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The Five Finger Revolution

Every few years there is an innovation that takes the world by surprise and changes our way of life to a great extent. Five Fingers is one of those revolutionary innovations that aims and promises to refashion our idea of footwear. These thin, lightweight, and glove-like protective coverings for your feet give you the pleasure of walking barefoot, while at the same time ensure that your feet remains safe from rough edges. 

The company has been in the shoe-business for more than 70 years now and is renowned for making high-quality soles. The Five Fingers are made of soft and elastic Polyamide soles that can be easily stretched to hug the outline of your feet. These soles can be washed in a machine so that you can keep them clean and fresh.

If you're one of those unfortunate people, who have to wear tightly laced shoes for long hours almost every day of your life, then you probably know how much of a burden shoes can become. The human feet have nothing less than 26 bones, 33 joints, 20 muscles, and hundreds of sensory receptors, tendons and ligaments within that little space. It is not for no reason, of course. Our feet is capable of great strength, quickness, and stamina-only, we have to hone their potential. Walking and running barefoot regularly ensures that our feet are exercised for improved performance. Shoes on the other hand change the shape of feet and make the muscles dormant. For runners and athletes, who wear running shoes, the calf muscles end up taking the burden of the pressure while the feet and the toe themselves are huddled up in foam.

Five Finger shoes were designed to reinvent the pleasure of 'barefooting.' It allows us to go back to the beginning, the way we were supposed to live. The moment we start using the Five Fingers to move bare feet, we experience a sudden resurge of a lot of abilities that we had lost due to years of forcing our body into unnatural movement. Gradually, we start to get back our original way of walking, the pain that we used to feel in our calf muscles or around our waist suddenly vanish, and most importantly we start feeling free and happy.

The Five Fingers was originally designed to increase the potential and performance of runners, athletes, and other people involved in adventure sports. These people invest a lot of time, money, and energy into good shoes and exercise for their feet, while totally unaware that barefoot is the best way of movement. Five Fingers have promoted the culture of barefoot running, and now many athletes world over find the Five Fingers their best choice. The increased grip, steadiness and quickness offered by barefoot moving with Vibram Five Fingers is useful for many, who have to make quick reflex-based movements or tackle rough terrain, muddy waters, slippery sands, and the like. The Five Fingers have already become popular among people involved in climbing, fitness training, martial arts, light trekking, yoga, canyoneering, running, pilates, sailing, boating, kayaking, surfing, flats fishing, travel, and Canoeing.

There are a few things in life that are just better in every aspect and leave no room for argument. Five Fingers is one of them. It is a product that is not dependent on its looks but on its performance. It might be a good idea to test one of them on your feet.

Your Step by Step Guide to Enzymes for Diagnostic and Drug Treatment Use (Part One)

Human body is a complex bioreactor where occurring thousands of metabolic reactions every day, in which various enzymes play a catalytic and regulative role. Therefore, it will be of significance to apply enzymes in diagnosing and treating diseases. In China, enzymes have been used as a drug to treat disease for thousands of years and the Chinese people discovered wheat koji’s (rich in a variety of enzymes) function of treating digestive disorders as early as 3,000 years ago. It was not until 1893 when Francis and his fellows found papain had good effects on curing diphtheria and tuberculosis that enzymes have attracted the attention of the whole medical field. In the following hundreds of years, with more kinds of enzymes were used as medicines, presenting its irreplaceable role in the clinical disease treatment, the theory of enzyme therapy was gradually formed. Enzymes function of diagnosis and treatment can be divided into the following three aspects: clinical diagnosis such as the use of transaminase to diagnose hepatitis, clinical treatment such as lysozyme for antibacterial anti-inflammatory and analgesic use, and metabolic waste cleaning by an extracorporeal circulation device containing a specific enzyme.

 

  1. Enzymes for Diagnostic Use

1.1 Diagnosis Based on Enzyme Activities in Body Fluid

The number of certain enzymes contained in a healthy human body fluid is relatively stable. If certain diseases or pathological changes occur, the vitality of some enzymes in the body fluid will change accordingly, relying on which certain diseases can be diagnosed by monitoring the enzymes’ changes.

