Connect with us

Health

Why was the human genome never completed?

Published

on

human genome

Why was the human genome never completed? So far, no human genome has been read completely. Scientists expect to pass this milestone for the first time this year.

Why was the human genome never completed?

Before the end of 2023, you should be able to read the complete human genome, which will be the story of a person; It will also provide insight into who he is and where he came from, and his future. The complete human genome is probably not very fun at first glance and it will be very, very long. But the online publication of the complete human genome without any flaws will be a very important moment.

At this point you may feel like you’ve heard this before: the human genome was published years ago. Was it not done perfectly?

In fact, the human genome had never been completely read. The first draft of the human genome was published in 2001, and then in 2003, a group of scientists from the Human Genome Project announced that they had completed the work. This sequence, which was prepared by combining DNA fragments from different people, became the reference sequence with which the DNA of other humans can be compared.

Compiling the human genome by combining the genomic information of several individuals was the best that scientists could do at the time, but it had significant flaws and errors. Later versions of the human genome improved, but many problems remained. Only in the last few years has technology advanced enough to read the entire human genome without gaps and with minimal errors.

But all human genome sequences published so far have been hybrid, using DNA from multiple individuals. This year, the entire genome of a person (a man named Leon Pushkin) is going to be published for the first time. This complete and single human genome will be a monumental technical achievement. It’s only been 70 years since Rosalind Franklin’s black-and-white image revealed the double helix structure of DNA and revolutionized scientists’ understanding of how genetic information is stored. Today, we have the ability to read the entire genetic book that gives rise to the unique characteristics of a human being.

But the project’s geneticists say this is just the beginning. They want to sequence the genomes of people from around the world to create a true picture of the genetic diversity of the human species. They want to find out what the previously unsequenced parts of the DNA do. They also want to introduce whole-genome sequencing into clinics to help doctors diagnose and treat diseases.

In short, the human genome will never be complete, and we will never finish reading it.

The first human genome

The Human Genome Major Project (HGP) was one of the largest scientific projects at a cost of about $3 billion. The goal of this project, which began in 1990, was to read the entire DNA that the average human carries in his cells. The first draft of the sequence was published a little over a decade later. In 2001, at the same time, another version of the human genome was published by Celera Genomics.

لئون پشکین

The human genome came with many promises. With the help of the human genome, we understand what genes do, especially genes that play a role in diseases. This enables personalized medicine where we receive treatments tailored to our genetic makeup.

The complete human genome also provides insights into our evolutionary origins: how exactly are we different from our closest living relatives, chimpanzees, and bonobos?

Some of these promises have happened and some have not yet been fulfilled. Our knowledge of the function of many genes and their roles in diseases ranging from breast cancer to schizophrenia has increased. However, most diseases are affected by hundreds of genes, so we are still a long way from genomic medicine.

Read More: Scientists have found a way to diagnose colon cancer more easily

A small number of inherited diseases are caused by a faulty gene, and the use of genetic screening to identify people at risk for rare diseases is largely limited to those at high risk.

Genetics has also changed our understanding of human evolution. For example, it has been shown that our ancestors interbred with other hominids such as Neanderthals.

Meanwhile, in the background of scientists’ efforts to provide a complete human genome, lies the unpleasant fact that the human genome has never been truly complete. While geneticists have been revising it since the first draft was published (last revised in February 2022), parts of the genome are still missing.

Repeated sequences

One of the problems is that parts of DNA are highly repetitive. In certain parts of the genome, the same sequences are repeated over and over, sometimes thousands of times.

Duplicated DNA often appears in similar parts of the genome. Our DNA is not stored as a long, continuous rope, but is divided into smaller pieces called chromosomes.

Chromosomes are X-shaped (except for the Y-shaped chromosome carried by males), and there are 23 pairs of them in human cells. Each of the chromosomes has duplicated DNA at the end of its four arms (telomeres) and at the central junction (centromeres), and both of these are important.

Telomeres act as protective caps and their damage is associated with aging. Meanwhile, centromeres are important for the process of cell division that underlies growth and reproduction. DNA rearrangements at centromeres play a role in the development of some cancers.

