Single Cell Sequencing: The Technology, Challenge and Future (IV)

Wolf Reik hopes that epigenetics technology can also reach the level of single cell detection as soon as possible.

What is more difficult than genome and transcriptome research is the epigenome study that attaches to the genome in the form of chemical markers and regulates gene expression. Although the current epigenetics technology has not yet reached the level of single-cell research (because traditional epigenetics research techniques will degrade DNA), researchers are still eager to see the epigenome of individual tumor cells. Tang’s research team has developed a new technology that can study the modification of DNA methylation within a single-cell genome (Genome Res. 23, 2126–2135, 2013). Tang believes that single-cell technology is also really required for epigenome research. Only in this way can researchers understand the difference between this tumor cell and the surrounding tumor cells, and this difference is caused by methylation modification. It is also caused by other mechanisms. The Wolf Reik team at the Wellcome Trust Sanger Institute in the UK analyzed the methylome of 50 to 100 cells, and he said he really wanted to go one step further.

2.6 Exploration to neuron cells

Neuron cells are the latest object used for single-cell research, and scientists are actually not quite sure what information and conclusions can be obtained through these studies. It was only recently that there was experimental evidence that neurons also have different genomes. Despite these research results, scientists are still confused about the diversity of neuronal cells. As early as 2001, Jerold Chun, who was still working at the University of California, San Diego, discovered chromosomal aneuploidy in the brain of mice, and then in human brain cells in 2005. The same phenomenon was found. According to McConnell, who was a graduate student in the Chun laboratory at that time, after getting these results, no one knew what to do next. They are equivalent to discovering the tip of the iceberg. If there is aneuploidy in the cell, there must be a lot of gene mutations, or genome mutations.

Almost at the same time, another group of researchers found that in the human genome, on average, each genome contains 80 to 100 potentially viable L1 elements (this is a kind of self-replication and self-pasting in the entire genome DNA elements), and in brain neuron cells, these L1 elements are active. This study, as well as some other research results, have proved that the genome is at least possible to have diversity, but no one can say clearly how great this variation is.

According to Thomas Insel of the US National Institute of Mental Health, they are just beginning to try to understand the molecular diversity of brain cells. The single-cell research technology in this field plays a key role, not only in determining the (classification) type of neuronal cells and glial cells, but also in helping us understand the experience and development of a certain area of the brain What is the role of gene expression.

Scientists can use several methods to detect single-cell genome variation. The Christopher Walsh team of Harvard Medical School conducted a single-cell L1 element insertion study on 300 neurons taken from the dead brain (Cell 151, 483–496, 2012). They only found a few L1 insertion elements, which indicates that L1 elements should not be the main cause of genomic diversity, but at least in cerebral cortex cells and caudate nucleus cells.

In 2013, several other research groups also conducted genome-wide scanning studies on single human neuronal cells. For example, in an article published in November 2013, a genome-wide sequencing study was performed on 110 frontal cortex neuron cells in the brains of three healthy people. The results were quite surprising. Large segments of CNV mutations (Science 342, 632–637, 2013). Studies on neuronal cells derived from healthy human skin cells have also found the same situation, and these neuronal cells have more CNV than skin cells from which they are derived, which shows that this neuron derived from iPS cells Cells are a very good research material, suitable for research work on cell diversity.

In fact, despite these discoveries, neuroscientists still have a headache because they do not know what these somatic mutations mean. Ira Hall, a geneticist at the University of Virginia, is also one of the collaborators of this article published in Science. He believes that these studies mean that the brain ’s resistance to influence and interference is very weak. In addition, genomic mosaicism can also affect people ’s risk of developing tumors and other diseases. To find out which parts of the brain are more susceptible to interference than other parts and how different the different parts of the brain are, researchers have to study more cells before they can find the answer. McConnell, who is currently engaged in research in this area, believes that he still knows nothing.

2.7 The further development

Although single-cell technology has the potential to solve many major problems in the life sciences, technological progress is far from over. For example, researchers must study how to distinguish true biological differences from the background noise of the test technology itself. Joakim Lundeberg of KTH Royal Institute of Technology in Sweden (who has developed tissue RNA sequencing technology in their laboratory) believes that single cell RNA and DNA sequencing technology is far from being powerful enough, he said that they also need to analyze more single cells in one experiment in order to solve the problem of background noise, which can at least deepen their understanding of the differences between different cells in the same tissue.