 

For example, the level of lipase in the blood can be regarded as a way to early diagnose acute severe pancreatitis. Lipase, mainly secreted by the pancreas, is an enzyme that hydrolyzes long-chain triglycerides (LCT), the amount of which will start to increase 4 to 8 hours after the induction of acute pancreatitis and reach the maximum level within 24 hours, which will last for 10 to 15 days. Amylase will increase parallelly at the same time. Currently, the accuracy of LPS in the diagnosis of acute pancreatitis can reach up to 82%~100%.

 

1.2 Diagnosis Based on Changes of Substances in Body Fluid by Enzymatic method

Certain substances in body fluid may indicate some diseases. Characteristics of high specificity and high catalytic efficiency allow enzymes to be used as a way to determine the content of certain substances in body fluids. For instance, the content of glucose in blood and urine, a clinical diagnosis basis for diabetes, can be detected by glucose oxidase combined with catalase. Moreover, the two enzymes can be immobilized into an enzyme reagent paper or an enzyme electrode for easier clinical detection.

 

Uricase is capable of rapidly oxidizing uric acid into allantoic acid, which will be no longer absorbed by the renal tubules, thereby being excreted out of the body. It has a good effect on hyperuricemia caused by nodular gout, urinary calculi or nephrotic failure. Measuring the amount of uric acid by uricase can diagnose gout. At present, the corresponding immobilized enzyme has been widely used in clinical diagnosis. Besides, cholinesterase and cholesterol oxidase have also been widely used to determine the amount of cholesterol in the blood to diagnose hypertension and cardiovascular disease.

 

  1. Enzymes in Medicines

2.1 Therapeutic Enzymes to Resist Inflammation and Tumor

Enzymes used as anti-inflammatory agents can enlarge the vascular permeability of inflammatory lesions, leukocyte migration, and the formation of granulation tissue. Clinically application in inflammation accompanied by edema can promote the reabsorption of exudate to accelerate the recovery process. The enzymes can be used internally by oral ingestion and externally to clean wounds and suppuration as the most common methods of drug administration.

 

Bromelain is capable of dissolving fibrin and blood clots in living organisms, which can not only be used to treat edema and various inflammations but also suitable for treating deep burns in small and medium areas without hurting normal tissues or affecting skin grafting as it can dissolve quickly. Lysozyme catalyzes the hydrolysis of the 1,4-β-glycosidic bond between the bacterial cell wall peptidoglycan N-acetylglucosamine and N-acetylmuramic acid.

 

Lysozyme can catalyze the hydrolysis of the 1,4-β-glycosidic bond between the peptidoglycan N-acetylglucosamine and N-acetylmuramic acid on the bacterial cell wall, causing the microbe to be killed by cell wall lysis. Therefore, it has obvious effects on the treatment of acute inflammatory diseases by lysozyme, such as acute pharyngitis, acute laryngitis, acute otitis media, etc.

To be continued in Part Two…

 

PNAS: New Study Shows that Remdesivir Prevents Coronavirus MERS-CoV Infection in Monkeys

In a new study, researchers from the National Institutes of Health reported that the experimental antiviral drug remdesivir (also known as GS-5734) successfully prevented rhesus monkeys infected with the Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) from becoming ill from this virus infection. Giving remdesivir before infection can prevent them from getting sick, while giving this drug after they are infected can improve their condition. The results were published online February 13, 2020 in the journal PNAS, entitled "Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection".

 

MERS-CoV is closely related to the 2019 novel coronavirus (SARS-CoV-2, also known as COVID-19, previously known as 2019-nCoV). Since the first case of 2019-nCoV infection was detected in Wuhan, China in December 2019, this virus has triggered a large-scale epidemic.

 

Remdesivir has previously been shown to protect animals from multiple viruses in laboratory experiments. Experiments have confirmed that the drug is effective in treating monkeys infected with Ebola virus and Nipah virus. It has also been studied in humans for the treatment of Ebola virus disease.

 

This new study involved three groups of monkeys: monkeys that were administered remdesivir 24 hours before infection with MERS-CoV; monkeys that were administered remdesivir 12 hours after infection (close to the peak time of MERS-CoV replication in these monkeys); and monkeys that were not administered remdesivir (as a control group).

 

The researchers observed the monkeys for six days. All control monkeys showed signs of respiratory disease. The monkeys that were given remdesivir before infection were in good health: they had no signs of respiratory disease, had markedly decreased levels of viral replication in the lungs, and had no lung damage compared with control monkeys. Monkeys that were given remdesivir after infection performed much better than control monkeys: the severity of the disease was lower than control monkeys, their lung virus levels were lower than control monkeys, and their lung damage was also milder.

