Cholesterol-25-Hydroxylase Inhibits Sars-Cov-2 And Other Viral Infection Mechanisms

The new coronavirus SARS-CoV-2 causes the 2019 Coronavirus Disease (COVID-19) and is now raging around the world. There is currently no US Food and Drug Administration (FDA) approved method for the treatment of COVID-19. Although several therapies are undergoing clinical trials, the current standard treatment methods are mainly to provide patients with convalescent serum and anti-fever drugs. In order to speed up the search for new COVID-19 treatments, scientists are testing reusable drugs (that is, drugs that have been safely used in the human body so that they can be quickly applied to the clinic) to reduce the ability of this virus infection.

In a new study, researchers from the University of California, San Diego School of Medicine found that cholesterol-25-hydroxylase (CH25H) can prevent the entry of  the coronavirus SARS-CoV-2 by removing cholesterol from the cell membrane.

According to existing research, it is known that the coronavirus will induce interferon (IFN) after infection. Interferon is an active protein (mainly glycoprotein) produced by monocytes and lymphocytes with multiple functions. They have broad-spectrum antiviral activities on the same cells, affecting cell growth, differentiation, and regulating immune function. After detecting the molecular pattern associated with viral infection, the IFN pathway is activated, prompting the further activation of hundreds of interferon-stimulating genes (ISG), thereby interfering with the virus life cycle process. Human type I interferons including IFN-α and IFN-β work through the ubiquitously expressed type I interferon receptor (IFNAR), while type III interferon IFN-λ and epithelial restricted type III interferon receptor Combine and function. In vitro and clinical studies have shown that SARS-CoV-2 is sensitive to type I IFN, and type I IFN treatment may be a promising treatment strategy for COVID-19.

Figure 1. 25-Hydroxycholesterol, produced by the interferon‐stimulated protein CH25H, inhibits the entry of SARS-CoV-2, SARS-CoV, and MERS-CoV.

Cholesterol 25-hydroxylase (CH25H) is a gene that encodes the enzyme that synthesizes hydroxysterol 25-hydroxycholesterol (25HC) from cholesterol. CH25H is an interferon-inducible gene that is strongly upregulated in SARS-CoV-2 infected lung epithelial cell lines and COVID-19 infected patients. Clinical studies have found that 25HC, the product of interferon-induced enzyme CH25H, has broad-spectrum antiviral activity against human coronaviruses. Previous studies have shown that 25HC can inhibit a variety of viruses from entering cells, including VSV, HIV, NiV, EBOV and ZIKV. However, the mechanism by which 25HC regulates virus entry is unclear. Existing studies have shown that the endoplasmic reticulum localization enzyme ACAT uses fatty acyl-coenzyme a and cholesterol as substrates to produce cholesterol esters, which are stored in cytoplasmic lipid droplets and induce the consumption of accessible cholesterol of plasma membrane through an unknown mechanism. When exploring the mechanism of 25HC function, the researchers found that 25HC triggers the consumption of accessible cholesterol in the plasma membrane by activating acyl coa:cholesterol acyltransferase (ACAT), thereby inhibiting the entry of viruses. Cholesterol has multiple effects on the lipid bilayer. The increase or decrease of cholesterol may be accompanied by changes in membrane fluidity, polarity, thickness, and inherent curvature. In addition, the change of cholesterol can affect the function of the entire membrane protein (including viral receptors or co-receptors) by changing its conformation or distribution on the plasma membrane. These changes will directly or indirectly affect the fusion of the virus and the cell membrane, and cell membrane fusion is critical to the release of the viral genome. Due to the importance of membrane cholesterol in virus-cell fusion, the mechanism of 25HC may extend to the cell membrane fusion process of other viruses.

2020 Nobel Prize in Chemistry Awarded to CRISPR-CAS9 Gene Editing Technology

On October 7, 2020, the Royal Swedish Academy of Sciences has decided to award the 2020 Nobel Prize in Chemistry to Dr. Emmanuelle Charpentier of the Max Planck Institute for Pathogenesis in Germany and Dr. Jennifer A. Doudna of the University of California, Berkeley, in recognition of them contributions in the field of genome editing.

Figure 1. Emmanuelle Charpentier and Jennifer A. Doudna. (Image source: NobelPrize.org)

Wonderful Encounter
The discovery and application of CRISPR-CAS9 gene editing technology has become a major revolution in the field of modern gene editing. The emergence of this technology has greatly simplified the process of gene editing. Its discovery process is also full of accidents like other scientific discoveries.
In an occasional conversation with a colleague who is engaged in microbiological research, Dr. Doudna learned that the same code of repeated DNA sequences in the genetic material of extremely different bacteria and archaea appear repeatedly, but they are separated by different sequences. These repeats are called clustered regularly interspaced short palindromic repeats, abbreviated as CRISPR. Because the unique non-repetitive sequences in CRISPR seem to match the genetic codes of various viruses, researchers believe that it is part of the ancient immune system of bacteria, which can protect bacteria and archaea from viruses. If the bacterium successfully resists the virus infection, it will add part of the virus’s genetic code to its genome as a memory of the infection. Although no one knew the molecular mechanism at the time, the basic hypothesis of the scientists at the time was that bacteria used the mechanism of RNA interference to neutralize viruses.