Due to various problems, such as cell separation, data calculation, and specificity issues when used in different fields, etc., Blainey hopes that single cell research technology can make greater progress in the next few years.

For newcomers to this field, which transcriptome sequencing technology they choose may be enough for them to have a headache for a long time. Regarding this issue, it should depend on the purpose of the research, such as whether you want to analyze multiple cells to find homologous transcripts, or you want to find low-abundance RNA. “But it’s always a good thing to have multiple methods to choose from,” Quake said. Quake ’s team found that if the reaction volume during pretreatment is controlled to be upgraded (they use the C1 system provided by Fluidigm), then the detection effect of single-cell qPCR technology and single-cell RNA sequencing technology is almost the same (Nat. Methods 11, 41–46, 2014).

With the introduction of commercial products, and the various laboratories who have summed up their “unique secrets” after years of practice, the choice of genome amplification technology is also improving. However, because everyone uses different techniques for genome amplification, it is difficult to directly compare different research results.

At the same time, researchers engaged in cancer research, brain neuroscience research, microbiological research, and drug development and other fields of research will also benefit from these technological advancements. And these technological advancements will also attract many outstanding talents to join the field of single-cell research, such as Reik, who has already made a lot of achievements in epigenetics research. Reik only participated in the single-cell academic conference for the first time last year, and has never been exposed to single-cell research before. Reik is very excited to see so many new technologies. He pointed out that at the beginning people will be excited by the technology itself, and it will not be long before people will use these new technologies to solve important life science problems, which will be more exciting.

To be continued in Part V…


First Direct Sequence of SARS-CoV-2 RNA Achieved

The Lachlan Coin and Sebastian Duchene teams and collaborators in the Department of Microbiology and Immunology, University of Melbourne, Australia, provided the first direct RNA sequence of the SARS-CoV-2, detailed the mRNA structure of the subgenome length of this coronavirus, and described various aspects of coronavirus evolutionary genetics revealed from shared data. Relevant articles were released on March 7 on the preprint server bioRxiv (all articles in bioRxiv were not peer reviewed).


SARS-CoV-2 is a positive single-stranded RNA virus of the family Coronaviridae that is associated with beta-coronaviruses that can infect mammalian and avian hosts, such as the MERS coronavirus and the SARS coronavirus.


To determine the structure of mRNAs of the subgenome length of SARS-CoV-2, researchers used a recently established direct RNA sequencing method based on highly parallel nanopore arrays. Briefly, nucleic acids were prepared from culture material with high levels of SARS-CoV-2 and sequenced on the GridION platform.


With this approach, the SARS-CoV-2 sample yielded 680,347 reads containing 860 Mb of sequence information in 40 hours of sequencing. Consistent with the genome of the cultured isolates of new coronavirus, partial reads belonged to coronavirus sequences (28.9%), including 367 Mb sequences distributed in 29,893 base genomes. Some of them are more than 20,000 bases in length, and the researchers also capture most of the genome on a single molecule.


Through data analysis, the investigators identified 42 sites with predictable 5-methylcytosine modifications that present consistent locations between mRNAs of subgenomic length.


In other positive single-stranded viruses, RNA methylation changes dynamically during infection, affecting host-pathogen interactions and viral replication. Once the data set is available for direct RNA sequences of the SARS-CoV-2, researchers may discover other modifications. Little is currently known about the apparent transcriptome modifications of coronaviruses.


The researchers believe that by using direct RNA sequence data, it helps to gain insight into the molecular biology of SARS-CoV-2 and may help construct a detailed view of the viral subgenome-length mRNA structure.

Somatic Cell Reprogramming (Part Three)

2.2. The replacement system iPS cell inducing factor and mechanism

In order to avoid the risk of tumorigenesis caused by the activation of c-Myc in i PS cells, members of the Myc family, L-Myc and N-Myc, can effectively replace c-Myc to induce human and mouse i PS cells, of which the ability of i PS cells to form chimeric mice with germline transmission can be improved, and the resulting mice do not develop tumors. The pluripotency-related factor Glis1 can also replace c-Myc to promote the reprogramming of human and mouse iPS cells, and the resulting chimeras produced by the mouse iPS cells also have the ability of germ line transmission. Glis1 promotes the induction of iPS cells by affecting a variety of reprogramming pathways, including the expression of genes Myc, Nanog, Lin28, Wnt, Essrb and the MET process.