 

These researchers point out that these promising findings support the use of remdesivir for clinical trials against MERS-CoV and 2019-nCoV. In China, several clinical trials are ongoing to treat patients with 2019-nCoV using remdesivir.

 

Gilead Sciences Inc. Developed the remdesivir and participated in this study.

 

In 2012, MERS-CoV emerged in Saudi Arabia. By December 2019, the World Health Organization had identified 2499 MERS-CoV cases and 861 deaths. Given that about one third of MERS-CoV cases are transmitted by infected individuals treated at health facilities, these researchers propose that remdesivir may be effective in protecting other patients, patient contacts, and health care workers from disease. They also noted that if this medication is taken shortly after the onset of symptoms, then it may help patients with a confirmed diagnosis of MERS-CoV or 2019-nCoV.

Creative Proteomics Scientists Share Precautions for Chromatin Immunoprecipitation Experiments

Eukaryotic genomic DNA exists as chromatin. Therefore, studying the interaction between protein and DNA in chromatin environment is the basic way to elucidate the gene expression mechanism of eukaryotes. Chromatin immunoprecipitation (CHIP) is currently the only method to study the interaction between DNA and proteins in vivo.

Its basic principle is to fix the protein-DNA complex in a living cell state and randomly cut it into small chromatin fragments within a certain length range, and then precipitate this complex by immunological methods to specifically enrich the target protein The bound DNA fragments are used to purify and detect the target fragments to obtain information about protein-DNA interactions.

CHIP can not only detect the dynamic effects of trans factors and DNA in vivo, but also can be used to study the relationship between various covalent modifications of histones and gene expression. In addition, the combination of CHIP and other methods has expanded its application scope. The CHIP-on-chip method established by combining CHIP and gene chips has been widely used for high-throughput screening of specific trans factor target genes; CHIP with in vivo footprinting methods is used to find the binding site of trans factor in vivo; RNA-CHIP is used to study the role of RNA in the regulation of gene expression. It can be seen that with the further improvement of CHIP, it will play an increasingly important role in the study of gene expression regulation.

Chromatin Immunoprecipitation (ChIP) is a method developed based on in vivo analysis, also known as binding site analysis. It has become the main method of epigenetic information research in the past decade. This technology helps researchers determine what histone modifications will occur at a particular location in the genome of the nucleus.

ChIP can not only detect the dynamic effects of trans factors and DNA in vivo, but also can be used to study the relationship between various covalent modifications of histones and gene expression. In recent years, this technology has been continuously developed and improved. Combining microarray technology to examine chromosomal activity in chromosomal gene expression regulatory regions is a very effective tool for in-depth analysis of major metabolic pathways for diseases such as cancer, cardiovascular disease, and central nervous system disorders.

Its principle is that while maintaining the combination of histone and DNA, through the use of a biological antibody corresponding to a specific histone tag, chromatin is cut into very small pieces and precipitated. IP is a method developed by utilizing the specific binding of an antigen protein and an antibody, and the phenomenon that "prorein A" of a bacterial protein specifically binds to an FC fragment of an immunoglobulin. At present, the multi-purpose refined prorein A is pre-bound and solidified on the beads of argarose, and after reacting with the solution and antibody containing the antigen, the prorein A on the beads can adsorb the antigen to achieve the purpose of purification.

The most important point of the experiment is the nature of the antibody. Different antibodies and antigen-binding capabilities are different, and dye-free binding may not be used in IP reactions. It is recommended to check the antibody manual carefully. In particular, the specificity of polyclonal antibodies is a problem.

Secondly, pay attention to the nature of the buffer in which the antigen is dissolved. Most antigens are cellular proteins, especially backbone proteins, which must be lysed by the buffer. For this reason, a buffer containing a strong surfactant must be used, although it may affect the binding of a part of the antigen-antibody.

On the other hand, if the cells are lysed with a weak surfactant, the cell proteins cannot be fully lysed. Even the dissolution also results in binding to other proteins, the epitope is blocked, affecting the binding to antibodies, and even if IP is successful, it is a tragic result of co-precipitation of many proteins and antibodies.

