OverviewWhat is DiabetesWhat is diabetes?

Diabetes happens when your body isn't able to take up sugar (glucose) into its cells and use it for energy. This results in a build up of extra sugar in your bloodstream.

Mismanagement of diabetes can lead to serious consequences, causing damage to a wide range of your body's organs and tissues including your heart, kidneys, eyes and nerves.

The process of digestion includes breaking down the food you eat into various different nutrient sources. When you eat carbohydrates (for example, bread, rice, pasta), your body breaks this down into sugar (glucose). When glucose is in your bloodstream, it needs help a "key" to get into its final destination where it's used, which is inside your body's cells (cells make up your body's tissues and organs). This help or "key" is insulin.

Insulin is a hormone made by your pancreas, an organ located behind your stomach. Your pancreas releases insulin into your bloodstream. Insulin acts as the key that unlocks the cell wall door, which allows glucose to enter your bodys cells. Glucose provides the fuel or energy tissues and organs need to properly function.

If you have diabetes:

Or

If glucose cant get into your bodys cells, it stays in your bloodstream and your blood glucose level rises.

The types of diabetes are:

Less common types of diabetes include:

Diabetes insipidus is a distinct rare condition that causes your kidneys to produce a large amount of urine.

Some 34.2 million people of all ages about 1 in 10 have diabetes in the U.S. Some 7.3 million adults aged 18 and older (about 1 in 5) are unaware that they have diabetes (just under 3% of all U.S. adults). The number of people who are diagnosed with diabetes increases with age. More than 26% of adults age 65 and older (about 1 in 4) have diabetes.

Factors that increase your risk differ depending on the type of diabetes you ultimately develop.

Risk factors for Type 1 diabetes include:

Risk factors for prediabetes and Type 2 diabetes include:

Risk factors for gestational diabetes include:

The cause of diabetes, regardless of the type, is having too much glucose circulating in your bloodstream. However, the reason why your blood glucose levels are high differs depending on the type of diabetes.

Symptoms of diabetes include:

Other symptoms

Type 1 diabetes symptoms: Symptoms can develop quickly over a few weeks or months. Symptoms begin when youre young as a child, teen or young adult. Additional symptoms include nausea, vomiting or stomach pains and yeast infections or urinary tract infections.

Type 2 diabetes and prediabetes symptoms: You may not have any symptoms at all or may not notice them since they develop slowly over several years. Symptoms usually begin to develop when youre an adult, but prediabetes and Type 2 diabetes is on the rise in all age groups.

Gestational diabetes: You typically will not notice symptoms. Your obstetrician will test you for gestational diabetes between 24 and 28 weeks of your pregnancy.

If your blood glucose level remains high over a long period of time, your bodys tissues and organs can be seriously damaged. Some complications can be life-threatening over time.

Complications include:

Complications of gestational diabetes:

In the mother: Preeclampsia (high blood pressure, excess protein in urine, leg/feet swelling), risk of gestational diabetes during future pregnancies and risk of diabetes later in life.

In the newborn: Higher-than-normal birth weight, low blood sugar (hypoglycemia), higher risk of developing Type 2 diabetes over time and death shortly after birth.

Diabetes is diagnosed and managed by checking your glucose level in a blood test. There are three tests that can measure your blood glucose level: fasting glucose test, random glucose test and A1c test.

Less than 100

Less than 140

Less than 5.7%

Gestational diabetes tests: There are two blood glucose tests if you are pregnant. With a glucose challenge test, you drink a sugary liquid and your glucose level is checked one hour later. You dont need to fast before this test. If this test shows a higher than normal level of glucose (over 140 ml/dL), an oral glucose tolerance test will follow (as described above).

Type 1 diabetes: If your healthcare provider suspects Type 1 diabetes, blood and urine samples will be collected and tested. The blood is checked for autoantibodies (an autoimmune sign that your body is attacking itself). The urine is checked for the presence of ketones (a sign your body is burning fat as its energy supply). These signs indicate Type 1 diabetes.

If you have symptoms or risk factors for diabetes, you should get tested. The earlier diabetes is found, the earlier management can begin and complications can be lessened or prevented. If a blood test determines you have prediabetes, you and your healthcare professional can work together to make lifestyle changes (e.g. weight loss, exercise, healthy diet) to prevent or delay developing Type 2 diabetes.

Additional specific testing advice based on risk factors:

Diabetes affects your whole body. To best manage diabetes, youll need to take steps to manage your risk factors, including:

You hold the keys to managing your diabetes by:

Checking your blood glucose level is important because the results help guide decisions about what to eat, your physical activity and any needed medication and insulin adjustments or additions.

The most common way to check your blood glucose level is with a blood glucose meter. With this test, you prick the side of your finger, apply the drop of blood to a test strip, insert the strip into the meter and the meter will show your glucose level at that moment in time. Your healthcare provider will tell you how often youll need to check your glucose level.

Advancements in technology have given us another way to monitor glucose levels. Continuous glucose monitoring uses a tiny sensor inserted under your skin. You don't need to prick your finger. Instead, the sensor measures your glucose and can display results anytime during the day or night. Ask your healthcare provider about continuous glucose monitors to see if this is an option for you.

Ask your healthcare team what your blood glucose level should be. They may have a specific target range for you. In general, though, most people try to keep their blood glucose levels at these targets:

Having a blood glucose level that is lower than the normal range (usually below 70 mg/dL) is called hypoglycemia. This is a sign that your body gives out that you need sugar.

Symptoms you might experience if you have hypoglycemia include:

You might pass out if your hypoglycemia is not managed.

If you have too much glucose in your blood, you have a condition called hyperglycemia. Hyperglycemia is defined as:

or

Treatments for diabetes depend on your type of diabetes, how well managed your blood glucose level is and your other existing health conditions.

Oral medications and insulin work in one of these ways to treat your diabetes:

Over 40 medications have been approved by the Food and Drug Administration for the treatment of diabetes. Its beyond the scope of this article to review all of these drugs. Instead, well briefly review the main drug classes available, how they work and present the names of a few drugs in each class. Your healthcare team will decide if medication is right for you. If so, theyll decide which specific drug(s) are best to treat your diabetes.

Diabetes medication drug classes include:

Many oral diabetes medications may be used in combination or with insulin to achieve the best blood glucose management. Some of the above medications are available as a combination of two medicines in a single pill. Others are available as injectable medications, for example, the GLP-1 agonist semaglutide (Ozempic) and lixisenatide (Adlyxin).

Always take your medicine exactly as your healthcare prescribes it. Discuss your specific questions and concerns with them.