In addition to being replaced by Klf1, Klf2 and Klf5, Klf4 can also be replaced by the orphan nuclear receptor Esrrb and the OS, two factors to achieve the induction of iPS cells. Esrrb mediates reprogramming by up-regulating pluripotent cell-specific genes. The bone morphogenetic protein Bmp4 can also replace Klf4 to promote reprogramming, and its mechanism of action is mainly to promote the MET process. Nanog and Lin28 can replace c-Myc and Klf4 to obtain human iPS cells. Nanog is one of the important factors to maintain the pluripotency of mouse embryonic stem cells. It cooperates with transcription factors such as Oct4 and Sox2 to regulate the pluripotency network, RNA binding protein Lin28 can indirectly regulate the expression of c-Myc to complete reprogramming.

Sox2 can be replaced by Sox1, Sox3, Sox15 and Sox18 of the Sox family. Rcor2 can effectively replace the exogenous Sox2 in the induction of mouse and human iPS cells, and the ogenogen factor Obox1 can replace Sox2 to obtain iPS cells. Obox1 can regulate the expression of cell cycle related genes, slow down the excessive proliferation of reprogrammed cells, and promote the MET process to promote somatic cell reprogramming. The ectoderm cell lineage specialization factor GMNN can also replace Sox2 to obtain human iPS cells. The histone variants TH2A and TH2B, which are abundantly expressed in oocytes, can replace Sox2 and c-Myc to promote the reprogramming of iPS cells. The mechanism is to enrich the X chromosome to inhibit X chromosome inactivation.

The orphan nuclear receptors Nr5a1 and Nr5a2 can replace Oct4 to induce iPS cells, respectively. The mechanism is to activate endogenous Oct4 expression. Oct4 can also be replaced by E-cadherin, the main regulator of epithelial cells. Overexpression of E-cadherin can affect the nuclear localization of β-catenin, thereby promoting the expression of pluripotency genes. In the induction of human iPS cells, the pluripotency-related factor TCL-1A can replace Oct4 and OM only to complete the reprogramming of human fibroblasts. The cells are similar and have the ability to differentiate into three germ layers. The transcription factor Brn4 can also replace Oct4 due to its homologous POU domain.

As the mechanism of each inducing factor in the induction process of iPS cells was gradually elaborated, the induction system in which the classic OSKM four factors were completely replaced also gradually appeared. Studies have shown that Sox2 can initiate the expression of pluripotency genes, including Sall4, Esrrb and Lin28, among which Sall4 can activate other pluripotency genes including Oct4. Therefore, two induction systems that completely replace OSKM are reported: Sall4, Esrrb, Lin28 and Dppa2 or Nanog composed of four factors can complete the induction of iPS cells. However, the induction efficiency of these two systems is low. The six-factor induction system that completely replaces OSKM includes Glis1, Sall4, Lrh1, Jdp2, Jhdm1b, and Id1, which can obtain iPS cells more efficiently.

2.3. The chemical induction system of iPS cells and mechanism

Since exogenous reprogramming factors are easily integrated into the cell genome, and multiple reprogramming factors are involved in the regulation of cancer-related signaling pathways, minimizing the number of transcription factors in the induction system is the first step to improve the biological safety of iPS cells. The use of small molecular compounds instead of reprogramming factors is one of the research directions for improving the safety of iPS cells and optimizing iPS cell technology. The establishment of a complete induction system using small molecule chemicals has revealed more signaling pathways and epigenetic mechanisms related to induced pluripotency.