Complex Molecular Mechanism Map

If bacteria are proven to have an ancient immune system, it will become a very important discovery in the scientific community. For this reason, Dr. Doudna began to learn about the CRISPR system out of curiosity. It turns out that in addition to the CRISPR sequence, there is also a special gene called CRISPR-related inside the bacteria, abbreviated as Cas. Dr. Doudna discovered that these genes are very similar to those that encode proteins that are specifically used to melt and cut DNA. Therefore, it has become a new problem to prove that Cas protein has the same function of cutting viral DNA.

A few years later, the research team led by Dr. Doudna succeeded in revealing the functions of several different Cas proteins. At the same time, the system has been discovered by other research groups. The bacterial immune system can take very different forms. The figure below shows the working mechanism of different types of CRISPR / Cas systems. The CRISPR/Cas system studied by Dr. Doudna belongs to Class I; this is a complex mechanism that requires many different Cas proteins to clear the virus. Interestingly, Type II systems are very simple, they require less protein. At the same time, Dr. Emmanuelle Charpentier happened to encounter a Type II system.

Dr. Emmanuelle Charpentier is a scientist with a wide range of interests. While working on pathogenic microorganisms, she is also very interested in small RNA molecules involved in gene regulation. In collaboration with researchers in Berlin, Charpentier et al. located small RNAs inside Streptococcus pyogenes. There is a large number of small RNA molecules that have not been reported before in this bacteria, and its genetic code is very close to the CRISPR sequence in the genome. By carefully analyzing their genetic code, Charpentier discovered that part of this new type of small RNA molecule partially matched the repetitive sequence in the CRISPR gene. Although Charpentier had never been exposed to the CRISPR system before. But her research team used a series of thorough microbiological tests to locate the CRISPR system in Streptococcus pyogenes. According to existing research, this system is known to belong to the class II CRISPR/Cas9 system, that is, only one Cas protein-Cas9 is needed to achieve the purpose of targeted lysis of viral DNA. Charpentier’s research also showed that an unknown RNA molecule (called trans-activated crisp RNA (tracrRNA)) is of decisive importance for the realization of CRISPR’s function. It can help the long RNA molecules produced by the transcription of CRISPR sequences in the genome to be processed into mature, active forms.

Figure 2. Streptococcus’ natural immune system against viruses: CRISPR/Cas9.(Image source: NobelPrize.org)

Dr. Emmanuelle Charpentier is a scientist with a wide range of interests. While working on pathogenic microorganisms, she is also very interested in small RNA molecules involved in gene regulation. In collaboration with researchers in Berlin, Charpentier et al. located small RNAs inside Streptococcus pyogenes. There is a large number of small RNA molecules that have not been reported before in this bacteria, and its genetic code is very close to the CRISPR sequence in the genome. By carefully analyzing their genetic code, Charpentier discovered that part of this new type of small RNA molecule partially matched the repetitive sequence in the CRISPR gene. Although Charpentier had never been exposed to the CRISPR system before. But her research team used a series of thorough microbiological tests to locate the CRISPR system in Streptococcus pyogenes. According to existing research, this system is known to belong to the class II CRISPR/Cas9 system, that is, only one Cas protein-Cas9 is needed to achieve the purpose of targeted lysis of viral DNA. Charpentier’s research also showed that an unknown RNA molecule (called trans-activated crisp RNA (tracrRNA)) is of decisive importance for the realization of CRISPR’s function. It can help the long RNA molecules produced by the transcription of CRISPR sequences in the genome to be processed into mature, active forms.

After in-depth and targeted experiments, Dr. Charpentier published his findings on tracrRNA in March 2011. Although she has many years of experience in microbiology, she hopes to cooperate with more professional scientists in continuing to study the CRISPR-Cas9 system. Dr. Jennifer Doudna therefore became a natural choice. When Charpentier was invited to a conference in Puerto Rico, the two scientists had a historic meeting.

Alliance Between Giants
After further communication, they hope to cooperate to complete the follow-up research. They speculate that bacteria need CRISPR-RNA to recognize the DNA sequence of the virus, and Cas9 is the scissors that ultimately cut the DNA molecule. However, when they tested in vitro, they did not get the expected results. After a lot of thinking and failed attempts, the researchers finally tried to add tracrRNA to their system. Previously, they believed that tracrRNA was only needed to cut CRISPR-RNA into its active form. When Cas9 obtained tracrRNA, the result everyone was waiting for finally appeared: the DNA molecule was cut into two parts. After that, the researchers simplified the “genetic scissors”. Using their new discoveries of tracr-RNA and CRISPR-RNA, they successfully fused the two into one molecule and named it “Guide RNA”. Using a simplified version of this genetic scissors, they successfully achieved the goal of cutting DNA at any position.

Figure 3. The CRISPR/Cas9 genetic scissors. (Image source: NobelPrize.org)

Upheaval in Life Sciences
Shortly after Emmanuelle Charpentier and Jennifer Doudna discovered the CRISPR/Cas9 gene scissors in 2012, several other research groups demonstrated that the tool can be used to modify the genomes of mouse and human cells, leading to their explosive development. Using CRISPR gene editing tools, researchers can in principle cut any genome they want. After that, it is easy to use the cell’s natural system to repair DNA, thereby realizing the “redefinition” of genes. Through the new discoveries of Emmanuelle Charpentier and Jennifer Doudna, life science has successfully entered a new era.