The Human Genome Project failed to sequence the duplicated DNA and did not attempt to do so. Their method could not solve this challenge. They didn’t read the entire genome at once, but instead divided it into small pieces a few hundred bases long, read them, and then put the sequences together using a computer. This method is not efficient for repeated segments, because the computer does not know in what order those segments are put together. “Eight percent of the copy that was officially completed in 2003 was missing,” says Adam Filippi, head of the genome informatics division at the National Human Genome Research Institute in Maryland.

Therefore, our duplicated DNA remained almost completely unread for 20 years. Then in 2021, Filippi and his colleagues announced that they had all read it.

What is the genome?

The genome is often compared to a book written in the DNA alphabet instead of the English alphabet. The DNA alphabet consists of only four letters: A, C, G, and T. Each of these letters represents different molecules called “bases” that are strung along the length of the DNA molecule. Any particular sequence of these letters constitutes a gene. The responsibility of translating this information lies with the molecular machines inside our cells. Some genes provide the information needed to make different proteins that have different functions in the body, while other parts of DNA have regulatory functions. What the Human Genome Project team achieved was the exact order of bases along the length of DNA; Something like CGATTTCCGAAAA and so on for over three billion characters.

Reading the human genome from beginning to end

The Telomere to Telomere (T2T) Consortium was not a big, famous, multi-billion dollar project. “It was really a public effort that took place during birth,” says Karen Miga, a geneticist at the University of California, Santa Cruz. In the eyes of many genomic experts, we appeared out of nowhere.

” A key advance was the ability to accurately read long stretches of DNA, says Evan Eichler, a professor of genome sciences at the University of Washington in Seattle. Previously, technologies capable of reading long sequences had been developed, but until recently they were not accurate enough. Therefore, improving the accuracy of these technologies was a key development. Also, the ability to read sequences that spanned over 100,000 bases was an important advance.

تشخیص بیماری های ژنتیکی

T2T’s first major breakthrough came in July 2020 when the project’s researchers published the complete sequence of the X chromosome. At the time, the best available sequence of the X chromosome had 29 gaps, and the T2T team filled in all the gaps. The following year, they published the complete sequence of chromosome 8. In 2021, they also published a preprint titled “The Complete Sequence of a Human Genome,” in which they filled in 8 percent of the missing sequence.

Reading repetitions

But the human genome had not been read completely yet. Ishler says the team used a little trick that some called cheating.

Most cells in our body have two copies of each chromosome: one from the mother and one from the father. This makes it more difficult to put the sequences together on the computer because the two versions differ very little. To solve this problem, T2T used abnormal cells that have two copies of the father’s DNA that are nearly identical. The mentioned cells were the result of a hydatiform mole (molar or baby-eating pregnancy), which is a type of failed pregnancy.

Eggs and sperm have only one copy of each chromosome, so when a sperm fertilizes an egg, the resulting embryo has two copies. However, sometimes the egg loses its DNA and is then fertilized. Then the egg cell, which has lost its DNA, replicates the sperm’s DNA. Hydatidiform moles form dangerous lesions that look somewhat like cancer and must be removed. This is what T2T sequenced. According to some researchers, they had read only half of the genome, because the complete genome has two copies of any particular sequence. Although overall, their sequence was a clear improvement over previous sequences and added more than 200 million letters and two thousand genes to the human genome.

Having a complete genome means finally being able to understand what the repetitive segments of DNA do, Miga says. “Now that we have these maps, I’m very excited to see what sequences are in these regions,” he says. But what is their main function? And if there is a problem in these areas, how can it contribute to our understanding of human disease and human health?”

Repetitive DNA contains many sequences that can move around the genome and are called “mobile DNA”. “Many of these elements have played a role in our recent evolution,” says Rachel O’Neill, a molecular geneticist at the University of Connecticut in Storrs. “Many evolutionary mutations, including placentation, loss of the tail, and some brain functions, can be attributed to this type of driver DNA.”

Meanwhile, Eishler refers to duplications, where long stretches of DNA that can contain multiple genes are duplicated at once. These sequences can evolve at an extraordinary rate. Ishler says: “The result of this phenomenon is the emergence of new genes that are specific to humans. “These genes contribute disproportionately to the differences that make us human.”

While the human and chimpanzee genomes are 99% identical, duplications are one of the ways in which important differences can arise between us and chimpanzees. The originally published human genome was largely devoid of these duplicated sequences.