Third, in order to prevent protein degradation and modification, the antigen-dissolving buffer must be added to each inhibitor of the protein, and the experiment should be performed at low temperature. Before each experiment, first consider the antibody / buffer ratio. Too little antibody will not detect the antigen, too much will not settle on the beads, and the supernatant will remain. Too little buffer will not dissolve the antigen, too much will dilute the antigen.

Creative Proteomics can provide custom experimental design and refine Co-IP parameters based on customers’ needs. The company promises reliable Co-IP analysis of protein-protein interactions with its experienced scientists and technicians. Established in 2004, Creative Proteomics has gradually developed into an integrated company that provides proteomics, metabolomics, glycomics, and bioinformatics analysis services to researchers in the pharmaceutical, biotechnology, agriculture and nutrition industries, as well as academic and government organizations.

 

TOR: The secret to maintaining longevity and healthy aging

From yeast to monkeys, calorie-restricted diets have been shown conducive to life extension and healthy life of all living organisms as long as there is no malnutrition. Although there is no long-term research to prove the benefits of limiting calories to human life, short-term studies have shown that it does improve health.

 

It might work like this. Our body monitors and senses the amount of nutrients through specific molecules in the cell. Depending on the amount of food we eat, these molecules regulate our metabolism to intervene how we use the available nutrients. One of these molecules is an enzyme called TOR. When there are many foods, the TOR enzyme will indicate cells to grow in the body. If there is less food, TOR will indicate cells to be vigilant - what scientists call "mild stress response."

 

Many experiments have shown that when animals eat a lot of food, especially for a long time, TOR will feel this, and their life will be shorter. But does all food have this effect on TOR?

 

The TOR enzyme is particularly active when cells sense large amounts of amino acids (components of proteins) or proteins. Protein-restricted diets do not cause malnutrition and can have the same effects on the metabolism and longevity of laboratory animals as calorie-restricted diets.

 

Age-related diseases

 

It is well known that age-related diseases are caused by genetic mutations, but is there a link between TOR, nutrition and senile diseases? We know that nutrition is associated with cancer and heart disease, and overactive TOR is associated with these diseases, but recent studies have shown that TOR is also directly associated with neurodegenerative diseases. For example, the TOR activity in the brain of Alzheimer's patients is much higher than in a healthy brain. In addition, mimicking these diseases in mice and other experimental animals suggests that removal of excess TOR can prevent brain cell death.

 

So, what we eat, how our body perceives, and the risk of neurodegenerative diseases may be linked. Scientists are exploring various possibilities to prevent neurodegeneration. If more protein means more active TOR, we can either safely adjust our diet or develop a drug that deceives our body and makes it think it consumes less protein.

 

Many laboratory studies have shown that caffeine and a drug called rapamycin actually can do that. Although cells are rich in proteins, their metabolism and longevity are similar to those of protein-restricted cells. We are currently conducting research on human neurons in this area, and the first result points in the same direction.

 

Not so simple

 

Does this mean that we should change our diet and protein intake? What about other nutrients like sugar? Unfortunately, as expected, things are not that simple. Many other molecules in our body are involved in the perception of nutrients, including carbohydrates, which affect lifespan and age-related diseases.

 

This is why we need to be very cautious. First, everyone's nutritional needs vary, depending on his/her stage of development, age, gender, or level of activity—only a few important factors are listed here. In addition, although there is growing evidence from the use of human cells and tissues in laboratories, we need to conduct large-scale population studies that record specific diets, including protein, fat, and carbohydrate intake, while analyzing related health or molecular markers. Such research will take decades to produce reliable data and valid conclusions.

 

Nevertheless, with the development of new technologies and scientific methods, we are taking steps to understand the underlying causes of aging and age-related diseases. Coupled with targeted clinical trials and population studies, we may be able to achieve healthy aging and longer life in the near future.

 

Creative Peptides is major supplier of cosmetic peptides, anti-aging peptides for both research and industrial use.

Blocks Important Enzymes

Antibiotics were once a "secret weapon" for humans to fight many diseases. In the late 19th and early 20th centuries, due to the discovery of a series of antibiotics, human lifespan was greatly improved. But it was less than a century since it was invented. Because of the increasing resistance of bacteria to antibiotics, antibiotics have gradually stepped down from the "sacred altar" and even become a major challenge in the field of medical and health care in the future.