The New Mechanism of Aging To Promote Cancer

As we age, the body will accumulate “garbage” in the body during the process of converting food into energy. Clinical statistics have found that the risk of cancer and related mortality in humans increases significantly with age from the age of 65. Is it because the body’s “junk” substances accumulated due to age cause cancer? Recently, a study published in the journal Nature discovered a special metabolic pathway or plays a potential role in the process of cancer. This A research finding illustrates the mechanism by which the aging process accelerates the development of lethal cancer in individuals, and also provides a new treatment idea for effectively blocking the occurrence of metastatic tumors.

In this study, the researchers focused on cancer metastasis. The metastasis of cancer is the process by which cancer cells break away from the original tumor site and form new tumors at other sites in the body. Through observation and analysis of metastatic cancer cells, the researchers discovered some interesting phenomena, namely, the metabolites of methylmalonic acid (MMA), a byproduct of malonate metabolism, seem to accumulate as the body ages. And as a mediator of tumor progression. In order to analyze whether MMA plays a key role in the process of cancer metastasis, the researchers conducted related studies on people under 30 and over 60. When lung cancer cells and breast cancer cells are exposed to the blood of these people,  the behavior of lung and breast cancer cells whether change. The results of the study showed that in 30 blood samples from young donors, cancer cells in 25 samples did not show any changes, but in 30 blood samples from elderly donors, cancer cells in 25 samples exhibits different characteristics, its migration and invasion capabilities are enhanced, and it also has a certain tolerance to two drugs that are often used to treat cancer.

Figure 1. The relation of MMA and Sox4.

More interestingly, when the treated cancer cells are injected into mice, they will produce metastatic tumors in the lung tissues of the mice. So does MMA induce these changes in cancer cells? Through experimental research, it is found that the key to the changes in cancer cells induced by MMA seems to be the existence of a special reprogramming mechanism, which turns on the expression of the SOX4 gene. Previous studies have shown that SOX4 can make cancer cells more aggressive and easier to metastasize.

To determine the correlation between MMA and SOX4, the researchers blocked the expression of this gene. It was found that after blocking the Sox4 gene, the induction effect of MMA could not be produced. In addition, blocking the function of SOX4 also inhibits the ability of cancer cells to develop resistance to the two cancer therapies. This provides new ideas for new therapies to reduce mortality in cancer populations by descreasing MMA levels.

At present, researchers still have a series of questions to be solved, including why MMA will continue to accumulate in the body as the body ages, and whether the mechanisms found in blood samples and mouse studies are the same in humans. In addition, the blood samples used in the existing research are all from men, and it is necessary to verify whether the same mechanism will also appear in the female body in the later period.

Existing studies have shown that the accumulation of MMA is related to the intake of a high-protein diet, so a low-protein diet may be helpful in the treatment of cancer patients. Theoretically, drugs that lower MMA levels may also work, that is, potentially reducing the malignant spread of cancer in the patient’s body.

SMS and MYC Synergistically Promote The Survival of Colorectal Cancer Cells

It has been clinically found that polyamine biosynthesis is often dysregulated in human cancers, especially in the development of early colorectal cancer. Recent research on polyamine metabolism has made new progress. Researchers at the University of Kentucky have discovered that spermine synthase (SMS) can promote the growth of colorectal cancer.

Studies have shown that SMS is an enzyme that produces spermine from spermidine. It is very important for cell growth. Because the excessive accumulation of spermidine can have a harmful effect on cell viability, the body has a precise mechanism to regulate the level of spermidine in the cell. However, the mechanism of  tumor cells maintain a relatively high level of spermidine and below the toxicity threshold to promote tumor growth remains unclear.

Recently, researchers at the University of Kentucky found that SMS is overexpressed in colorectal cancer (CRC) and plays an important role in balancing the level of spermidine in cells. In this study, the researchers discovered that SMS and MYC together inhibit the important function of the apoptotic protein Bim by a unique regulatory pathway, and found that the down-regulation of Bim is a key survival signal for promoting CRC tumor growth.

Figure 1. A model for SMS and MYC cooperation in CRC tumorigenesis.

Studies have shown that SMS overexpression plays an important role in balancing cell spermidine levels, and low spermidine levels are a necessary condition for the occurrence of CRC. In normal body cells, polyamine homeostasis is necessary for cell growth and tissue regeneration. With age, in model organisms and in humans, due to the decline in polyamine biosynthesis, the concentration of spermidine in tissues also decreases. In contrast, oncogenic signals induce the up-regulation of the biosynthetic activity of polyamine generating enzymes (such as ODC, SRM, SAMDC, and SMS), which in turn leads to an increase in the level of polyamines, which are thought to play an important role in tumor development. However, excessive polyamine accumulation itself can have a serious and harmful effect on cell activity. Similar to these findings, SMS is overexpressed in CRC during the entire tumorigenesis process, which can destroy the excessive accumulation of spermidine, and then make the spermidine in CRC cells at a tolerable level. Under normal physiological conditions, normal cells with properly regulated polyamine homeostasis can tolerate a certain level of spermidine, which is beneficial to their proliferation. In CRC cells, since the up-regulation of polyamine biosynthesis enzymes is much higher than in normal cells, spermidine is converted into spermine to prevent the level of spermidine from exceeding the toxicity threshold. Therefore, SMS gene knockout completely inhibits the function of SMS and induces excessive accumulation of spermidine, which leads to the inhibition of the growth of CRC.