There are many types of insulins for diabetes. If you need insulin, you healthcare team will discuss the different types and if they are to be combined with oral medications. To follow is a brief review of insulin types.

There are insulins that are a combination of different insulins. There are also insulins that are combined with a GLP-1 receptor agonist medication (e.g. Xultophy, Soliqua).

Insulin is available in several different formats. You and your healthcare provider will decide which delivery method is right for you based on your preference, lifestyle, insulin needs and insurance plan. Heres a quick review of available types.

Yes. There are two types of transplantations that might be an option for a select number of patients who have Type 1 diabetes. A pancreas transplant is possible. However, getting an organ transplant requires taking immune-suppressing drugs for the rest of your life and dealing with the side effects of these drugs. However, if the transplant is successful, youll likely be able to stop taking insulin.

Another type of transplant is a pancreatic islet transplant. In this transplant, clusters of islet cells (the cells that make insulin) are transplanted from an organ donor into your pancreas to replace those that have been destroyed.

Another treatment under research for Type 1 diabetes is immunotherapy. Since Type 1 is an immune system disease, immunotherapy holds promise as a way to use medication to turn off the parts of the immune system that cause Type 1 disease.

Bariatric surgery is another treatment option thats an indirect treatment for diabetes. Bariatric surgery is an option if you have Type 2 diabetes, have obesity (body mass index over 35) and considered a good candidate for this type of surgery. Much improved blood glucose levels are seen in people who have lost a significant amount of weight.

Of course other medications are prescribed to treat any existing health problems that contribute to increasing your risk of developing diabetes. These conditions include high blood pressure, high cholesterol and other heart-related diseases.

Although diabetes risk factors like family history and race cant be changed, there are other risk factors that you do have some control over. Adopting some of the healthy lifestyle habits listed below can improve these modifiable risk factors and help to decrease your chances of getting diabetes:

No. Type 1 diabetes is an autoimmune disease, meaning your body attacks itself. Scientists arent sure why someones body would attack itself. Other factors may be involved too, such as genetic changes.

Chronic complications are responsible for most illness and death associated with diabetes. Chronic complications usually appear after several years of elevated blood sugars (hyperglycemia). Since patients with Type 2 diabetes may have elevated blood sugars for several years before being diagnosed, these patients may have signs of complications at the time of diagnosis.

The complications of diabetes have been described earlier in this article. Although the complications can be wide ranging and affect many organ systems, there are many basic principles of prevention that are shared in common. These include:

If you have diabetes, the most important thing you can do is keep your blood glucose level within the target range recommended by your healthcare provider. In general, these targets are:

You will need to closely follow a treatment plan, which will likely include following a customized diet plan, exercising 30 minutes five times a week, quitting smoking, limiting alcohol and getting seven to nine hours of sleep a night. Always take your medications and insulin as instructed by your provider.

If you havent been diagnosed with diabetes, you should see your healthcare provider if you have any symptoms of diabetes. If you already have been diagnosed with diabetes, you should contact your provider if your blood glucose levels are outside of your target range, if current symptoms worsen or if you develop any new symptoms.

Sugar itself doesn't directly cause diabetes. Eating foods high in sugar content can lead to weight gain, which is a risk factor for developing diabetes. Eating more sugar than recommended American Heart Association recommends no more than six teaspoons a day (25 grams) for women and nine teaspoons (36 grams) for men leads to all kinds of health harms in addition to weight gain.

These health harms are all risk factors for the development of diabetes or can worsen complications. Weight gain can:

Most people with diabetes see their primary healthcare provider first. Your provider might refer you to an endocrinologist/pediatric endocrinologist, a physician who specializes in diabetes care. Other members of your healthcare team may include an ophthalmologist (eye doctor), nephrologist (kidney doctor), cardiologist (heart doctor), podiatrist (foot doctor), neurologist (nerve and brain doctor), gastroenterologist (digestive tract doctor), registered dietician, nurse practitioners/physician assistants, diabetes educator, pharmacist, personal trainer, social worker, mental health professional, transplant team and others.

In general, if you are being treated with insulin shots, you should see your doctor at least every three to four months. If you are treated with pills or are managing diabetes through diet, you should be seen at least every four to six months. More frequent visits may be needed if your blood sugar isn't managed or if complications of diabetes are worsening.

Although these seem like simple questions, the answers are not so simple. Depending on the type of your diabetes and its specific cause, it may or may not be possible to reverse your diabetes. Successfully reversing diabetes is more commonly called achieving remission.

Type 1 diabetes is an immune system disease with some genetic component. This type of diabetes cant be reversed with traditional treatments. You need lifelong insulin to survive. Providing insulin through an artificial pancreas (insulin pump plus continuous glucose monitor and computer program) is the most advanced way of keeping glucose within a tight range at all times most closely mimicking the body. The closest thing toward a cure for Type 1 is a pancreas transplant or a pancreas islet transplant. Transplant candidates must meet strict criteria to be eligible. Its not an option for everyone and it requires taking immunosuppressant medications for life and dealing with the side effects of these drugs.

Its possible to reverse prediabetes and Type 2 diabetes with a lot of effort and motivation. Youd have to reverse all your risk factors for disease. To do this means a combination of losing weight, exercising regularly and eating healthy (for example, a plant-based, low carb, low sugar, healthy fat diet). These efforts should also lower your cholesterol numbers and blood pressure to within their normal range. Bariatric surgery (surgery that makes your stomach smaller) has been shown to achieve remission in some people with Type 2 diabetes. This is a significant surgery that has its own risks and complications.

If you have gestational diabetes, this type of diabetes ends with the birth of your child. However, having gestational diabetes is a risk factor for developing Type 2 diabetes.

The good news is that diabetes can be effectively managed. The extent to which your Type 1 or Type 2 diabetes can be managed is a discussion to have with your healthcare provider.

Yes, its possible that if diabetes remains undiagnosed and unmanaged (severely high or severely low glucose levels) it can cause devastating harm to your body. Diabetes can cause heart attack, heart failure, stroke, kidney failure and coma. These complications can lead to your death. Cardiovascular disease in particular is the leading cause of death in adults with diabetes.

Although having diabetes may not necessarily increase your risk of contracting COVID-19, if you do get the virus, you are more likely to have more severe complications. If you contract COVID-19, your blood sugars are likely to increase as your body is working to clear the infection. If you contract COVID-19, contact your healthcare team early to let them know.