 

The effectiveness of antibiotics is generally declining. The reason is that antibiotic-resistant bacteria are spreading rapidly, and resistant bacteria are not killed by certain antibiotics, and then they are no longer restricted and their resistance is even transmitted. Because of the resistance to antibiotics, some common pathogens are becoming so-called "super bacteria". Once antibiotics fail, our lives are fraught with danger-minor abrasions can lead to death, and minor ear infections can cause deafness.

 

The problem of antibiotic resistance has increasingly become a problem that plagues countries around the world. The World Health Organization has published a report saying that by 2050, bacterial resistance to antibiotics will kill 10 million people every year, equivalent to one person losing their life every 3 seconds, and the harm will exceed cancer. At the same time, the most worrying thing is that this harmfulness is increasing year by year. For example, the treatment of E. coli is usually effective with ordinary antibiotics, but in recent years, many countries have reported that some patients even use the most powerful antibiotics to no avail. At present, approximately 25,000 people are killed each year in Europe, causing 1.5 billion euros in annual medical costs and economic losses in the EU. Globally, about 700,000 people die from various drug-resistant infections every year, and 230,000 Newborns died as a result.

 

Antibiotic resistance has become one of the world's difficult problems. But recently, good news came from the Technical University of Munich, Germany. A team of chemists at the school proposed a new method: they have identified important enzymes in the metabolism of S. aureus. Breaking these enzymes can starve the pathogen.

 

"Many bacteria have developed resistance to broad-spectrum drugs, and an important goal of this research is to find new points of attack." Professor Stephan Sieber of the Technical University of Munich, together with his doctoral student Annabelle Hoegl and others, developed an isolation And the method of metabolizing enzymes, which can control the metabolic process, and if they are blocked, they can "starve" the pathogen more or less.

 

Their latest study tested S. aureus. This bacterium is made up of thousands of proteins and is ubiquitous in the world, and some S. aureus species are resistant to antibiotics. Stephan Sieber said that being able to isolate needles with characteristic properties in a haystack, identifying them, and investigating their functionality is a real challenge.

 

In this experiment, researchers used vitamin B6 to accelerate intracellular chemical reactions. A key component of vitamin B6 is pyridoxal phosphate (PLP). Without pyridoxal phosphate-dependent enzymes, the bacterial metabolism would Interruptions can cause bacteria to starve to death.

 

The team then used chemically modified pyridoxal phosphate to detect PLP-dependent enzymes, and the labeled molecules were placed in the nutrient solution of S. aureus. Because the nutrient solution does not contain natural pyridoxal phosphate, PLP-dependent enzymes will bind to these labeled molecules, and the researchers will then use an ultrasonic device to break down these bacteria and pick out the enzymes that carry the label. The basic principle of molecular selection is also called "protein profiling". This is not a new technology, but scientists at the Technical University of Munich have used this method for the first time to investigate and analyze PLP-dependent enzymes.

 

Stephan Sieber said: "We can confirm that this method is very effective and feasible. Many important enzyme substances in staphylococci rely on pyridoxal phosphate. We selected and isolated 73% of the enzymes and identified them by mass spectrometry.

 

In addition, the researchers discovered previously unknown PLP-dependent enzymes and deciphered their function. Stephan Sieber sees the search for new antibiotic targets as a treasure trove.

 

This discovery can be used to develop new antibacterial active drugs. In the next step, researchers hope to study the functionality of enzymes in more detail and determine how to block the metabolism of bacteria in a targeted manner without harming the health of human cells. 

Introduction to Glyco-engineering Antibody

What is glycosylation?

Various modes of action of monoclonal antibodies include neutralization of antigens, complement-dependent cytotoxicity (CDC), and antibody-dependent cytotoxicity (ADCC). Neutralization of antibodies relies on the binding of the Fab region to epitopes on antigens, thereby preventing disease progression due to biomolecular interactions. Monoclonal antibodies acting through CDC and ADCC depend on two stages of action—the first stage: binding of the antibody Fab region to the epitope of the antigen. In the second stage, various types of immune cells can bind to the Fc region of the antibody, thereby triggering the recruitment of "effector" cells in the immune system. These immune cells secrete some cytokines that can ultimately lead to the death of antibody-binding cells. CDC and ADCC involve different death modes of receptors, effector cells and target cells, but both are significantly affected by the type of glycosylation in the Fc functional region of the antibody.