Further studies have found that the targeted destruction of SMS in CRC cells leads to the accumulation of spermidine, which transfers to the nucleus and transcriptionally induces the expression of the pro-apoptotic protein Bim. However, myc-driven expression of miR-19a and miR-19b inhibited the production of Bim, thereby attenuating this induction. In CRC cells lacking SMS, drugs or genes inhibiting MYC activity can significantly induce Bim expression and apoptosis, leading to tumor regression, but these effects will be significantly reduced after Bim is silenced. These findings reveal the key survival signals in CRC that inhibit the expression of Bim through the aggregation of different signals and myc-mediated signaling pathways.

Therefore, inhibiting SMS and MYC signaling is a promising treatment strategy for CRC. The Cancer Genome Atlas data shows that MYC-dependent transcription is activated in almost all CRCs. Therefore, MYC is an attractive therapeutic target for CRC. Although there is no clear ligand-binding domain small molecule drug for MYC, emerging evidence suggests that small molecule compounds (such as JQ1) that target the epigenetic readers of the bromodomain and terminal extradomain (BET) family act as inhibitors of MYC activity. However, the efficacy of BET inhibitors in CRC is generally moderate, indicating that CRC tumors are inherently resistant to BET inhibition. However, the combined use of SMS inhibitors and BET inhibitors may have broad prospects in the treatment of CRC that is abnormally affected by SMS and MYC-mediated signaling pathways.

Prealbumin Blood Test

A prealbumin blood test measures prealbumin levels in your blood. Prealbumin is a protein synthesized in your liver. It helps carry thyroid hormones and vitamin A through your bloodstream as well as regulate energy consumption in the body. If your prealbumin levels are lower than normal, it may be a sign of malnutrition, a condition which your body lacks the calories, vitamins, and/or minerals needed to support normal functions.Determining the level of prealbumin, a hepatic protein, is a sensitive and cost-effective method of assessing the severity of illness resulting from malnutrition in patients who are critically ill or have a chronic disease. Prealbumin levels have been shown to correlate with patient outcomes and are an accurate predictor of patient recovery. In high-risk patients, prealbumin levels determined twice weekly during hospitalization can alert the physician to declining nutritional status, improve patient outcome, and shorten hospitalization in an increasingly cost-conscious economy.

Although the association between poor nutrition and illness has long been recognized, there is a lack of reliable, objective, short-term screening methods to evaluate nutritional risk. Determination of the prealbumin level is a cost-effective and objective method of assessing severity of illness in patients who are critically ill or have a chronic disease.

A prealbumin test may be used to:

* Find out if you are getting enough nutrients, especially protein, in your diet

* Help diagnose certain infections and chronic diseases

Low prealbumin scores mean that you are likely to need a nutritional assessment. Low prealbumin scores may also be a sign of liver disease, inflammation, or tissue death (tissue necrosis). High prealbumin scores may be a sign of long-term (chronic) kidney disease, steroid use, or alcoholism.

TABLE 1 Prealbumin Risk Stratification

PREALBUMIN LEVEL RISK LEVEL
<5.0 mg per dL (<50 mg per L) Poor prognosis
5.0 to 10.9 mg per dL (50 to 109 mg per L) Significant risk; aggressive nutritional support indicated
11.0 to 15.0 mg per dL (110 to 150 mg per L) Increased risk; monitor status biweekly
15.0 to 35.0 mg per dL (150 to 350 mg per L) Normal

Source:  Prealbumin in Nutritional Care Consensus Group. Nutrition 1995;11:170.

Test results may vary depending on your age, gender, health history, the method used for the test, and other things. Your test results may not mean you have a problem. Ask your healthcare provider what your test results mean for you.

Creative Diagnostics can support your testing by providing native prealbumin antigens and several monoclonal antibodies. Our new high-affinity mouse monoclonal antibodies have been validated both in ELISA and Immunoturbidimetric. Clones can be paired with each other and are suitable for the development of diagnostics assays.

Featured Prealbumin/TTR Antigens and Antibodies

Antibody
CABT-L5122 Anti-Human Prealbumin/TTR Mab ELISA, Immunoturbidimetric
CABT-L5123 Anti-Human Prealbumin/TTR Mab ELISA, Immunoturbidimetric
CABT-L5124 Anti-Human Prealbumin/TTR Mab ELISA, Immunoturbidimetric
Antigen
DAGC251 Native Human Prealbumin Purity > 96%

 

Novel Chimera Targeting Lysosomes Can Degrade Extracellular Proteins

When clinical studies find that there are potentially dangerous proteins on cells, researchers hope to shrink themselves into mini-surgeons, specifically eliminate problematic protein molecules, and keep healthy cells intact. In a recent new study, researchers from Stanford University in the United States discovered tools that can excise individual proteins from the surface of cells. The relevant research results were published online in the journal Nature on July 29, 2020.