Neuroscientists have shown that some duplicated genes are important in brain function. But geneticists couldn’t study them precisely because they were in repeats that didn’t occur in older genomes.

The T2T sequence was finally published in a special issue of the journal Science in March 2022. But at that time the team was moving forward.

The remaining large gap was the Y chromosome, which is only present in males. Sperm usually carry only one sex chromosome (either an X chromosome or a Y chromosome). Because the hydatidiform mole DNA used by T2T came from sperm that contained an X chromosome, the Y chromosome was not sequenced. The team needed a male donor to finish their work, so they used Pushkin.

DNA donor

Pushkin is a systems biologist at Harvard Medical School in Boston, Massachusetts. Much of his research focuses on understanding the mechanisms of aging and how to slow them down. He believes that the human life span has no limit and can be increased. Genomics is a big part of his work. Pushkin has donated his DNA to a number of major sequencing projects.

Pushkin’s first donation was to the Personal Genome project, which was launched in 2015. The goal of the project was to attract volunteers who were willing to share their DNA publicly to enable faster and more efficient research, as well as to overcome fears about the potential misuse of genomic data.

A decade later, Pushkin’s DNA was again used by the GIAB project. The goal of the project was to sequence the genomes of cell lines that could be grown indefinitely in the laboratory and make it easier to study the effects of mutations. Pushkin’s genome was favorable because he had also enrolled his parents in the project and provided them with information on his mother, father, and son.

تعیین توالی ژنوم

Pushkin does not regret his choices, although he points to an unpleasant consequence. “I can’t go to labs that work with my cells, because if my immortal cells somehow get into my body, my immune system won’t recognize them, and there’s a chance that the immune system will go into overdrive, and it’s a dangerous situation,” he says. come.” He is delighted to have his DNA sequenced again by T2T, this time in full.

In December 2022, T2T published another preprint paper describing the complete sequence of Pushkin’s Y chromosome. Since this chromosome has many repetitions and complications, more than half of the chromosome was not present in the past genomes. The new sequence added more than 30 million characters including dozens of genes.

The team is now working on Pushkin’s complete genome, including both copies of each chromosome. “We’ve finished sequencing and reconstructing it,” says Filippi. The resulting genome is complete and without defects and takes duplications into account. All that remains is the review. Filippi says there are a handful of errors we can check. He says their final genome should be published this year.

Pan Genome

Will the human genome be completed this year? The answer is no because there is no single human genome. Each person’s DNA is different and these differences are important. We won’t really understand the genome unless we have a record of how it differs between different populations.

The initial HGP project attempted to address this problem by drawing its sample from a few individuals, all from New York. The sequence that was released was a combination of all of them. Indeed, they tried to provide an average genome, but an American city cannot represent the full spectrum of human genetic diversity. This is why many members of the T2T Consortium are also enrolled in another project: the Human Pangenome Reference Consortium. The goal of this project is to sequence the genomes of hundreds of people from all over the world.

The project’s genomes will not be complete, as they will lose some degree of completeness in exchange for using automated methods that allow them to include more people in the study. In July 2022 the team published a preprint describing the 47 sequenced genomes that they had combined to create a draft “pangenome.”

They are now collaborating with researchers from around the world. “We don’t want this to be done exclusively in one place,” says Ishler. I think it is better to have genomes, especially from populations whose genetic diversity we have not identified well, and to do this in their own communities and by their own people.”

Pangenome’s effort has already paid off. Filippi is a co-author of a study published in January that identified a mechanism for a genetic abnormality. About 1 in 1,000 babies have a Robertsonian translocation, in which two chromosomes fuse together. If the genetic material is not destroyed, the health of the person is not affected, but in some cases, it can lead to conditions such as Down syndrome.

There appears to be a conserved sequence of DNA (that is, a sequence that is the same across species) that is found on multiple chromosomes. This can confuse the cellular mechanisms of DNA replication and cause chromosomes to fuse together. The critical sequence is located in a region that is both repetitive and highly variable between individuals, so it cannot be studied without multiple complete genomes.

Such findings explain why many project researchers want whole genome sequencing to be done in hospitals as well. “My ultimate goal is to be able to replicate T2T genomes in the clinic for any disease,” says Filippi. The methodology we have developed is in this direction. The cost of genome sequencing has fallen dramatically over the decades, so much so that the cost of the T2T project was much less than the cost of the original HGP.