Histone deacetylase inhibitors (HDACi) Valproic acid (VPA) can effectively replace c-Myc to complete the reprogramming of mouse fibroblasts, and can even replace c-Myc and Klf4 Reprogramming of human fibroblasts. Vitamin C can be reprogrammed with histone demethylase Jhdm1a instead of Klf4, c-Myc and Jhdm1b instead of Sox2, Klf4, c-Myc Inhibiting TGF-β signaling can activate the expression of endogenous Nanog to replace Sox2 and c-Myc. Since the activation of TGF-β plays an important role in the ME differentiation process, inhibiting TGF-β can replace Sox2 induction system. Protein methyltransferase inhibitor BIX01294 and calcium channel agonist BayK8644 can also effectively replace Sox2. Although there have been reports of many systems in which small molecule chemicals are used instead of transcription factors for induction, there has been no progress in screening small molecule compounds to replace Oct4. Using a small molecule combination system of TGF-β inhibitors, HDAC inhibitors, MEK inhibitors and phosphoinositide-dependent kinase PDK1a. Human iPS cells can be induced with a single transcription factor Oct4.

Although both SCNT and iPS cells can bring the cells to a pluripotent state, the ways to achieve pluripotency are different. iPSC technology is to reprogram cells into pluripotent cells similar to ES cells using certain transcription factors or small molecular compounds, while SCNT technology is to transfer donor cells to enucleated oocytes to achieve that donor cells are reprogrammed to a totipotent state similar to fertilized eggs.

The reprogramming speed of SCNT and iPSC technology is also very different. The SCNT reprogramming process occurs very quickly and can be completed within a few hours. The rapid histone replacement driven by egg histones may be the reason for this rapid reprogramming. In contrast, the process of iPS cell reprogramming is relatively slow, and the ectopic expression of exogenous inducing factors first causes a gradual change in cell morphology, and then the expression of pluripotency markers such as alkaline phosphatase and SSEA-1 Before the gradual increase, the expression of somatic cell-specific genes decreased. After a few days or even weeks, the stable expression of endogenous Oct4 and Nanog indicates the completion of the reprogramming process.


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[2] Liu W, Liu X, Wang C, Gao Y, Gao R, Kou X, et al. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing. Cell Discov 2016; 2:16010.

[3] Wen D, Banaszynski LA, Rosenwaks Z, Allis CD, Rafii S.H3.3replacement facilitates epigenetic reprogramming of donor nuclei in somatic cell nuclear transfer embryos. Nucleus 2014;5 (5) :369-75.

[4] Redmer T, Diecke S, Grigoryan T, Quiroga-Negreira A, Birchmeier W, Besser D.E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming. EMBO Rep 2011;12 (7) :720-6.

Will Severe Asthma Be the Next Hot Antibody Drugs (I)?


About Omalizumab

Omalizumab was first FDA approved in 2003 to treat adults and children 12 years of age and older with moderate to severe persistent allergic asthma which is not controlled by inhaled steroids. Since its U.S. approval, more than 200,000 patients older than 12 with allergic asthma have been treated. In August 2017, CFDA officially approved Novartis Tall® (Omalizumab) for the treatment of moderate to severe persistent allergic asthma, which means that China’s asthma treatment has entered the era of antibody drugs. Then in September 2018, a new prefilled syringe formulation of this drug was approved by the FDA.

Omalizumab has been listed for 17 years, and in 2016 brought Novartis nearly $ 800 million in annual sales. It is estimated that there are about 340 million asthma patients in the world, and the asthma drug market is about 16 billion US dollars and will continue to grow.

The role of antibody drugs in the treatment of asthma, especially severe uncontrollable asthma, is becoming more and more important, and its market share will also increase.

Facing the complicated immune system and asthma pathology, which targets are more likely to become the next market contenders?

About Asthma

Asthma is a chronic inflammatory disease of the respiratory tract. The pathogenesis is more complicated. It is generally believed that it is caused by genetic and environmental factors. The main features of the disease are airway hypersensitivity, reversible airflow obstruction, bronchospasm muscle spasm, and airway inflammation. Common symptoms include wheezing, difficulty breathing, coughing, and chest tightness.

The 2014 Global Asthma Report shows that the incidence of asthma is about 4.3%, which means that about 334 million people worldwide have asthma of varying degrees. It is expected that by 2025, this number will increase by 100 million to 434 million.

Asthma imposes a great financial burden on society and individuals. It is estimated that the annual expenditure of asthma in advanced economies accounts for 1% to 2% of their total medical expenditure, which is higher than the sum of tuberculosis and AIDS.