Blood vessels are located throughout our bodys tissues and organs. They surround our bodys cells, providing a transfer of oxygen, nutrients and other substances, using blood as the exchange vehicle. In simple terms, diabetes doesnt allow glucose (the bodys fuel) to get into cells and it damages blood vessels in/near these organs and those that nourish nerves. If organs, nerves and tissues cant get the essentials they need to properly function, they can begin to fail. Proper function means that your hearts blood vessels, including arteries, are not damaged (narrowed or blocked). In your kidneys, this means that waste products can be filtered out of your blood. In your eyes, this means that the blood vessels in your retina (area of your eye that provides your vision) remain intact. In your feet and nerves, this means that nerves are nourished and that theres blood flow to your feet. Diabetes causes damage that prevents proper function.

Unmanaged diabetes can lead to poor blood flow (poor circulation). Without oxygen and nutrients (delivered in blood), you are more prone to the development of cuts and sores that can lead to infections that cant fully heal. Areas of your body that are farthest away from your heart (the blood pump) are more likely to experience the effects of poor blood flow. So areas of your body like your toes, feet, legs and fingers are more likely to be amputated if an infection develops and healing is poor.

Yes. Because unmanaged diabetes can damage the blood vessels of the retina, blindness is possible. If you havent been diagnosed with diabetes yet but are experiencing a change in your vision, see primary healthcare provider or ophthalmologist as soon as you can.

Scientists dont have firm answers yet but there appears to be a correlation between hearing loss and diabetes. According to the American Diabetes Association, a recent study found that hearing loss was twice as common in people with diabetes versus those who didnt have diabetes. Also, the rate of hearing loss in people with prediabetes was 30% higher compared with those who had normal blood glucose levels. Scientists think diabetes damages the blood vessels in the inner ear, but more research is needed.

Yes, its possible to develop headaches or dizziness if your blood glucose level is too low usually below 70 mg/dL. This condition is called hypoglycemia. You can read about the other symptoms hypoglycemia causes in this article. Hypoglycemia is common in people with Type 1 diabetes and can happen in some people with Type 2 diabetes who take insulin (insulin helps glucose move out of the blood and into your bodys cells) or medications such as sulfonylureas.

Yes, its possible for diabetes to cause hair loss. Unmanaged diabetes can lead to persistently high blood glucose levels. This, in turn, leads to blood vessel damage and restricted flow, and oxygen and nutrients cant get to the cells that need it including hair follicles. Stress can cause hormone level changes that affect hair growth. If you have Type 1 diabetes, your immune system attacks itself and can also cause a hair loss condition called alopecia areata.

People with Type 1 diabetes need insulin to live. If you have Type 1 diabetes, your body has attacked your pancreas, destroying the cells that make insulin. If you have Type 2 diabetes, your pancreas makes insulin, but it doesnt work as it should. In some people with Type 2 diabetes, insulin may be needed to help glucose move from your bloodstream to your bodys cells where its needed for energy. You may or may not need insulin if you have gestational diabetes. If you are pregnant or have Type 2 diabetes, your healthcare provider will check your blood glucose level, assess other risk factors and determine a treatment approach which may include a combination of lifestyle changes, oral medications and insulin. Each person is unique and so is your treatment plan.

You arent born with diabetes, but Type 1 diabetes usually appears in childhood. Prediabetes and diabetes develop slowly over time. Gestational diabetes occurs during pregnancy. Scientists do believe that genetics may play a role or contribute to the development of Type 1 diabetes. Something in the environment or a virus may trigger its development. If you have a family history of Type 1 diabetes, you are at higher risk of developing Type 1 diabetes. If you have a family history of prediabetes, Type 2 diabetes or gestational diabetes, youre at increased risk of developing prediabetes, Type 2 diabetes or gestational diabetes.

Diabetes-related ketoacidosis is a life-threatening condition. It happens when your liver breaks down fat to use as energy because theres not enough insulin and therefore glucose isnt being used as an energy source. Fat is broken down by the liver into a fuel called ketones. The formation and use of ketones is a normal process if it has been a long time since your last meal and your body needs fuel. Ketones are a problem when your fat is broken down too fast for your body to process and they build up in your blood. This makes your blood acidic, which is a condition called ketoacidosis. Diabetes-related ketoacidosis can be the result of unmanaged Type 1 diabetes and less commonly, Type 2 diabetes. Diabetes-related ketoacidosis is diagnosed by the presence of ketones in your urine or blood and a basic metabolic panel. The condition develops over several hours and can cause coma and possibly even death.

Hyperglycemic hyperosmolar nonketotic syndrome (HHNS) develops more slowly (over days to weeks) than diabetes-related ketoacidosis. It occurs in patients with Type 2 diabetes, especially the elderly and usually occurs when patients are ill or stressed. If you have HHNS, you blood glucose level is typically greater than 600 mg/dL. Symptoms include frequent urination, drowsiness, lack of energy and dehydration. HHNS is not associated with ketones in the blood. It can cause coma or death. Youll need to be treated in the hospital.

This means your kidneys are allowing protein to be filtered through and now appear in your urine. This condition is called proteinuria. The continued presence of protein in your urine is a sign of kidney damage.

A note from Cleveland Clinic

Theres much you can do to prevent the development of diabetes (except Type 1 diabetes). However, if you or your child or adolescent develop symptoms of diabetes, see your healthcare provider. The earlier diabetes is diagnosed, the sooner steps can be taken to treat and manage it. The better you are able to manage your blood sugar level, the more likely you are to live a long, healthy life.

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The Federal Reserve just raised rates. Heres what it could mean for inflation and interest rates in the new year.

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The use of stem cell therapy in treating patients with autism spectrum disorder (ASD) is not recommended and its promotion and advertisement will be considered professional misconduct, the National Medical Commission (NMC) has said.

The NMCs ethics and medical registration board set up a committee to look into the prescription, recommendation, or administration of such treatment after doctors and parents complained about the mushrooming stem cell therapy centres and their advertisements promising cure for autism. The committee submitted its report on December 6 and it was uploaded on December 14.

The medical fraternity has welcomed the order, saying, there is not scientific basis to this line of treatment for which patients have been shelling out for years.

Dr Samir Dalwai, developmental-behavioural paediatrician, Nanavati Max Hospital, Vile Parle, said, Autism is a chronic condition and the results are slow. And there is a stigma attached to it. However, desperate parents fall for such promotional gimmicks as the therapy is marketed well not just in the city but across the country.

Every week, the hospital gets at least one patient in the 7-8 age group whose parents have tried stem cell therapy to treat autism, but in vain. Because of the stigma or being told by doctors that there wont be much, or slow improvement in their child, parents fall for the stem cell advertisements. Many a time, patients stopped the treatment midway and opted for the therapy hoping to see the miracle cure it promises, he said, adding each therapy reportedly costs 3- 4 lakh.