Clearly, there is still much to learn from our genome. As new techniques reveal more secrets of the genome and make it possible to sequence more genomes, there is no end in sight. “As long as humans exist, the Human Genome Project will continue,” says O’Neill.

Health

Discovering the 100% effectiveness of a drug in preventing HIV

Published

on

By

HIV

Scientists say that a new drug for the prevention of the HIV virus (HIV) has shown 100% effectiveness in a clinical trial.

Discovering the 100% effectiveness of a drug in preventing HIV

A large clinical trial in South Africa and Uganda has shown that a twice-yearly injection of a new HIV prevention drug before exposure gives young women complete protection against infection from the virus.

The trial tested whether a six-month injection of the drug, called lenacapavir, provided better protection against HIV infection than two other drugs, both pills that must be taken daily. It should be mentioned that all three of these drugs are preventive drugs (PrEP).

Linda Gale Becker, medical scientist and deputy director of the Desmond Tutu Center for HIV at the University of Cape Town’s Institute of Infectious Diseases and Molecular Medicine and the study’s principal investigator, explains what makes this breakthrough so important and what to expect in the future.

He talks about this trial and what has been considered and achieved. This trial was conducted with 5,000 participants in three locations in Uganda and 25 locations in South Africa to test the effectiveness of lenacapavir and two other drugs.

Lenacapavir is a capsid inhibitor that interferes with the capsid of HIV. The capsid is a protein shell that protects the genetic material of HIV and the enzymes needed for its replication. This medicine is injected under the skin every six months.

The randomized controlled trial, sponsored by the drug’s developers at Gilead Sciences, tested several subjects.

The first was whether a six-month injection of lenacapavir is safer than daily Truvada F/TDF, which has been widely used and available for more than a decade and can provide better protection against HIV infection as a preventive measure. For women between the ages of 16 and 25 years old or not.

Second, the trial also tested whether taking a newer pill called Descovy F/TAF daily was as effective as Truvada.

Descovi pill is newer and has superior properties than Truvada. Pharmacokinetics refers to the movement of drugs into the body. The Descovi pill is also smaller than Truvada.

This experiment was conducted randomly and in a double-blind manner. This means that neither the participants nor the researchers knew which participants received which drug until the end of the clinical trial.

HIV and AIDS

Human immunodeficiency virus (HIV) is a type of slow-spreading virus (retrograde virus with low replication speed) and the cause of AIDS. HIV attacks the vital cells of the body’s immune system, including helper T lymphocytes (CD4+ T), macrophages, and dendritic cells, and is a virus that disrupts the function and destroys a type of cells responsible for immune coordination, leading to the failure of the human body’s immune system, which It is called AIDS.

It may take between 6 months and 15 years from the time HIV enters the body until the onset of AIDS. During this time, although a person may appear to be healthy, the virus may spread from him to others.

HIV is mainly transmitted through unprotected sexual intercourse, transfusion of infected blood and contaminated needles, and from mother to child during pregnancy, childbirth, or breastfeeding. Some body fluids such as saliva and tears cannot transmit HIV. Prevention of HIV mainly through safe sex and needle exchange programs is considered as a solution to prevent the spread of this disease.

AIDS or Acquired Immune Deficiency Syndrome is also a type of disease that is caused by the entry of HIV and the attack of the immune system by this virus. The disease caused by HIV has three main stages. In the first stage (acute infection), a person may experience an influenza-like illness for a short period, which, of course, does not happen to all people, for this reason, the disease is usually followed for a long period without any symptoms, which leads to this stage of The disease is called the incubation period.

As the AIDS disease progresses, the body’s immune system becomes weaker and people suffer from more infections such as infections and opportunistic cancers and tumors. Of course, it usually shows a lower incidence in people whose immune system works well. Finally, the disease will enter the third stage, or AIDS when the number of CD4+ T cells reaches less than 200 cells per microliter.

Young women in East and Southern Africa are the population bearing the brunt of new HIV infections. Also, taking medications daily is challenging for them due to social and structural reasons.

During this clinical trial, none of the 2,134 women who received Lancapavir became infected with HIV, which means that the drug was 100% effective.

In comparison, 16 of 1,068 women (1.5 percent) who took Truvada and 39 of 2,136 (1.8 percent) who received Descovi became infected with HIV.