Glycosylation refers to the process or reaction of covalent binding of non-sugar biomolecules to sugar under the action of glycosyltransferases. Glycosylation can be divided into O-linked glycosylation and N-linked glycosylation according to the linkage mode. Due to the differences in the length of the variable region of the antibody, incomplete statistics of the amino acid sequences of the recombinant antibodies published so far revealed that the length of the heavy chain could be between 441 and 454 amino acids, and the glycosylation sites correspondingly varied between Asn-291 and Asn-304.

According to its structure, glycosides can be divided into three types: high mannose type, heterozygous type and complex type. Recombinant monoclonal antibodies can generally contain about 20-30 glycosides, and random pairing of different glycosides of antibody heavy chain can form more than 400 glycoforms. Studies have shown that glycosylation of recombinant monoclonal antibodies can have important effects on antibody-dependent cytotoxicity, complement-dependent cytotoxicity, half-life and immunogenicity of antibody drugs, and can be engineered for glycosylation of recombinant antibody drugs.

Glyco-engineering antibody

 

Figure1. Engineering antibody

Antibody Fc-segment glycans are glycan chains composed of various monosaccharides, such as acetylglucosamine, mannose, galactose, fucose, N-hydroxyacetylneuraminic acid, N-acetylneuraminic acid, etc. Thesemonosaccharides make up polysaccharides through different combinations. Therefore, the antibodies produced by cell fermentation are actually a mixture of antibodies with the same amino acid sequence and different glycan chains. Most of the currently marketed antibody drugs are produced by the expression of CHO cells and NS0 cells. Since these two cells are murine-derived cells, the various glycosylation forms of antibodies expressed by them are exogenous to human. Therefore, these antibody molecules will produce immunogenicity in vivo. Using gene editing technology to alter glycosylation-related genes in antibody-expressing cells can produce glyco-engineering antibodies with higher activity and lower immunogenicity.

  1. Removal of fucose side chains enhances antibody ADCC effect

The majority of Fc-segment glycan chains of recombinant antibodies contain Fuc side chains, and studies have demonstrated that antibodies lacking Fuc have 10 to 100 times the ADCC effect of antibodies with Fuc side chains on glycan chains. Fuc-deleted antibodies can be obtained by overexpressing GMP3 and Golgimannosidase2 glycosylation-related enzymes in host cells, and this modification can greatly improve the activity of antibodies in vivo.

  1. Trigeminal glycan chains enhance antibody ADCC effects

The majority of antibody glycosylation expressed by CHO cells is a bifurcated structure lacking GlcNAc branches. Antibodies glycosylated into trigeminal structures can be obtained by introducing GnTIII enzyme into CHO cells, and antibodies with trigeminal glycosyl chains have higher ADCC activity. Researchers in Switzerland have adapted a host cell for the production of antibodies against neuroblastoma using this technology, and the resulting antibody activity has also been greatly improved.

  1. Overexpression of glycan chain sialic acid can improve the anti-inflammatory activity of antibodies

Fc glycan chains with sialic acid are only a small proportion of recombinant antibodies produced by cell engineering, and the expressed antibodies can be hypersialylated by overexpressing sialyltransferase in host cells. For antibodies used to neutralize in vivo factors, hypersialylation of antibodies can inhibit the immune response of patients after injection of antibodies.

 

Introduction to the Applications of Immune Repertoire (Part Two)

2. In ovarian cancer

The researchers analyzed primary and metastatic immune repertoire sequencing (T-cell receptor beta sequencing) in five ovarian cancer patients. From the characteristics of the TIL repertoire of 93 samples, it was found that whether it is the primary site, metastatic tissue of the same patient, or TIL in different parts of the same tissue, or TIL of different patients, the repertoire features are similar. The characteristics of T cell repertoire in peripheral blood are very different, and the TIL CDR3 rearrangement rate of invading tumors is very low. These results indicate that ovarian cancer induces adaptive immunity of TIL, so that the human immune system still maintains a certain response to ovarian cancer. The basic measurement of TIL cells includes the total number, which is expected to be used as a prognostic indicator, which is applicable to various cancers including ovarian cancer.