The neuro-developmental paediatric chapter of Indian Academy of Paediatrics had also written to all government authorities to take action in this regard.

Parul Kumtha, trustee, Forum for Autism, said the therapy neither had enough proof of efficacy nor did it have the Food and Drug Administrations approval.

Many parents have mortgaged their jewellery, property to bear the cost of treatment. If any medical centres are doing research on stem cell therapy, then they should not charge the patients. Also, as the NMC order says, there is no cure for autism and it is medically unethical to promise it as a cure, she said.

Dr Milan Balakrishnan, psychiatrist and member of Bombay Psychiatric Society, said it has been observed that after the therapy, there is a rise in irritability and aggression in the patient.

Recently, a 15-year-old boy with autism was brought to us. He had a lot of anger, and he was beating up his parents too. We had to manage the outbursts with medication. It was during the course of treatment that the parents told us about stem cell therapy and the aggression their son developed as a new symptom after the procedure, he said.

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Live Cell Imaging Market accounted for US$ 1.8 billion in 2022 and is estimated to be US$ 5.0 billion by 2032 and is anticipated to register a CAGR of 9.2% - By PMI  India Shorts

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Is your child suffering from severe headaches and dysfunctional motor skills, it can be a sign of Childhood Leukaemia  APN News

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Is your child suffering from severe headaches and dysfunctional motor skills, it can be a sign of Childhood Leukaemia - APN News

GD2 enriches for breast CSCs. We recently reported that, following the induction of EMT, human mammary epithelial cells show functional properties similar to those of human bone marrowderived MSCs (13). Therefore, we hypothesized that the cell markers expressed on the surface of MSCs could also be expressed on the surface of breast CSCs. To test this hypothesis, we analyzed for the expression of several known MSC cell surface markers (i.e., CD105, CD90, CD106, CD166, CD73, CD271, MSCA-1, and GD2) on HMECs that had been experimentally transformed to become tumorigenic using oncogenic V12-H-Ras (HMLER cells) (21). Absolute expression of most of the markers analyzed could not divide HMLER cells into two distinct subpopulations (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI59735DS1), similar to CD44hiCD24lo cells (12). However, ganglioside GD2, one of the cell surface markers for MSCs, was able to separate HMLER cells into GD2+ (4.5% 2.4%) and GD2 (92.7% 3.8%) populations (Figure 1A and Supplemental Figure 1). Strikingly, GD2+ HMLER cells isolated using FACS appeared spindle-shaped, with limited cell-cell contacts; conversely, the GD2 cells displayed cobblestone epithelial morphology (Figure 1B). Moreover, the GD2+ HMLER cells proliferated approximately 5-fold slower than the GD2 HMLER cells (Figure 1C).

GD2 identifies CSCs in breast cancer. (A) HMLER cells were stained with anti-GD2 antibody by indirect staining and analyzed on an LSR II flow cytometer. GD2+/ gates were drawn based on IgG2a isotype control. FSC, forward scatter. (B) GD2+/ HMLER cells were cell sorted and cultured in cell culture dishes for 4 days. Scale bars: 50 m. (C) 2 104 GD2+/ HMLER cells were cultured in 6-well cell culture dishes in triplicate. Total cells were counted on days 2, 4, and 6 using a Vi-CELL (Beckman Coulter) cell counter. (D) HMLER or MDA-MB-231 cells (1 103) were sorted into each well of 24-well ultra-low attachment dishes containing mammosphere growth medium using the FACSAria II cell sorter. Cells were cultured for 12 days, and the photos were taken using a light microscope. Scale bars: 100 m. (E and F) Number of mammospheres formed from GD2+/ HMLER (E) and MDA-MB-231 (F) cells. The experiment was performed in triplicate. P < 0.01 (G) GD2+/ MDA-MB-231 cells were sorted (1 cell or 5 cells/well) into 96-well ultra-low-attachment dishes containing mammosphere growth medium. Cells were cultured for 12 days, and mammospheres were counted using a light microscope. Scale bars: 200 m. (H) Number of mammospheres formed from single GD2+/ MDA-MB-231 cells. *P < 0.002. (I) Size of mammospheres measured using a hemocytometer. *P < 0.0001.

To further investigate the functional properties of GD2+ and GD2 cells, we sorted HMLER and MDA-MB-231 cells based on GD2 expression and examined them by mammosphere assay. Interestingly, the GD2+ cells from HMLER and MDA-MB-231 cells formed 2-fold more mammospheres compared with GD2 cells (Figure 1, DF, P < 0.01). Direct sorting of GD2+ and GD2 MDA-MB-231 cells into low-attachment 96-well plates at either 1 or 5 cells per well also resulted in a 2-fold increase in sphere formation by GD2+ cells regardless of the number of cells per well compared with GD2 cells (Figure 1, G and H). In addition, the mammospheres generated by GD2+ cells were 3 times larger than those generated by GD2 cells (Figure 1, G and I), indicating that the GD2+ cells are capable of growing better in suspension cultures.

CSCs are known to be more migratory and invasive (1, 3). To examine the migration and invasion potential of GD2+/ cells, we fractionated HMLER cells into GD2+ and GD2 cells and analyzed them for migration and invasion using Boyden chamber Matrigel invasion assays. After 24 hours of incubation, GD2+ HMLER cells migrated to a more than 4-fold greater extent compared with GD2 cells, indicating that GD2+ cells are highly migratory (Supplemental Figure 2). The hallmark of CSCs is their ability to initiate tumor better than their bulk tumor counterparts (1, 2). To determine the tumor-initiating potential of GD2+ cells, we sorted GD2+ and GD2 MDA-MB-231 cells and transplanted them subcutaneously into the flank of NOD/SCID mice at limiting dilutions. At lower cell numbers including 100 or 10 cells/site, the GD2+ cells generated 2- and 5-fold more tumors, respectively, compared with the GD2 fraction (Table 1). However, at higher cell numbers (10,000 or 1,000 cells/site), there were no significant differences in tumor initiation between GD2+ and GD2 cells. These data firmly established that GD2 is a marker of cells capable of initiating tumors at a higher frequency than cells without GD2.

Generation of tumors by GD2+/ cells in vivo

Percentage of GD2+ cells is highest in cell lines with a basal molecular signature. On the basis of gene expression profile (22), breast cancer cell lines have been classified into 3 groups: luminal, basal A, and basal B. We randomly selected 12 breast cancer cell lines representing these 3 subgroups and analyzed them for GD2 expression. Interestingly, the majority of these lines, independent of the subgroup, contained a subpopulation of GD2+ cells at variable levels (Table 2). However, basal cell lines contained a much greater number (mean 9%, range 1.2%17%, n = 6) of GD2+ cells compared with luminal cell lines (median 0.2%, range 03%, n = 6, Table 1, P = 0.00237). Since basal-derived cell lines show greater tumor initiation potential and contain more CSCs based on the previously reported CD44hiCD24lo profiles (23), this finding once again confirms GD2 as a stem cell marker.

