Sand, O., Thieffry, D., and van Helden, J. (2012). RSAT peak-motifs: motif analysis in full-size ChIP-seq datasets. Nucleic acids research 40, e31.
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CD Genomics, the world leading genomic sequencing company, is able to offer the genotyping of 11 all HLA loci, including HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 and deliver a thorough and detailed report.

As known, the HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in a large part of the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Hence CD Genomics introduces an accurate and reliable HLA genotyping service, which uses NGS technology and amplification methods to address limitations of traditional Sanger sequencing assays.

HLA Genes have an important/integral role in the human adaptive immune system. For example, classical class I (HLA-A, -B, and -C) and class II (HLA-DR, -DP, and -DQ) HLA gene starts to function by presenting foreign antigens to T cells to trigger immune responses. Those HLA genes display amazing sequence diversity on human body. Let’s say it in another way, imagining there are >4,000 known alleles for the HLA-B gene alone. The genetic diversity in HLA genes in which different alleles have different efficiencies for presenting different antigens is believed to be a result of evolution conferring better population-level resistance against the wide range of different pathogens to which humans are exposed.

Since the first description of an association between HLA and human disease, HLA molecules have proven to be central to physiology, protective immunity and deleterious, disease-causing autoimmune reactivity. It is reported that HLA has been associated with more than hundreds of different diseases, including various autoimmune disorders, transplantation etc. However, although it plays an important role in human health, people do not routinely have their HLA genes typed. With the current trend toward precision medicine, knowing their HLA types will be crucial in early diagnosis and management of many diseases.

CD Genomics HLA Typing can be done at different levels of resolution. We offer a worldwide service, with our high throughput service to process hundreds of samples at a time. We can only accept genomic DNA. Our aim is to deliver a high confidence and responsive service to our clients at realistic cost.”

About CD Genomics HLA typing Service

The service is offered for R&D / non-diagnostic purposes only and results must not be utilized to inform patient management decisions. It is featured with: first, comprehensive assay; second, unambiguous results; and third, sample-to-report solution. The service has complete workflow, covering from sample preparation, sequencing, data analysis, and reporting.

For more information, please visit https://www.cd-genomics.com/HLA-Typing.html 

About CD Genomics

CD Genomics was established in 2004, the company is aiming at providing the research community with high quality Next Generation Sequencing, high throughput microarray services. Due to the demand for our services has being increased; CD Genomics has already updated its technology platform to mainstream NGS and microarray instruments.

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The introduction to non-coding RNAs

Non-coding RNAs (ncRNAs) used to be considered as transcription noises or byproducts of RNA processing, but increasing evidence suggests that a majority of them are biologically functional and regulate various activities in the cells. The ncRNAs are roughly classified into two categories according to their sequence length: small ncRNAs (<200 bp) and long ncRNAs (200 bp or more). The categories of ncRNA are listed in Table 1.

Table 1. Overview of ncRNA (Fu 2014).

ncRNAs

Full name

Function

Housekeeping ncRNAs

rRNA

Ribosomal RNA

Translational machinery

tRNA

Transfer RNA

Amino acid carriers

snRNA

Small nuclear RNA

RNA processing

snoRNA

Small nucleolar RNA

RNA modifications

TR

Telomere RNA

Chromosome end synthesis

Regulatory ncRNAs

miRNA

MicroRNAs

RNA stability and translation control

endo-siRNA

Endogenous siRNA

RNA degradation

rasiRNA

Repeat-derived RNA

Transcriptional control

piRNA

Piwi-interacting RNA

Silencing transposon and mRNA decay

eRNA

Enhancer-derived RNA

Regulation of gene expression

PATs

Promoter-associated RNA

Transcription initiation and pause release

lncRNA

Long non-coding RNA

Imprinting, epigenetics, nuclear structure

As shown in Table 1, ncRNAs can be roughly divided into two classes: housekeeping ncRNAs and regulatory ncRNAs. Housekeeping ncRNAs, involving rRNA, tRNA, snRNA, snoRNA, and TR, are considered “constitutive” since they are ubiquitously expressed in all cell types and offer essential functions to the organisms. Regulatory ncRNAs, involving miRNA, endo-siRNA, rasiRNA, piRNA, eRNA, PATs, and lncRNA, have received increasing attention from the research community due to their regulatory function in gene expression, imprinting, and epigenetics. RNA-seq is an advanced technique to illustrate the ncRNA species. Here, we made a summary of the bioinformatics tools for ncRNA analysis with data from NGS.