Most existing treatments for individual proteins rely on interactions with specific activity regulation of the target protein, such as enzyme inhibition or ligand blockade. However, there are some treatment-related proteins whose activities are unknown or unavailable, so the strategies above cannot be used to treat some proteins. In addition, strategies involving protein degradation platforms such as proteolytic targeting chimeras and other platforms (such as dTAGs, Trim-Away, chaperone-mediated autophagy targeting, and SNIPERs) have been developed for proteins that are generally difficult to target. However, these methods require the use of intracellular protein degradation mechanisms, so they are limited to proteins containing intracellular domains, and ligands can bind to these domains and recruit necessary cellular components.

Figure 1. LYTACs using CI-M6PR traffic proteins to lysosomes.

Studies have found that extracellular proteins and membrane-associated proteins account for 40% of all protein-coding genes and are key factors in cancer, aging-related diseases and autoimmune diseases. Therefore, the general strategy of selective degradation of these proteins may improve human health . In this study, the researchers used a conjugate that binds to the lysosomal shuttle receptor on the cell surface and the extracellular domain of the target protein to determine a targeted degradation strategy for extracellular and membrane-associated proteins. These researchers have developed a new class of molecules that can shuttle unwanted proteins from the cell surface or the surrounding environment to the lysosome, which is the cell compartment dedicated to protein degradation. These molecules are called lysosomal targeted chimeras (LYTACs).

Mechanism of action of lysosome targeted chimera (LYTAC)
Existing studies have shown that cation-independent mannose-6-phosphate receptor (CI-M6PR) is involved in the biochemical pathway that mediates the internalization of cell line cargo. LYTAC uses this mechanism to transport labeled targeting molecules to the lysosome for degradation. The LYTAC molecule consists of an oligosaccharide peptide group (which binds to the cell surface receptor CI-M6PR) and an antibody that binds to a specific transmembrane protein or extracellular protein. The antibody can also be replaced by a small protein binding molecule. When combined with CI-M6PR and target protein at the same time, the resulting complex is swallowed by the cell membrane to form a transport vesicle. This brings this complex to the lysosome, after which the target protein is degraded and the receptor CI-M6PR is recycled. By degrading treatment-related proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71, and programmed death ligand, it proves that the platform has a wide range of effects.

The key to this tool’s function lies in its dual-function design. One side of the LYTAC molecule can be customized to bind to any protein of interest. On the other side of it is a short amino acid sequence, or peptide, inlaid with a sugar called mannose-6-phosphate. This sugar acts as a label for the cell. When a cell contains proteins that are sent to the lysosome for degradation, it adds this sugar to ensure they reach their destination. There are receptors on the cell surface that interact with this sugar. When they grab the LYTAC molecule and pull it into the cell, the labeled protein will also be dragged in with it. In the process of attaching this tag to the protein, LYTAC hijacks a natural cell shuttle mechanism designed to escort newly synthesized lysosomal proteins to their new home. However, lysosomal proteins are tough enough to survive in the presence of degrading enzymes encountered in lysosomes, and most proteins cannot do this, so those that are labeled proteins by the LYTAC method are usually destroyed. This selective degradation can help scientists study and treat diseases such as cancer and Alzheimer’s disease related to surface proteins by targeting and degrading proteins that play an important role. More interestingly, the tethered end of LYTAC can be anything that binds to proteins, such as antibodies or existing drugs, so in the future, many other proteins and diseases can be target attacked.

Mitochondrial Metabolites May Be Involved In the Lifespan Adjustment of The Body

Existing research results indicate that the body’s metabolic homeostasis is closely related to body aging. The process of cellular energy production affected by metabolic stress will in turn affect the bioenergy state of the cell and the adaptability of the entire organism. In addition, studies have also found that metabolic stress in early life seems to be involved in chromatin reorganization, epigenetic changes that have a lasting impact on subsequent, and may even affect the aging process.

Studies have found that the regulatory center of these epigenetic changes is located in the mitochondria. Mitochondria not only produce a large amount of 5′-adenosine triphosphate (ATP) through tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) to maintain cell homeostasis, they also participate in the biosynthesis of some biological molecules, such as lipids and heme , Iron-sulfur clusters and intermediate metabolites, these intermediate metabolites can be used as signals to participate in the regulation of the rest of the cell, so that mitochondria become the center of a variety of biological processes. Through the continuous signal communication between the energy control center mitochondria and the nucleus, cells and organisms can monitor and integrate the body’s nutrient utilization and energy demand information, thereby ensuring the body’s metabolic stability.

Although the complete loss or nearly complete loss of mitochondrial function is unfavorable for cells, studies have found that partial inhibition of mitochondrial activity can promote the longevity of worms, flies, and mice. Studies on Caenorhabditis elegans indicate that in the early stages of life, the reduction of electron transport chain (ETC) activity in the mitochondria leads to extensive chromatin reorganization, which is essential for activating the mitochondrial unfolded protein response (UPRmt). This pathway can promote the restoration of mitochondrial protein homeostasis and stress. Specifically, the overall chromatin compaction induced by mitochondrial stress is manifested by the use of histone H3K9 methyltransferase SETDB1/MET-2 and its cofactors ATF7IP/LIN-65 to methylate non-essential genes to avoid the transcription of non-essential genes under stress conditions. The activation of stress-related UPRmt genes is demethylated and activated by histone H3K27 demethylases JMJD-1.2 and JMJD-3.1, thereby causing a sustained response and prolonging lifespan.