Expression of GD2 in breast cancer cell lines

GD2 identifies the CD44hiCD24lo population in breast cancer cell lines and patient samples. Since we found that GD2, similar to previously reported CD44 and CD24 cell surface markers, is capable of separating cancer cells into two populations with differing tumor-initiating potential (7), we hypothesized that GD2 would be mostly expressed in the CD44hiCD24lo cancer cell fraction. To test this, we initially analyzed the expression of CD44hiCD24lo cells in GD2+ HMLER cells and found that more than 85% (85% 3.5%) of GD2+ HMLER cells also displayed a CD44hiCD24lo CSC profile, whereas less than 1% (0.7% 0.2%) of GD2 HMLER cells were CD44hiCD24lo (Figure 2A). In addition, through reverse gating analysis of CD44hiCD24lo HMLER cells, we noted that more than 84% (84% 2.5%) of CD44hiCD24lo HMLER cells were also positive for GD2 (Figure 2B), whereas less than 5% of CD44loCD24hi HMLER cells were GD2+ (4.3% 1.2%). To further determine the correlation between the expression of GD2 and the CD44/CD24 profiles, we sequentially gated HMLER cells into GD2hi, GD2lo, and GD2neg cells. This analysis revealed that GD2 expression levels correlated strongly with the CD44hiCD24lo phenotype (Supplemental Figure 3A). Moreover, by determining the MFI, we found that GD2 expression levels correlated positively with CD44 expression (correlation index, r2 = 0.85; P < 0.0003; Supplemental Figure 3B). To validate the coexpression of GD2 on CD44hiCD24lo cells, we used anti-GD2 antibody from a different source (Abcam, clone 2Q549) to stain HMLER cells in a 4-step staining procedure as explained before (24), along with anti-CD44 and anti-CD24 antibodies. Analysis of GD2+ cells revealed that these cells coexpress CD44hiCD24lo, confirming our initial findings with the 14G2a clone (Supplemental Figure 4).

GD2 identifies CD44hiCD24lo stem cell phenotype in breast cancer cells. (A) HMLER cells were stained with anti-GD2 antibody and with CD44-APC and CD24-FITC using the 4-step staining protocol described in Methods. Cells were electrically gated on GD2+/ cells and displayed in a pseudocolor dot plot with CD44 on the y axis and CD24 on the x axis using FlowJo data analysis software. (B) In an identical experiment, CD44hi/loCD24lo/hi cells were displayed on a pseudocolor dot plot with GD2 on the y axis and FSC on the x axis. (C) Primary breast tumor samples were processed as described in Methods, and the single cells in suspension were stained with anti-GD2, CD44-APC, CD24-FITC, CD45-FITC, and DAPI using the 4-step staining protocol. Cells were initially gated on DAPI-negative cells to exclude dead cells, and the cells were then gated on CD45 cells to exclude hematopoietic cells. GD2+CD45 cells were displayed on a dot plot, with CD44 on the y axis and CD24 on the x axis. Analysis was perfumed using an LSR II flow cytometer. Data were analyzed using FlowJo software.

To further investigate the correlation between GD2 expression and the CD44hiCD24lo profile, we also analyzed primary breast tumor samples (n = 12, Table 3). Using multi-parameter flow cytometry, we excluded CD45+ inflammatory and other hematopoietic cells from dissociated tumor samples. The non-hematopoietic CD45 fraction was then analyzed for the expression of GD2, CD44, and CD24. This analysis of the CD45 fraction revealed that GD2 was expressed, at variable levels from 0.5% to 35% (median 4.35%, range 0.5%35.8%), in tumor samples (Table 3). Importantly, similar to what we observed in cell lines, more than 95.5% (95.5% 2.7%) of GD2+CD45 tumor cells also co-segregated with the CD44hiCD24lo phenotype (Figure 2C). In contrast, only 2.4% (2.4% 0.4%) of GD2CD45 cells exhibited the CD44hiCD24lo phenotype (Figure 2C). Together these findings clearly indicated that GD2 is a marker of a subset of cancer cells with stem cell properties. To validate that the identified GD2+ cells are in fact tumor and not MSCs, we stained human breast tumor tissues with anti-GD2 and epithelial-specific antipan-cytokeratin antibodies. We found coexpression of GD2 and cytokeratin in some of the breast cancer cells, suggesting that GD2 identifies breast tumors cells (Supplemental Figure 5, A and B).

Breast cancer patient samples analyzed: patient number, tumor type, percentage of GD2+ cells

GD2+ and CD44hiCD24lo cells have similar gene signatures. Since we found that GD2 is capable of independently enriching for CSCs as a single marker compared with the previously known double marker CD44hiCD24lo, we compared the global gene expression profiles in these two populations isolated from HMLER cells using microarray analysis. We initially compared the GD2+ fraction with the GD2 fraction of cells (GD2 set) and the CD44hiCD24lo with the CD4loCD24hi fraction (CD44 set) and identified gene signatures specific to the GD2+ and CD44hiCD24lo fractions (Figure 3A). Comparison of the top 600 differentially expressed genes in the GD2 set (GEO GSE36643) and the CD44 set (GSE36643) identified 231 genes as being identical in the two sets (Supplemental Table 1). In addition, we applied Pearsons 2 test with a Yates continuity correction to assess the association between these two cell types and found that the identified 231 genes correlated (100%) between the two groups described above (Figure 3B). This gene expression analysis along with the cell surface protein analysis shown in Figure 2 indicated that GD2+ cells share not only functional properties but also a gene signature with CD44hiCD24lo cells.

GD2+ and CD44hiCD24lo cells have a similar gene signature. (A) Heat maps derived from microarray analysis of CD44hi/loCD24lo/hi and GD2+/ populations of HMLER cells. (B) Two hundred thirty-one genes of the top 600 differentially expressed genes were identical in GD2+ versus GD and CD44hiCD24lo versus CD44loCD24hi groups. These genes were cross-classified in a 2-by-2 table by GD2+ up-/downregulation and CD44hiCD24lo up-/downregulation. Pearsons 2 test with a Yates continuity correction was applied to assess the association. Statistical significance was assessed at the 0.05 level. (C) Biosynthesis reaction of GD2. (D and E) To measure the expression of GD2S/GD3S mRNA, CD44hiCD24lo or CD44loCD24hi and GD2+/ cells from HMLER (D) or MDA-MB-231 cells (E) were FACS sorted, and mRNA was analyzed using qRT-PCR. *P < 0.001.