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Figure 1. ncRNAs as integrated parts of gene network (Fu 2014).

Small ncRNA analysis

Small RNAs play a crucial role in transcriptional regulation and are essential to fully understand the entire scenario of transcriptional regulation. Their aberrant expression profiles are considered to be associated with cellular dysfunction and disease. Therefore, many researches are focused on detection, prediction, or expression quantification of small RNAs, particularly miRNAs, to better understand human health and disease. The available computational tools for small RNA sequencing data are summarized in Table 2.

Table 2. Computational tools for small ncRNA analysis

Tools

Descriptions

DARIO

Quantify and annotate ncRNAs with access to several ncRNA public databases.

CPSS

Quantify and annotate ncRNAs, with special emphasis on miRNAs.

ncPRO-seq

Detect known small ncRNAs in an unbiased way and discover novel ncRNA species.

CoRAL

Divide small ncRNA into functional categories based on biologically interpretable features other than sequence;
Annotate ncRNA in less well-characterized organisms.

RNA-CODE

Combine secondary structure with de novo assembly.
Applicable to ncRNA annotation lacking reference genomes.

miRDeep

Used to detect both known and novel miRNAs in small RNA sequencing data.

Circular RNA detection

CircRNAs are a novel type of RNA that form a covalently closed continuous loop. Most of them are generated from exonic or intronic sequences, and RNA-binding proteins (RBPs) or reverse complementary sequences are necessary for their biogenesis. CircRNAs are mostly conserved, and function as miRNA sponges, regulator of splicing and transcription, or modifiers of parental gene expression. Increasing evidence suggests the potential significance of circRNA in human diseases, such as atherosclerotic vascular disease, neurological disorders, and cancer. Among all the presented tools for circRNA detection, CIRI, CIRCexplorer, and KNIFE exhibit a balanced performance between precision and sensitivity. The available computational tools for circRNA sequencing data are summarized in Table 2.

Table 3. Computational tools for circular RNA detection.

Method

Approach

dependencies

CIRI

Segmented read-based

Bwa, peri

CIRCexplorer

Segmented read-based

STAR, bedtools, python (pysam, docopt, Interval)

KNIFE

Candidate-based

Bowtie, Bowtie2, tophat2, samtools, perl

LncRNA investigation

LncRNA is a type of non-coding RNA with more than 200 nucleotides, such as lincRNAs and macroRNAs. LncRNAs function as a platform for the interaction with mRNA, miRNA, or protein. They have emerged as vital regulators in diverse aspects of biology, including transcriptional regulation, post-transcriptional regulation, and chromatin remodeling. Increasing researches suggest misexpression of lncRNAs contributes to tumor initiation, growth, and metastasis. LncRNAs hence become a promising target for cancer diagnosis and therapy. The combination of lncRNA sequencing and matched computational tools is a powerful approach for this purpose.

Table 4. Computational tools for lncRNA investigation.

Tools

Applications

Reference

lncRScan

Detect lncRNA from the complex assemblies; Distinguish lncRNA from mRNAs

(Sun et al., 2012)

iSeeRNA

Accurately and quickly detect lincRNA from large datasets

(Sun et al., 2013)

Annocript

Detect lncRNA by leveraging public databases and sequence analysis software to verify high non-coding potential

(Musacchia et al. 2015)

LncRNA2Function

Annotate lncRNA based on the theory that similar expression patterns across diverse conditions may share similar functions and biological pathways.

(Jiang et al. 2015)

References:

1. Choudhuri S. Small noncoding RNAs: biogenesis, function, and emerging significance in toxicology. Journal of biochemical and molecular toxicology, 2010, 24(3): 195-216.

2. Fu X D. Non-coding RNA: a new frontier in regulatory biology. National science review, 2014, 1(2): 190-204.

3. Gao Y, Wang J, Zhao F. CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome biology, 2015, 16(1): 4.

4. Jiang Q, Ma R, Wang J, et al. LncRNA2Function: a comprehensive resource for functional investigation of human lncRNAs based on RNA-seq data//BMC genomics. BioMed Central, 2015, 16(3): S2.

5. Musacchia F, Basu S, Petrosino G, et al. Annocript: a flexible pipeline for the annotation of transcriptomes able to identify putative long noncoding RNAs. Bioinformatics, 2015, 31(13): 2199-2201.