The results of an independent review by the Data Safety Monitoring Board led to the recommendation that the “blind” phase of the trial should be stopped and that all participants should be given a choice.

This panel is an independent committee of experts who are formed at the beginning of the clinical trial. They monitor unblinded data at specific times during the trial to monitor progress and safety and ensure that the trial is continued or stopped if there is clear harm or benefit in one group over the others.

What is the importance of these tests?

This success gives great hope because it shows that preventive drugs can be effective in protecting people against HIV.

Last year, 1.3 million new HIV infections were reported worldwide. Although this number is lower than the 2 million infections observed in 2010, it is clear that at this rate we will not reach the target of reducing HIV prevalence to less than 500,000 people worldwide by 2025, and may not even reach We will not reach the goal of ending AIDS by 2030.

Preventive drugs are not the only means of prevention

Preventive medications should be provided alongside access to condoms, screening and treatment for sexually transmitted infections, and access to contraception for women of reproductive age.

In addition, young men should also be circumcised for health reasons.

Despite these options, we are not yet at the point where we can stop new infections, especially among young people, the researchers say.

For young people, taking the pill daily or using a condom or taking the pill during intercourse can be challenging.

HIV scientists and activists hope that young people will embrace it, given that it only needs to be injected twice a year. For a young woman who for social reasons cannot take the pill every day, just two injections a year is an option that can save her from contracting HIV.

What is the next plan?

The plan is to continue the trial now in a non-double-blind fashion. This means that participants will have a choice between using any of those three drugs.

Another trial is also being planned. The test is being conducted among transgender people in a number of regions, including some parts of Africa.

It is important to conduct trials between different groups because researchers have observed differences in the effectiveness of drugs. Also, the type of sexual intercourse is important and may affect the effectiveness of the drugs.

Read more: Skin cancer: symptoms, prevention and treatment

How much time is left until the release of the drug?

Gilead’s press release stated that within the next few months, the company will submit the case with all the results to a number of national authorities, particularly those in Uganda and South Africa.

The World Health Organization will also review this data and may issue recommendations based on it.

The researchers say, we hope that after that this new drug will be used in the guidelines of the World Health Organization and countries.

We also hope to see the drug tested in more studies to better understand how it might combine in different real-world settings, the researchers say.

Of course, the price of this drug is a vital factor to ensure its access and distribution in the public sector.

Gilead Sciences has said it offers licenses to companies that make generic drugs, another important way to keep prices down.

In an ideal situation, governments could purchase the drug at an affordable price and make it available to everyone who wants it and needs protection against HIV.

Continue Reading

Health

A miracle cure for cancer with one pump

Published

on

By

cancer

A miracle cure for cancer with one pump. New experiments by Swedish and Austrian researchers show that injecting drugs into the tumor with an electronic pump can significantly increase the effectiveness of cancer treatment.

A miracle cure for cancer with one pump

When low doses of cancer drugs are continuously injected near malignant brain tumors using a technology called iontronic, the growth of cancer cells is greatly reduced.

According to Mirage News, researchers from “Linköping University” (Linköping University) in Sweden and “Med Uni Graz” (Med Uni Graz) in Austria showed this result in an experiment on bird embryos. Their results are one step closer to providing new and effective treatments for a variety of aggressive cancers.

Malignant brain tumors often recur despite surgery, chemotherapy, and radiation therapy. This is because cancer cells can hide deep in tissue and then grow back. Even the most effective drugs cannot cross the blood-brain barrier that surrounds the blood vessels of the brain and prevents the entry of many substances into the blood. As a result, there are very few treatment options for invasive brain tumors.

In 2021, a group of researchers from Linköping University and the Medical University of Graz demonstrated how to use an electronic pump to locally inject drugs and inhibit cell growth in a malignant and aggressive type of brain cancer called glioblastoma. At that time, experiments were performed on tumor cells in petri dishes.

Read more: Artificial intelligence identifies cancer killer cells

The same research group has now taken the next step to use this technology in the clinical treatment of cancer. This new treatment method can be tested on living tumors. The researchers showed that with the continuous use of low doses of powerful drugs such as “Gemcitabine” by an electronic pump that is located directly in the vicinity of the brain tumor, the growth of cancer cells is reduced.