Among the genes differentially expressed between GD2+ and GD2 populations, GD3S, a key enzyme involved in the biosynthesis of GD3 (an intermediate for GD2, Figure 3C), was found to be upregulated approximately 9-fold in GD2+ compared to GD2 cells (Supplemental Table 2). The microarray data were validated by qRT-PCR (Figure 3D). However, expression of the gene encoding GD2S, which is involved in conversion of GD3 to GD2, was not altered (Figure 3D). Expression of a number of genes involved in migration and invasion, including MMPs (MMP2, MMP7, and MMP19), and EMT-associated markers, including N-cadherin and vimentin, were expressed at higher levels, whereas E-cadherin was expressed at low levels in GD2+ cells (Supplemental Table 2). We confirmed these findings by qRT-PCR (Supplemental Figure 6). In addition, CD44 mRNA was upregulated and CD24 mRNA downregulated in GD2+ relative to GD2 cells, which was confirmed by FACS analysis (Figure 2A). In addition, the stem cell marker nestin was also found to be upregulated in GD2+ cells compared with GD2 cells (Supplemental Figure 7). These and other genes that were differentially expressed in GD2+ versus GD2 cells are listed in Supplemental Table 2. Conversely, as in GD2+ versus GD2 cells, GD3S was overexpressed more than 10-fold in CD44hiCD24lo compared with CD4loCD24hi cells (Supplemental Figure 8), but no significant difference was found in the expression of GD2S between CD44hiCD24lo and CD4loCD24hi cells. This again demonstrates that the expression of GD3S and GD2 strongly correlates with the CD44hiCD24lo phenotype. Similar to HMLER cells, GD2+ cells from MDA-MB-231 cells expressed GD3S at a more than 5-fold-higher level than GD cells, and consistent with our earlier finding, no significant differences in GD2S expression were observed (Figure 3E).

GD2 cells can spontaneously generate GD2+ cells. Since we observed only a 2-fold difference in mammosphere formation and a 2- to 5-fold difference in tumor initiation between GD2+ and GD2 populations, we investigated whether this was due to the generation of GD2+ cells from GD2 cells. In fact, GD2+ and GD2 cells were sorted from HMLER (Figure 4A) and MDA-MB-231 cells (Figure 4B) and cultured in vitro for 12 days in their respective growth media. Surprisingly, approximately 10% of GD2+ HMLER cells had become GD2, and 15% of GD2 cells had spontaneously generated GD2+ cells, and this proportion was almost identical to that in the unfractionated original HMLER cells (Figure 4A). Similarly, the GD2+ and GD2 cells from MDA-MB-231 cells also generated 81% (81% 2.5%) of GD2 and 12% of GD2+ cells, respectively, again reflecting the percentage of GD2+ cells within the parental MDA-MB-231 cell composition. To investigate the generation of GD2+ cells from GD2 cells and vice versa in vivo, GFP-labeled MDA-MB-231 cells were sorted into GD2+ and GD2 fractions, and 1 106 GD2+ and GD2 cells (GD2+/ cells) were subcutaneously transplanted into NOD/SCID mice. Four weeks later the tumors were dissected, and single-cell suspensions were prepared as described in Methods. The cells were then stained with anti-GD2 antibody and analyzed by flow cytometry. Tumors generated by GD2+ cells consisted of nearly 91% 4.5% GD2 cells, whereas 2.4% 1.1% of cells in GD2 derived tumors were positive for GD2. These findings indicate that GD2+ cells can spontaneously achieve a GD2 phenotype and vice versa in vivo (Supplemental Figure 9, A and B).

GD2-depleted cells are able to generate GD2+ cells in culture: a possible role of EMT. (A and B) GD2+/ cells from HMLER (A) MDA-MB-231 (B) cells were FACS sorted and cultured in MEGM medium for 10 days. After incubation, the cells were stained with GD2 antibody (BD) and analyzed on an LSR II flow cytometer. Note the regeneration of GD+ cells in a GD2-depleted population. (C) To determine the possible role of EMT, HMLER cells transduced with two known EMT inducers (Twist and Snail) were stained with anti-GD2 antibody and analyzed on an LSR II flow cytometer. Expression of GD2 is shown on the y axis and FSC on the x axis. Lower panels in A and C represent antibody staining controls without primary antibody for each cell line. (D) Graphic representation of percentage of GD2+ vector control or Twist- or Snail-transduced HMLER cells. (E and F) mRNA expression analysis of GD3S (E) and GD2S (F) in vector- or Twist- or Snail-transduced cells was performed by real-time TaqMan qRT-PCR. *P < 0.001.

Induction of EMT generates GD2+ cells. Since we recently reported that the induction of EMT in HMLER cells results in the acquisition of stem cell properties (12), we also examined the expression of GD2 on HMLER cells induced to undergo EMT by the ectopic expression of either Twist or Snail. Strikingly, we found that the induction of EMT by Snail or Twist resulted in a significant increase in the percentage of GD2+ populations from the initial 18% (control) to 40% in HMLER-Snail cells and 100% in HMLER-Twist cells (Figure 4, C and D). Corroborating our previous data suggesting a correlation between GD3S and CSCs, we also found that the expression of GD3S mRNA increased in the EMT-derived HMLER cells following induction of EMT by 2.5-fold in Snail cells and 8-fold in Twist cells (Figure 4E), which correlates with the total percentage of GD2+ cells in their respective population (40% in Snail and 100% in Twist cells). In contrast, we found no significant difference in the expression of GD2S (Figure 4F), supporting the hypothesis that GD3S is the key regulator in the biosynthesis of GD2.