3. In small lesions

Leukemia minimal residual desease (MRD) refers to a state in which a small amount of leukemia cells remain in the body after complete remission of leukemia induction chemotherapy (or after bone marrow transplantation treatment). These residual leukemia cells have become the source of leukemia recurrence, and the treatment after relapse is very difficult, mainly because the relapsed leukemia cells have developed drug resistance, so the detection of small residual leukemia is of great significance in predicting early recurrence and treatment at the same time. So the examination of minimal residual leukemia cannot be ignored. Patient samples were used to assess minimal residual disease (MRD) after 29 days of treatment; HiSeq sequencing of T cell receptor C and T cell receptor B targeting potential VJ rearrangement combined mutation regions after multiplex PCR; The sample was a cloned T cell receptor (TCR) complementarity determining region 3 (on CDR3) sequence, and the paired prognostic samples were used to evaluate MRD; abnormal T lymphoblasts were found by multi-parameter flow cytometry compared with the above method. High-throughput sequencing of the receptors TCRB and TCRG existed in most of the case groups and subsequent MRDs. Clones were also found (31 T-cell receptors B in 43 and 27 T-cell receptors G in 43). As expected, high-throughput sequencing TCRB and TCRG were able to detect MRDs that were not detectable in flow cytometry (25 / 35HTS vs. 13/35), which highlights the low-throughput sequencing technology in defining a lower detection threshold MRD has the potential to influence clinical diagnosis and treatment decisions. The study found that Immune Repertoire sequencing may become a novel and personalized way to guide clinical diagnosis and treatment of diseases, in which T cell activity is the main determinant. Therefore, the sequencing of lymphoid receptor genes may improve the clinical diagnostic efficacy and the potential of MRD to monitor lymphoproliferative diseases.

4. In hematopoietic stem cell transplantation

The high morbidity and mortality of chronic graft-versus-host disease (GVHD) covers the well-organized different parts of allogeneic hematopoietic stem cell transplantation (HCT) and the use of immune groups in the peripheral blood repertoire. As the allogeneic reaction is mainly a cross-reaction that is randomly distributed throughout the T cell repertoire, the diversity of T cell receptors (T cell receptors) and the allogeneic hematopoietic stem cell transplantation (all -HSCT) is associated with an increased risk of cancer recurrence. Due to technical limitations, the diversity of T cell receptors can be measured by allogeneic hematopoietic stem cell transplantation. By combining 5- RACE PCR and deep sequencing, the T cell receptors of 28 allogeneic hematopoietic stem cell transplant recipients can be quantified using a single oligonucleotide. Repeated blood sample analysis confirms that the frequency of individual temperature coefficients of resistance can be accurately determined. After 6 months, the diversity of T cell receptors in umbilical cord blood recipients and healthy people was similar, while the diversity of CD4 and CD8 T cells in peripheral blood hematopoietic stem cell transplant recipients with T cell depletion was lower 28 times and 14 times. After 12 months these defects have improved in CD4, but not in the CD8 T cell compartment. Overall, this method provides a groundbreaking view of resuscitation in a T cell repertoire, which can be used to identify high-risk groups of infection or relapse after allogeneic hematopoietic stem cell transplantation.

5. In rheumatoid arthritis

The central link in the onset and development of rheumatoid arthritis (RA) has been considered to be antigen-specific T cell activation, using a new next-generation sequencing method to detect multiple cells in a potentially self-reactive cloned T cell repertoire. Recent onset (early) or RA in peripheral blood of each joint and patient. Patients who have recently developed (early) RA (<6 months) (n = 6) or established RA (> 18 months) (n = 6) and screened T cell clones for sequencing over 10,000 T cell receptors (T cell receptors). For each sample, comparative analysis was performed from paired blood samples of T cells. Synovial T cells from two patients were obtained from multiple expanded joints. The degree of each clone is based on the frequency of unique CDR3 sequences within a sample. Cloning more than 0.5% is considered highly cloned (HFC). Results In early RA synovium, the T cell repertoire was mainly 35HEC (median, range 270), accounting for 56% of T cell receptor sequencing. In patients with synovial T cell clones, it was significantly higher than the established RA [11 (in the established RA synovial membrane accounts for 9.8% of T cells 5 to 24), the median, P <0.01]. 34% of highly cloned T cells (range 28% to 40%) are in different joints in the same patient, and only 4% of synovium and blood (P = 0.01) are compared (0% -8%). The results show that RA is a systemic autoimmune disease, and a special T cell clone region is formed in the inflamed and swollen synovium, especially in the early stage of the disease. This indicates that at least RA should address the problem of autoreactive T cells in inflamed and swollen synovial tissue, preferably in the early stages of the disease.