Figure 1. Illustration diagram showing that acetyl-CoA availability is nutrient dependent and dynamically regulates histone acetylation levels.

UPR mt is a transcriptional response that can induce the expression of mitochondrial chaperones and proteases, and then repair or degrade the misfolded proteins in mitochondria, and ultimately restore protein homeostasis in mitochondria. In addition, studies also found that UPR mt can also promote the reconnection of cell metabolism to reduce mitochondrial stress and promote cell survival. In Caenorhabditis elegans, when mitochondrial protein homeostasis is disrupted, UPR mt is induced, resulting in a decrease in the efficiency of mitochondrial entry, so that the transcription factor (TF) ATFS-1 that would otherwise enter the mitochondria for degradation enters the nucleus. Another TF and chromatin remodeling factor DVE-1 is also involved in UPR mt signal transduction, but unlike ATFS-1, its nuclear translocation does not depend on the efficiency of mitochondrial entry.

With the deepening of research, people have increasingly realized that many metabolic intermediates such as folic acid, acetyl-CoA (acetyl-CoA) and α-ketoglutarate (α-KG) not only affect the energy and material metabolism of cells , It also has a profound and dynamic impact on the overall chromatin modification. These intermediate metabolites can serve as substrates for enzymes that modify signal proteins, metabolic enzymes, and chromatin proteins. As the main source of epigenetic regulation of metabolites, such as, acetyl-CoA which is the source of histone and protein acetylation, and s-adenosylmethionine is the epigenetic methyl donor. In addition, α-KG produced in the TCA cycle of mitochondria is an essential cofactor for histone demethylase and DNA methylase. In contrast, succinic acid and fumaric acid inhibit these α-KG-dependent enzymes. In short, mitochondrial disturbances can change the nuclear epigenome by affecting various mitochondrial-derived metabolites, and ultimately affect cell proliferation and aging.

Recently, scientists have studied TF DVE-1, which is essential for UPR mt and contains homology domains, and identified nucleosome remodeling and histone deacetylases ( NuRD) complex that mediate the subcellular localization of DVE-1 in response to mitochondrial stress. The results show that after the mitochondrial function is disturbed, the level of mitochondrial-derived acetyl-CoA decreases, and the level of histone acetylation decreases, so that the NuRD complex can achieve overall chromatin reorganization. Restoring acetyl-CoA levels by providing the substrates and nutrients needed to produce acetyl-CoA can prevent the reduction of histone acetylation and inhibit chromatin reorganization under mitochondrial stress. In general, acetyl-CoA is a signal of mitochondrial dysfunction, which regulates body aging through NuRD-mediated chromatin remodeling.

The Dual Characteristics of CD95 on Tumor Growth

Tumor is one of the major challenges that modern medicine must face. In order to find effective diagnosis and treatment methods, researchers actively explore the process and molecular basis of its occurrence and development. It is worth noting that some biomolecules found in the research process that the role of the tumor is not simply to promote or inhibit the tumor, but to play a diametrically opposite function as the environment or tumor development changes.
CD95 (also known as Fas and APO-1) is a classic death receptor, mainly by inducing apoptosis to maintain tissue homeostasis in the immune system. Existing studies have shown that during cancer progression, CD95 is often down-regulated in tumor cells or makes cells resistant to apoptosis, thereby allowing tumor cells to escape apoptosis. However, complete loss of CD95 is rarely seen in human cancer. Many cancer cells express large amounts of CD95 in vitro and are highly sensitive to CD95-mediated apoptosis. In addition, cancer patients often have elevated levels of CD95 and CD95L. These data indicate that CD95 may have its non-apoptotic activity to promote tumor growth.

Figure1. Graphical summary of the role of CD95/CD95L in cancer.

To test the function of endogenous CD95 in tumor cells, CD95-specific short hairpin (sh) RNA lentivirus was used to reduce the expression of CD95 in various human cancer cell lines. Infection with CD95-mediated lentiviral shRNA to the CD95-mediated apoptosis in vitro CD95 overexpressing ovarian cancer cell line HeyA8 greatly reduced the CD95 protein and surface expression, resulting in decreased CD95 apoptosis sensitivity. Interestingly, the abolition of CD95 expression also resulted in a substantial reduction in cancer cell growth. This has been confirmed by another shRNA targeting CD95 (R4) and another ovarian cancer cell line SKOV3.ip1, which expresses a very small amount of CD95 and is completely resistant to CD95L-induced apoptosis. Rebuilding SKOV3.ip1 CD95 knockdown cells and short-term anti-interference (si) RNA CD95 cells to endogenous levels can restore the growth of SKOV3.ip1 cells. In addition, growth inhibition of CD95 expression was also found in cell lines derived from colon cancer (HCT116), renal cancer (CAKI-1), breast cancer (MCF7), and liver cancer (HepG2). It is worth noting that 6 days after lentivirus infection, knockdown of CD95 in MCF7 cells with R6 virus resulted in growth arrest. However, 26 days after infection, the growth of cells expressing moderate CD95 levels was partially restored. However, neither MCF7 nor CAKI-1 cells benefited from the overexpression of CD95, indicating that regardless of absolute levels, cancer cells maintain CD95 expression at a level sufficient to promote optimal growth based on CD95 expression.