GD3S is necessary for CSC properties. To investigate the functional role of GD2 in CSCs, we suppressed the expression of GD3S, the critical enzyme involved in the biosynthesis of GD2, in MDA-MB-231 cells using a lentiviral-based shRNA expression vector and achieved more than 80% knockdown (Figure 5A). As expected, GD3S knockdown reduced the percentage of GD2+ cells from 12.3% (12.3% 1.7%) to 5.5% (5.5% 0.8%) in MDA-MB-231 cells (Figure 5, B and C). Since GD3S is known to regulate a-series gangliosides including GM3, we tested whether knockdown of GD3S could induce the expression of GM3 in MDA-MB-231 cells. Flow cytometric analysis revealed that expression of GM3 was increased from 0.4% 0.3% (control cells) to 15% 1.4% (in GD3S knockdown [GD3S-KD] cells), suggesting that knockdown of GD3S was efficient in these cells (Supplemental Figure 10, A and B). In addition, functional analysis revealed that GD3S-KD-MDA-MB-231 (GD3S-KD-MDA231) cells migrated approximately 3-fold less in Transwell Matrigel invasion assays (Figure 5D) and formed 3-fold fewer mammospheres compared with controls (Figure 5E). To further investigate the effects of suppression of GD3S on tumor formation, we subcutaneously injected MDA-MB-231cells expressing either control shRNA or the GD3S shRNA into the flank of NOD/SCID mice. Strikingly, even after 8 weeks, 1 106 GD3S shRNA cells had not formed tumors, whereas the control shRNA cells had formed tumors in 4 of 4 mice (Figure 5F). The growth rate (tumor size) was also dramatically altered, as plotted in Figure 5G.

Knockdown of GD3S reduces cell proliferation, mammosphere formation, and tumor initiation in MDA-MB-231 cells. (A) To measure knockdown of GD3S, vector control, or GD3S-KD-MDA231 cells were analyzed for mRNA expression of GD3S by real-time TaqMan RT-PCR. Relative expression of GD3S is shown. *P < 0.0001. (B) To measure levels of GD2 on the cell surface, vector control or GD3S-KD-MDA231 cells were stained with anti-GD2 antibody and analyzed on an LSR II flow cytometer (BD). GD2 expression is shown on the y axis and FSC on the x axis. (C) Percentage of GD2+ cells in vector control and GD3S-KD-MDA231 cells. *P < 0.01. (D) To measure cell migration, vector control and GD3S-KD-MDA231 cells were cultured in the presence or absence of 30% serum in a Transwell migration chamber. The average number of cells per microscopic field is shown. *P < 0.001. (E) Mammosphere formation assay using either vector control or GD3S-KD-MDA231 cells was performed by seeding 1,000 cells per well in 24-well cell culture dishes containing mammosphere growth medium. After 10 days, the mammospheres were counted under a light microscope. Scale bar: 200 m. Numbers of mammospheres formed from either vector control or GD3S-KD-MDA231 cells are shown. *P < 0.0001. (F) To examine tumor initiation potential, 1 106 vector control or GD3S-KD-MDA231 cells were transplanted subcutaneously into flanks of NOD/SCID mice. At the end of the ninth week, mice were shaved to remove excess hair on the tumors, and photographs were taken. (G) The tumors size was measured between 4 and 9 weeks. P < 0.000001.

Triptolide, a small molecule inhibitor, inhibits GD3S expression and CSC properties. Triptolide, a small molecule anti-inflammatory drug, has been shown to inhibit GD3S in a melanoma cancer cell line (25). Therefore, we investigated whether triptolide could inhibit GD3S in breast cancer cell lines as well. MDA-MB-231 and SUM-159 cells were treated with different concentrations of triptolide for 24 hours. Triptolide inhibited GD3S mRNA expression in both cell types in a dose-dependent manner, with greater than 95% inhibition at 125 nM (Figure 6, A and B). To test whether inhibition of GD3S by triptolide also inhibited GD2 expression, we treated MDA-MB-231 cells with different concentrations of triptolide for either 24 or 48 hours. Absolute cell counts were measured using flow cytometry. A dose- and time-dependent decrease in GD2+ cells was observed after triptolide treatment, indicating the successful inhibition of GD3S by triptolide (Figure 6C). Of note, a decrease in GD2+ cells was seen under conditions that induced apoptosis in less than 5% of cells.

Triptolide inhibits the expression of GD3S, induces apoptosis in MDA-MB-231 cells, and blocks tumor growth in NOD/SCID mice. MDA-MB-231 cells (A) or SUM-159 cells (B) (5 105/well) of 6-well cell culture dishes were treated with 25, 50, 75, 100, or 125 nM triptolide for 24 hours. /, no treatment. Total RNA was extracted, and GD3S expression was measured by qRT-PCR. (C and D) To measure GD2+ cell growth inhibition, 5 105 MDA-MB-231 (C) and SUM-159 (D) cells were plated in each well of 6-well cell culture dishes and treated with 25, 50, 75, 100, or 125 nM triptolide for 24 or 48 hours. After incubation, the cells were detached with trypsin and stained with anti-GD2 antibody and Sytox Red (for dead cells; Invitrogen). The stained cells were analyzed on an LSR II flow cytometer. Absolute numbers of live cells were calculated by measuring 1,000 events for Trucount beads as explained in Methods. (E) To determine the inhibition of tumor growth, 1 106 MDA-MB-231 cells were subcutaneously transplanted into NOD/SCID mice (n = 8; 4 mice/group). A group of the mice were treated with 0.15 mg/kg/d triptolide, and the control group was treated with PBS every day by i.p. injection. At the end of 8 weeks, mice were sacrificed, and tumors were dissected out and photographed. (F) Tumor sizes from the mice in experiment in E were measured every week after tumor engraftment, and the measurements are shown. P < 0.001, week 3. (G) The survival analysis was based on Kaplan-Meier estimation, and groups were compared by the log-rank test. Control (n = 4, black line) and triptolide (n = 4, blue line) were analyzed for cumulative survival. Survival was defined as the time (in weeks) from transplantation until death. P = 0.015.

To further examine whether triptolide could also inhibit tumor growth in vivo, we introduced 1 106 MDA-MB-231 cells subcutaneously into NOD/SCID mice (2 injections per mouse and 4 mice per group). After the tumors reached 50 mm3, we randomly divided the mice into two groups and treated half of the mice with triptolide (0.15 mg/kg/d) and the other half with PBS (control mice) every day by i.p. injection. Interestingly, after 4 weeks, triptolide-treated animals showed a dramatic decrease in tumor growth compared with control mice. Fifty percent of triptolide treated breast tumors were completely tumor free, and there was a more than 8-fold reduction in tumor volume in 25% of mice (Figure 6D). In addition, tumors in triptolide-treated mice were 3-fold smaller in size and 4-fold lighter by weight (Figure 6E and Supplemental Figure 11). Moreover, in a repeat, identical experiment, triptolide significantly prolonged survival of the treated mice (log-rank, control vs. triptolide, P = 0.0015) (Figure 6F). These findings indicate that GD3S plays a major role in regulating GD3S expression and the resulting GD2+ population. Specifically, it affects cell proliferation and tumor initiation of GD2+ breast cancer cells and when inhibited, greatly diminishes tumor growth and increases metastasis-free survival of breast cancerbearing mice.

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JCI - Ganglioside GD2 identifies breast cancer stem cells and promotes ...