 

A Huge Leap of the mAb Drug Against Tumor—Humanized Monkey Antibody

It’s a common sense among the professionals working in the field of anti-tumor medicine that the antibody drugs outperform traditional ones in efficacy on a large scale, of which the humanized mAbs are a pillar rock in midstream.

 

Overview of antibody humanization

Original mAb drugs come from immunization in mice, like Muromonab-cd3, the first new monoclonal antibody drug approved by FDA in history, which have high immunogenicity and are liable to induce human body produce corresponding antibodies, and thus neutralized the drugs themselves. As a result, since the 1980s, scientists have been dedicated to cutting down immunogenicity in the mAbs by increasing the proportion of sequences from human body in the antibody sequence, which was the first attempt of humanization. Yet the humanized antibody whose host is rodent still can not achieve satisfactory efficacy as there are still alloplasmic sequences. As technology advances, methods in this stream have evolved too. Creative Biolabs, as a conscientious CRO in the field of antibody humanization, has worked out a way to humanize antibodies from monkey (non human primate, NHP), leading the whole industry.

 

The notion of adopting monkeys' antibody originates from their close relationship with human beings, and the unsatisfactory test results of rodent animals. In order to accelerate the research steps in the whole field, a humanization platform has been designed that accepts not simply rabbit, dog, chicken, but also monkey antibodies for humanization, on which two crucial NHP’s 3D antibody prototypes are built so as to detect mutations.

 

Major techniques adopted during the NHP antibody humanization process:

 

CDR Grafting & SDR Grafting—complementarity-determining region grafting combined with specificity-determining residue grafting to ensure lessening the immunogenicity of the resulting antibody as much as possible, even in the residues.

 

Chain Shuffling Strategybased on construction and screening of two chimeric phage display libraries to orderly shift the light chain and heavy chain of antibody for further screening, which enables full humanization.

 

Humanized IgG Library Screening—a mammalian cell surface display approach allowing selecting humanized antibodies in a full-size IgG format, and support protein synthesis.

 

NHPs are involved in the study of more than 70 kinds of human infectious diseases, including pathogens like bacteria, viruses, fungi, parasites and prions; infectious diseases like global infectious diseases, children's infectious diseases, tropical infectious diseases, sexually transmitted diseases, new infectious diseases, zoonoses, potential bioterrorism, etc. Creative Biolabs’s NHP humanization solutions can solve most of the problems encountered by researchers and medicine manufactures that large-scale experiments on NHPs are costly and confined to high experimental standards, that systematic fundamental immunological information of NHPs’ gene, protein metabolites and signaling pathways are lacked. Furthermore, the application of NHP humanized antibodies in immunotherapy is hopeful to become more common to fix with the high risk of immune reaction stimulated by humanized rodent antibodies. More details can be reached at https://www.creative-biolabs.com/.

Things to Consider in Creating a Fitness Boot Camp Marketing Plan

Have you been wondering lately what fitness boot camp marketing plan to take into consideration? Since there are numerous marketing tactics available, it is always hard to know which one can work for your particular business. So to help you out, I have list some few pointers to consider in creating your final camp plan.

But before we go into that part, keep in mind that your business is about getting people to be healthy or back into shape. Therefore, you must finalize your fitness program and what are the benefits they could hope for in your camp. In addition, the place or location is what you also need to think about carefully as this could create an impact on the people decision to join your camp.

That is right! These two factors can make or break your fitness camp marketing plan. So if you wish for a better profit and camp, it does not have to be boring and routine. After all, it is a camp so everything is possible, and anything can be done to help them lose weight. Keep these thoughts into account when you finally sat down and plan your marketing strategy.

Now other things you need to consider in your fitness boot camp marketing are listed down below:

* Time and Day - some fitness camp fail to take notice that sometimes the day or time of their camp do not coincide with the most number of people schedule. So in creating your fitness boot camp marketing plan considers the days when you will hold the workout, this can also be useful in promoting your camp. Try to full blast your campaign when the day and time coincides on the time that they are available to listen and read about your offers.

* Age Group - It can really help your fitness boot camp marketing if you can create ads that would target the specifics age bracket. For example, if the camp is for young overweight kids make your ads fun and especially for them. Now for older people, tap their need to get back into shape in order to age well.

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