The Market Share of Asthma

The treatment of asthma generally follows the step-by-step treatment guidelines of the Global Initiative for Asthma (GINA). Commonly used drugs include inhaled / oral glucocorticoids (ICS / OCS), leukotriene receptor antagonist (LTRA), long-acting β2 receptor agonist (LABA), short-acting β2 receptor agonist (SABA), Long-acting muscarinic receptor antagonist (LAMA), theophylline, etc. Antibody drugs have also been included in treatment guidelines since 2005.

Due to the large number of patients and the long course of illness, asthma drugs have always been the mainstay of respiratory system drugs. The global asthma drug market in 2022 is likely to reach US $ 22 billion.

From the perspective of drugs, long-acting β2 receptor agonists + glucocorticoid combined inhalation (LABA + ICS), or both single-use drugs dominate the market, accounting for almost half of the market. Considering that there are some ultra-long-acting β2 receptor agonists (ultra-LABA) that are used once a day in the research and development, the market position of these drugs will be further consolidated in the next five years.

LAMA occupies the second place in the market (18%). As a commonly used rapid asthma relieving agent, SABA can account for approximately 6.5% of the market. As the classic combination of LABA + ICS faces patent expiration and other issues, new combination dosage forms such as LAMA + LABA, LAMA + LABA + ICS and other new combinations run into the market to replace classic combination.

An obvious trend is the rapid rise of antibody drugs. Although there are only four asthma antibody drugs in the global market, it is certain that this proportion and market share will continue to grow rapidly. It is expected to reach 2.2 billion US dollars in 5 years.

Antibody drugs: hope for patients with severe uncontrollable asthma

Most asthma patients can be well controlled under the existing treatment guidelines, but about 5% to 10% have severe or uncontrollable asthma. However, the medical expenses of this small group of patients can account for more than 60% of the entire asthma treatment expenditure, because the deterioration rate and hospitalization rate of this part of the patients are relatively high, occupying more outpatient and emergency resources.

Individually, patients with severely uncontrollable asthma spend more than type 2 diabetes, stroke, or chronic pulmonary obstruction (COPD).

The following factors have led to the limitations of existing guidelines for the treatment of severe asthma:

First, the curative effect is limited. Patients with mild to moderate asthma can be well managed according to the guidelines, but the mortality and morbidity of severe asthma patients have not been effectively improved.

Second, adverse reactions. Inhaled drugs have good safety record, and oral drugs should be more noticeable. For example, oral glucocorticoids are more likely to cause systemic adverse reactions and potential morbidity; theophylline has a narrow therapeutic window and poor tolerance.

Third, the patient’s compliance is poor. It is estimated that approximately 50% of children and 30-70% of adults cannot strictly follow the treatment regimen of the asthma guidelines. There are many reasons for this, such as improper use of inhalants, complicated treatment plans, and patients’ concerns about adverse reactions.

Fourth, there are comorbidities. Many asthma patients have obesity, cardiovascular disease, allergies, smoking and other comorbidities, which bring more challenges to treatment. Usually, comorbidities and asthma form a vicious circle.

Because antibody drugs directly target the immune mechanism of asthma, it has brought hope to many severely uncontrollable patients. In order to better understand the targets of antibody drugs, let’s first understand the inflammatory mechanism of severe asthma.

The inflammatory response of asthma is very complicated, involving a series of immune cells and inflammatory molecules in the respiratory cavity.

Triggering of allergic asthma begins when respiratory epithelial cells secrete interleukins 25, 33 (IL-25, IL-33) and thymic stromal lymphopoietin (TSLP) after exposure to allergens, thereby activating dendritic cells (DC).

The immunogen is presented to helper T cells (Th0) after DC treatment, and the latter secretes IL-4 to activate Th2 helper cells. Th2 cells are activated to release more IL-4 and IL-13, thereby promoting B cells to produce immunoglobulin E (IgE).

IL-4 and transforming growth factor β (TGFβ) secreted by Th2 cells can also activate Th9 helper cells to secrete IL-9, thereby promoting the growth of mast cells.

Mast cells bound by IgE can bind antigens, causing the cells to degranulate and release a large amount of chemical mediators, such as histamine, prostaglandins, leukotrienes, etc., which induces the contraction of bronchial smooth muscle and further stimulates the inflammatory response. Th2 cells can also secrete IL-5 to ensure the survival and growth of eosinophils.