Further research shows that stimulation of CD95 in 22 tumor cell lines does not lead to increased proliferation. However, incubating cells with neutralized CD95L monoclonal antibody (mAb) NOK-1 reduced cell growth, indicating that the small amount of CD95L produced by tumor cells can help their growth. To further test this hypothesis, the researchers used three independent shRNAs specific to CD95L based on lentivirus to knock down CD95L. As a result, it was found that when the knockout efficiency was comparable, these viruses caused different degrees of growth inhibition, from reduced growth to complete loss of growth. In addition, knocking out CD95L will also reduce the growth of all other cancer cell lines, suggesting that CD95L is essential for the growth of many tumor cells.

In short, the CD95/CD95L system can promote the growth of cancer. CD95 activates neuronal stem cells and acts as a tumor promoter for glioblastoma by activating Src kinase. In addition, it was recently discovered that mice expressing only soluble CD95L can induce large histiocytic sarcoma in the liver, which may be due to the lack of apoptosis induction and the carcinogenic activity of CD95L. The data also shows that CD95 plays a role in promoting growth mainly through the pathways involving JNK, Jun, Erg1 and Fos.

JNK-mediated Destruction of Bile Acid Homeostasis Promotes Intrahepatic Cholangiocarcinoma

Liver cancer is the fifth most common cancer in the world and the second leading cause of cancer death. Cholangiocarcinoma is the second most common liver cancer. It is a malignant tumor of bile duct epithelium. It is clinically asymptomatic and the global incidence is increasing. Due to the lack of early markers for diagnosis, most patients with cholangiocarcinoma are identified as advanced and metastatic, and eventually die.

Existing studies have found that elevated bile acid (BA) can cause liver cell inflammation, apoptosis and liver cell necrosis, so long-term elevated BA levels in patients are considered to be risk factors for the development of liver cancer. In fact, changes in serum BA may be more useful in the diagnosis of cholangiocarcinoma. Therefore, as a key factor that mediates bile acid homeostasis, JNK has been focused on research. Previous studies have determined that liver c-Jun NH2 terminal kinase (JNK) deficiency can inhibit cholangiocarcinoma cell proliferation and oncogenic transformation in a p53/Kras-induced cholangiosarcoma model; and it is deficient in hepatocellular carcinoma with diethylnitrosamine and NEMO promotes cholangiocarcinoma in the syndrome model. Interestingly, recent studies have found that liver JNK alone is not enough to develop cholangiocarcinoma.

Figure 1. Cholic acid (CA) and chenodeoxycholic acid (CDCA) are absorbed in the ileum via the apical sodium-dependent bile acid transporter (ASBT).

Through the search, scientists discovered an important regulator involved in lipid and BA liver metabolic homeostasis, the receptor α (PPARα) activated by peroxisome proliferators. The study found that JNK can inhibit the activation of PPARα in liver cells, thereby affecting lipid metabolism and steatosis through the liver factor FGF21 (encoded by the PPARα target gene). Further research found that JNK-mediated inhibition of PPARα leads to changes in BA homeostasis, thereby inhibiting cholangiocyte proliferation. Therefore, JNK deficiency will stimulate the proliferation of cholangiocarcinoma cells and promote the development of cholangiocarcinoma. Increased proliferation is caused by changes in BA metabolism and increased liver expression of FXR / FGF15 / FGFR4, which triggers ERK activation in cholangiocellular cells, which in turn activates downstream proliferation-related genes. The activation of FXR by BA triggers human fibroblast growth Factor 15/19 (FGF15 / FGF19) secretion. Clinical studies have found that FGF15/19 is expressed in the liver during the development of hepatocellular carcinoma and intrahepatic cholangiocarcinoma. It is worth noting that FGF15/19 improves blood glucose response and reduces hepatic steatosis, while also promoting liver cancer. Therefore, the steady state change of BA causes FGF15/FGF19 to promote the development of cholangiocarcinoma.Through the search, scientists discovered an important regulator involved in lipid and BA liver metabolic homeostasis, the receptor α (PPARα) activated by peroxisome proliferators. The study found that JNK can inhibit the activation of PPARα in liver cells, thereby affecting lipid metabolism and steatosis through the liver factor FGF21 (encoded by the PPARα target gene). Further research found that JNK-mediated inhibition of PPARα leads to changes in BA homeostasis, thereby inhibiting cholangiocyte proliferation. Therefore, JNK deficiency will stimulate the proliferation of cholangiocarcinoma cells and promote the development of cholangiocarcinoma. Increased proliferation is caused by changes in BA metabolism and increased liver expression of FXR / FGF15 / FGFR4, which triggers ERK activation in cholangiocellular cells, which in turn activates downstream proliferation-related genes. The activation of FXR by BA triggers human fibroblast growth Factor 15/19 (FGF15 / FGF19) secretion. Clinical studies have found that FGF15/19 is expressed in the liver during the development of hepatocellular carcinoma and intrahepatic cholangiocarcinoma. It is worth noting that FGF15/19 improves blood glucose response and reduces hepatic steatosis, while also promoting liver cancer. Therefore, the steady state change of BA causes FGF15/FGF19 to promote the development of cholangiocarcinoma.