“Daniel Simon” (Daniel Simon), a professor at Linköping University, said: We have already shown the effectiveness of this concept. Now we’re using a living tumor and we can see that the pump delivers the drug very effectively. So, although this is a simplified model of humans, it can be more confidently said that it works.

The basic concept of the future treatment of glioblastoma involves implanting an electronic device directly in the brain near the tumor. This method allows the use of low doses of strong drugs while bypassing the blood-brain barrier. Accurate dosing in both location and time is critical for effective treatment. This method can minimize side effects because it does not need to circulate throughout the body.

Beyond brain tumors, the researchers hope that iontronic technology can be used in many cancers that are difficult to treat.

Theresia Arbring Sjöström, a researcher at Linköping University, explained: “This is a very stable treatment that the tumor cannot hide from. Even if the tumor and its surrounding tissue try to remove the drug, the materials and control systems used in Untronic can continuously deliver a high concentration of the drug to the tissue adjacent to the tumor.

Linda Waldherr, a researcher at the Medical University of Graz and visiting researcher at Linköping University, said: “In bird embryos, certain biological systems, such as the formation of blood vessels, act similar to those in living animals.” However, we don’t need to implant any devices in them yet. This suggests that although there are still many challenges to be addressed, the concept is viable.

Researchers believe that human trials will be possible within the next five to 10 years. The next steps include further development of materials that allow the implantation of iontronic pumps. Further tests will be conducted on mice and larger animals to further evaluate this treatment.

This research was published in “The Journal of Controlled Release”.

Continue Reading

Health

Making a small version of the intestine in the laboratory

Published

on

By

Making a small version of the intestine in the laboratory

Making a small version of the intestine in the laboratory. A lab-grown small intestine could help provide personalized treatment for Crohn’s disease.

Making a small version of the intestine in the laboratory

Scientists have found a way to reveal the severity of intestinal diseases through epigenetic changes, which could help develop a new treatment plan for patients.

For decades, biomedical researchers have been looking for ways to develop a standard treatment for patients with Crohn’s disease and irritable bowel disease (IBD).

Now, scientists at the University of Cambridge have discovered a way to grow a small intestine in the lab from cells taken from a patient for more precise and personalized treatments.

Professor Matthias Zilbauer, professor of pediatric gastroenterology at the University of Cambridge and Cambridge University Hospitals, explained: “The actual model of this small intestine was made more than a decade ago by a scientist named Hans Clovers. Together with a group of scientists, he discovered structural units called intestinal epithelial stem cells.

He added that the scientists combined this with what is needed for cells to continue growing and dividing after they leave the gut.

Focusing on children with Crohn’s disease

Inspired by this model that grows organoids from humans, researchers in this new study found specific epigenetic findings in patients, especially children and adolescents, with Crohn’s disease.

Crohn’s disease is a chronic inflammatory bowel disease whose cases are increasing worldwide, especially among children. This disease significantly affects the quality of life of patients and can lead to severe complications.

A new pathway called major histocompatibility complex class I (MHC class I) was observed, which appears to be regulated by changes in epigenetic programming.

Scientists have discovered a way to reveal the severity of diseases through epigenetic changes, which could help develop a new treatment plan for patients.

“What we found was that patients with significant epigenetic changes had a more severe disease course,” says Seelbauer.

Drug treatment for the small intestine in vitro before administration to the patient

Scientists hope to develop new drugs that can be tested on this small lab intestine before being given to a patient.

Conventional treatments are only effective 60% of the time, so the vast majority of patients may not respond to them and may even be exposed to severe side effects.

In the future, scientists hope to grow these organoids from patients for drug testing and, if a drug works on the small intestine, administer it to the patient.

The study found that the cells that make up the inner lining of the intestine in patients with Crohn’s disease show increased activity of major histocompatibility complex class I, which are proteins found on the surface of nearly all nucleated cells in the body and are critical for the immune response.

Read more: Artificial intelligence identifies cancer killer cells

This high activity can lead to inflammation by activating immune cells to more easily recognize antigens such as toxins or other foreign substances. Antigens may include molecules from food or gut microbiota that trigger an immune response and contribute to the inflammation characteristic of Crohn’s disease. This is the first time that stable epigenetic changes have been shown to explain intestinal epithelial abnormalities in Crohn’s patients.

The team of researchers is currently working on finding drugs that can modify this pathway.

Continue Reading

Popular