In addition, Th17 helper T cells are an important role in the pathogenesis of non-eosinophilic asthma, which can produce IL-17 to recruit and expand neutrophils.

To be continued in Part II…


The Importance of Ceramide to The Skin in Spring

The recent weather has been cold and hot. I believe that many sensitive partners have unstable skin temperature and environmental changes, which cause facial skin, allergies, redness, peeling, and even some small rashes. A bad word is Began to rotten. So how can friends with sensitive muscles prevent allergies in the more sensitive season of spring?

The formation of red blood cells is mostly caused by improper skin care, which results in too thin stratum corneum, which leads to the loss of skin barrier protective factor ceramide. If ceramide is insufficient, the barrier function is reduced, the skin cannot lock in nutrients and moisture, and it is easy to become red, hot, and dry. Frequent skin allergies. How to cure the red blood on the face, the most effective method is to replenish the ceramide lost from the skin.

The main symptoms of red blood cells are the damage to the skin barrier, and the disorder of the skin’s metabolic function makes the skin unable to operate normally. How to cure the red blood on the face, and the formation of red blood is not a matter of two or three days, but also the result of long-term accumulation.

Since you don’t want the symptoms of rotten skin, you need to base your skin. Only healthy skin can cope with the change of harsh environment, then we have to talk about this ceramide. Ceramide is naturally occurring in our skin and is a lipid. It forms a waterproof barrier on the surface of the skin and helps the skin retain moisture. It is also an important part of the stratum corneum, and plays an important role in maintaining the moisture balance of the stratum corneum. Therefore, ceramide can not only help the skin lock water, but also promote the skin barrier to repair itself and regulate skin cells. Unfortunately, as you get older, the ceramides in your skin are gradually lost.

Ceramide is a gospel for people with sensitive muscles. It can not only help thicken the stratum corneum, increase the tolerance of the entire skin, protect it from harmful substances from the outside world, avoid allergies, and repair red bloodshot blood. In addition, ceramide has a very good anti-aging, whitening and antioxidant effects. And more than one type of ceramide in our skin is formed by the reconciliation of multiple ceramides, and different ceramides have different effects.

Sufficient ceramide in the skin can resist external stimuli, like having a strong barrier. But if it is missing or not, the skin loses its natural protective effect, and it has no defense ability against all external physical and biological damage. Therefore, timely supplementation of ceramide is very imminent.

Don’t want your skin to become dry with age, lose elasticity or even have an allergic reaction, just prepare a ceramide skin care product!

Proteins Involved in DNA Repair May Contribute to the Suppression of Cancer

Every day, cells in the body undergo numerous divisions. New cells are used to replace old, damaged or dead cells. However, before a cell divides, DNA is copied first to make a precise copy and passed on to the new cell.

To start the replication process, the DNA double helix unfolds first, so that each strand can be used as a template for the synthesis of new DNA. Scientists call the stretched DNA strand fragments replication forks. As this highly complex replication process proceeds, both strands of the original DNA may break or be damaged thousands of times.

“In fact, DNA sequences suffer considerable “damage” during replication, which is why complex mechanisms are needed to ensure that the replication process is protected.”


Accumulating evidence suggests that the pathway for repairing DNA breaks is called homologous recombination (HR). The key factor in HR is a protein called Rad51 that binds to a single DNA strand at the broken end and supports the replication fork by transforming the fork into a structure similar to a chicken foot.

In a recent study, the authors constructed mutants of RAD51 to better understand its key function at the replication fork.

“We created a recombination-deficient variant of RAD51 that still binds DNA, but it does not search for complete copies of DNA or perform strand exchange,” said the authors. “The more we learn about this process, the more likely we are to figure out how it creates problems in cancer, and we can help improve future treatment strategies.”

In a recent study published in Nature Communications, Mason found that the strand-exchange activity of Rad51 is not required for the repair process, but that it is important to restart replication after removing the barrier.

“The field wants to understand the role of this pathway in cancer and the role of each participating factor,” Mason said.

Other scientists in the field of cancer biology could apply the RAD51 gene mutation to their own studies to help further elucidate the replication process and better understand ways to repair breaks, Mason says.