In this process, liver PPARα is an important mediator of this regulatory cascade. PPARα deficiency significantly inhibits the phenotype caused by JNK deficiency. However, the role of PPARα in liver cancer remains unclear. Although some studies indicate that PPARα activation may promote liver cancer, other studies indicate that PPARα activation may be neutral or inhibit the development of liver cancer. This may be due to the different experimental conditions used in these studies. Recent studies have shown that the oncogenic effect of PPARα activation is partly due to changes in BA metabolism that drives ERK activation, which indicates that PPARα activation is a key factor in the development and progression of cholangiocarcinoma. The role of JNK /PPARα/ FGF signaling in lipid metabolism suggests that this pathway may represent a therapeutic target for steatosis and obesity. However, the potential risk of this pathway for carcinogenic progression indicates a serious problem with long-term treatment. Therefore, in the clinical treatment of steatosis and obesity using JNK inhibitory treatment strategies should consider the potential risk of cholangiocarcinoma.

Cytokine Storm: Symptoms and Diagnosis

Cytokine storm, also known as cytokine storm syndrome (CSS) or hypercytokinemia, refers to the phenomenon that multiple cytokines, such as TNF-α, IL-1, IL-6, IL-12, IFN-α, IFN-β, IFN-γ, MCP-1, and IL-8, are rapidly and massively produced in the body fluid when the body’s immune system is over-activated. This is an important cause of acute respiratory distress syndrome and multiple organ failure. Once a cytokine storm occurs, it can quickly cause single organ or multiple organ failure, and eventually becomes life-threatening.

Symptoms of a cytokine storm

The daily work of the immune system is to clear the infection, but if the immune system is activated to the limit or loses control, it will harm the host. An extreme immune attack is a cytokine storm.

The cytokine storm is a signal for help, and the purpose is to ask the immune system to fight tooth and nail at once. This kind of suicide attack can damage the virus, but will also leave a lot of wounds to the host. The blood vessels withstood the most important offensive, in which the cytokine storm makes the blood vessel wall easier to penetrate. Therefore arteries, veins, and capillaries all begin to leak blood and plasma. The cytokine storm also triggers a massive release of nitric oxide, which will further dilute the blood and destroy blood vessels. All of these factors combine to lower blood pressure to a dangerous level, so the patient does not die from blood loss, but from a symptom similar to severe septic shock.

Cytokine storm plays an important role in the chronicles of human diseases

The concept of cytokine storm first appeared in graft-versus-host disease (GvHD) in 1993. GvHD shows the symptom that immune cells in the transplant treat the host as a foreign body and attack the host cells. Later, humans gradually discovered that viruses (such as SARS virus and MERS virus) and bacterial infections can also cause cytokine storms. At this time, it is the patients’ own immune cells that attack the host cells.

COVID-19 can trigger a cytokine storm

When SARS-CoV-2 infects humans for the first time, the human immune system has no ability to recognize this virus. Once the virus invades into normal cells, the immune system will not be able to distinguish between friends and foes. When the virus multiplies rapidly and the immune system becomes intolerable and chooses to work hard to clear the virus, a cytokine storm may break out. From this point of view, a virus’s toxicity depends on how destructive the immune response it induces.

Human ACE2 stable cell line is frequently used in research of SARS-CoV-2 since studies have shown that SARS-CoV-2 enters the cell through angiotensin-converting enzyme 2 (ACE2), so the lung tissue with high expression of ACE2 and easy access has become the main invasion target of these viruses. After invading the lungs, the immune system sends a large number of immune cells to the lung tissue to kill the enemy, thus forming pneumonia, and some symptoms appear such as fever, coughing, and expiratory dyspnea.

However, because these immune cells are not capable of destroying the virus accurately, they can only attack indiscriminately and summon more immune cells to kill the enemy, leading to the result that more and more immune cells and cytokines gather together. Once a cytokine storm is formed, the immune system may not be able to kill these viruses, but it will definitely kill a large number of normal cells in the lungs, severely destroying the ventilation function of the lungs, and large white shadows will appear on the lung CT scan, which is known as “white lung”. The patient will have respiratory failure until death from hypoxia.

Diagnosis of cytokine storm

The diagnosis of cytokine storm mainly depends on the detection of inflammatory factors in the blood. However, in fact, different viruses do not trigger cytokine storms through exactly the same mechanism, so they will cause different cytokine changes. For example, SARS-related cytokine storms are mainly related to IL1B, IL6, IL12, IFNγ, IP10, and MCP1, while MERS CoV-induced cytokine storms are mainly related to IFNγ, TNFα, IL15, and IL17. The performance of COVID-19 is different from the above two. On January 24, 2020, The Lancet published a retrospective study of 41 COVID-19 pneumonia in Wuhan University Zhongnan Hospital. In this study, compared with patients with mild symptoms, the expression levels of multiple plasma pro-inflammatory factors (IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, TNFα) in severe cases were significantly higher, and these inflammatory indicators suggested that cytokine storms occurred in patients with severe COVID-19.