6. Qu S, Yang X, Li X, et al. Circular RNA: a new star of noncoding RNAs. Cancer letters, 2015, 365(2): 141-148.

7. Su Y, Wu H, Pavlosky A, et al. Regulatory non-coding RNA: new instruments in the orchestration of cell death[J]. Cell death & disease, 2016, 7(8): e2333.

8. Sun K, Chen X, Jiang P, et al. iSeeRNA: identification of long intergenic non-coding RNA transcripts from transcriptome sequencing data. BMC genomics, 2013, 14(2): S7.

9. Sun L, Zhang Z, Bailey T L, et al. Prediction of novel long non-coding RNAs based on RNA-Seq data of mouse Klf1 knockout study. BMC bioinformatics, 2012, 13(1): 331.

10. Veneziano D, Nigita G, Ferro A. Computational approaches for the analysis of ncRNA through deep sequencing techniques. Frontiers in bioengineering and biotechnology, 2015, 3: 77.

11. Yang G, Lu X, Yuan L. LncRNA: a link between RNA and cancer. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2014, 1839(11): 1097-1109.

12. Zeng X, Lin W, Guo M, et al. A comprehensive overview and evaluation of circular RNA detection tools. PLoS computational biology, 2017, 13(6): e1005420.

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Microorganisms widely exist in nature and are closely related to human life and production. They are generally divided into fungi, actinomycetes, bacteria, spirulina, rickettsia, chlamydia, mycoplasma and viruses. Microbial whole genome sequencing is an important tool for mapping genomes of novel organisms, finishing genomes of known organisms, or comparing genomes across multiple samples. Sequencing the entire microbial genome is important for generating accurate reference genomes, for microbial identification, and other comparative genomic studies. Comparative genomic analysis based on whole genome sequencing plays an irreplaceable role in studying pathogenic mechanism of pathogenic microorganism, evolution of pathogenic genes and screening of novel, efficient drug targets. Microbial whole genome sequencing can be widely used in various fields.

Diseases

Pathogenic microorganism includes all kinds of microorganisms that cause human diseases, food corruption, animal infection in animal husbandry and breeding industry, and plant diseases. Researches focus on disease-related genes, regulatory and interaction systems, metabolic systems, genetic variation, laboratory diagnosis and specific prevention, drug resistance genes, virulence genes and so on. Specifically, microbial whole genome sequencing can be applied to:

· Diagnosis and identification of pathogens

· Epidemiological investigation and tracing

· Rapid identification of pathogen character

· Analysis and prediction of disease prevalence

· Vaccine variation monitoring and efficacy evaluation

· Surveillance of foodborne pathogens

· Drug targets discovery

Foods

Whole genome sequencing of microorganisms in food and bioinformatics analysis of data can help people predict genes that play an important role in the fermentation process or product quality, providing information about the metabolic pathways of microorganisms and their interaction with the environment. Microbial whole genome sequencing opens up the possibility of modifying microorganisms to make them more efficient in the production of vinegar, liquor, yoghourt, and many other fermentation processes.

Agriculture

Microorganism in agriculture involves in planting and breeding, processing of agricultural products, agricultural biotechnology, agricultural ecology and other research and application. Microbial whole genome sequencing can make people have a better understanding of agriculture microbe from genomic level, and the subsequent studies on genome structure and function lay an important foundation for agromicrobiology in the field below:

· Establish agricultural microbial gene bank

· Soil microorganism (including root microorganism)

· Plant nutrition

· Biological nitrogen fixation

· Microbial pesticide

· Microbial fertilizer

· Feed additive

· Biogas fermentation

Environment & Industry

Microorganism is one of the important factors to maintain the energy and material circulation in the ecosystem, it plays an important role in the degradation of various pollutants and harmful substances, and has great application value in energy production and renewable utilization. Some environmental microorganisms can adapt to special environments, such as high temperature, low temperature, high pressure, acid, alkali, heavy metal, and high substrate concentration.

Microbial whole genome sequencing allows people to know about the secrets of these microorganisms’ adaptation to extreme environments, and provides a lot of assistance in pollution control, environmental protection, oil exploitation, preservation and transportation of food and medicine, biofuel, fermentation industry and many other fields in environment and industry.

At CD Genomics, our expert team with extensive experience can help you fully understand microbial communities and take advantage of them. For this purpose, we provide the following services:

16S/18S/ITS Amplicon Sequencing
Metagenomic Shotgun Sequencing
Viral Metagenomic Sequencing
Metatranscriptomic Sequencing
Microbial Whole Genome Sequencing
Viral Genome Sequencing

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