Review of Siddhartha Mukherjees The Song of the Cell: Life is cell deep  The Hindu

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Review of Siddhartha Mukherjees The Song of the Cell: Life is cell deep - The Hindu

What is diabetes mellitus?

Diabetes mellitus is a disease of the pancreas, a small organ located near the stomach. The pancreas has two different types of cells that have very different functions. One group of cells produces the enzymes necessary for proper digestion. The other group, called beta cells, produces the hormone insulin, which regulates the level of glucose (sugar) in the bloodstream and controls the delivery of glucose to the tissues of the body. In simple terms, diabetes mellitus is caused by the failure of the pancreas to regulate blood sugar.

The clinical signs of diabetes mellitus are related to elevated concentrations of blood glucose and the inability of the body to use glucose as an energy source.

The four main symptoms of diabetes mellitus are increasedthirst, increased urination, weight loss, and increased appetite. Because of the nature of cats, these signs may go unnoticed, especially in the early stages of disease or if a cat spends a lot of time outdoors. Cats that are fed canned or semi-moist diets receive much of their water intake from their food, and increased water intake will be harder to recognize.

Diabetes mellitus is usually classified into three types of disease:

Type I diabetes mellitus results from total or near-complete destruction of the beta cells. This appears to be a rare type of diabetes in the cat.

Type II diabetes mellitus is different because some insulin-producing cells remain, but the amount of insulin produced is insufficient, there is a delayed response in secreting it, or the tissues of the cat's body are relatively insulin-resistant.Obesity is a predisposing factor in type II diabetes, which appears to be the most common type of diabetes in the cat.

Type III diabetes results from insulin resistance caused by other hormones and can be due to pregnancy or hormone-secreting tumors.

Diabetes mellitus is the second most common endocrine disease in cats. It is seen more frequently in middle-aged to senior cats and is more common in males than females. While the exact incidence is unknown, the number of diabetic cats is increasing at an alarming rate due to the tremendous increase in the number of overweight and obese cats. It is important to note that a cat three pounds over its ideal weight is considered obese, and that means the average domestic cat weighing 13 pounds or more is at high risk for developing type 2 diabetes mellitus.

Diabetes mellitus is diagnosed by the presence of the typical clinical signs (excess thirst, excess urination, excess appetite, and weight loss), a persistently high level of glucose in the blood, and the presence of glucose in the urine.Diabetes is the most common disease that will cause the blood glucose level to rise substantially.

To conserve glucose within the body, the kidneys do not filter glucose out of the blood stream into the urine until an excessive level is reached. This means that cats with normal blood glucose levels will not have glucose in the urine. Diabetic cats, however, have excessive amounts of glucose in the blood, so it spills into the urine. Once blood glucose reaches a certain level, the excess is removed by the kidneys and enters the urine. This is why cats and people with diabetes mellitus have sugar in their urine (glucosuria).

Definitive confirmation of feline diabetes mellitus may require a specialized test called a serum fructosamine test. This test tells us average blood glucose levels over the past 7 -14 days.

Diabetes mellitus is a treatable condition. Although long-term treatment requires commitment and dedication, it can be rewarding to manage this condition successfully in a beloved cat.

Initial steps in treating a diabetic cat include removing potential predisposing causes for the diabetes. For example, some medications such as corticosteroids predispose cats to develop diabetes, and withdrawal of these drugs may lead to resolution of the condition. Obesity is a risk factor for diabetes in cats, so weight normalization may actually lead to resolution of diabetes in some cats.

All cats with diabetes mellitus benefit from being fed a well-balanced diet, and your veterinarian is the best source for guidance about which nutrient profile will best benefit your cat. Many cats with diabetes mellitus benefit from a diet that is high in protein and relatively low in carbohydrates because a relatively low carbohydrate diet decreases the amount of glucose absorbed from the intestinal tract and lowers the requirement for insulin. Unfortunately, while nutrition is a critical element of diabetes management success in cats, it is generally not as easy as making a simple nutritional choice.

Most cats require regular insulin injections to control the diabetes mellitus, at least initially. Your cat may require several hospital visits until an appropriate insulin dosage is determined. New technology has allowed the adoption of home glucose monitoring with the use of a simple device, such as an AlphaTrak 2. Additional home monitoring can involve the evaluation of urine for the presence of glucose, although this is not a very sensitive way to monitor glucose levels and insulin changes should not be made based on urine glucose levels. Most cats will achieve initial stabilization within a few days to a few weeks, and will require once or twice daily injection of a small dose of insulin. Very small needles are available which cause no pain to the cat, and within a short time the procedure becomes routine. Insulin pens are now available which make it even easier to give your pet an insulin injection. Your veterinarian will determine the appropriate administration frequency, dosages, and type of insulin that your cat requires.

Yes, it is important to monitor treatment of diabetes mellitus to be sure the cat is doing well. Home monitoring of blood glucose is becoming more popular and more common, although part of treatment monitoring will involve periodic blood samples collected by your veterinarian.

To assist in the care of your cat, it is particularly valuable to keep accurate records of the following information:

Daily record:

Weekly record:

Although urine test strips cannot be used to guide insulin dose it may be valuable to monitor the quantity of glucose passed in the urine to identify need for further testing including full glucose curves or other laboratory tests.

To collect cat urine, it is usually easiest to replace the normal cat litter with specially designed urine collecting pellets or with clean and washed aquarium gravel overnight. These materials will not soak up any urine, which can then be collected into a clean container for testing. Your veterinarian may provide you with test strips to dip into the urine and measure the sugar level. If there is a marked change in the amount of glucose in the urine or in blood glucose levels, this may indicate the need to modify the insulin dose, but you should never change the dose of insulin without first discussing it with your veterinarian. Changes in insulin doses are usually based on trends in blood glucose levels, as there is normally some day-to-day variation.

If a cat receives too much insulin, it is possible for the blood sugar level to drop dangerously low (hypoglycemia). For this reason, it is important to be very careful to ensure the cat receives the correct dose of insulin.

Clinical signs displayed by a cat with a very low blood sugar level include weakness and lethargy, shaking, unsteadiness and even convulsions. If a diabetic cat shows any of these signs it is important to take a blood glucose reading if you have a home monitoring device, and seek immediate veterinary attention. In mild cases of hypoglycemia, you may observe wobbling or a drunken walk, or the cat may seem sedated when you call or pet them. Low blood sugar is a medical emergency! Your veterinarian can advise you about specific emergency treatment of low blood sugar in your cat that you can deliver at home until the cat can be seen by a veterinarian.

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diabetes-mellitus-in-cats-overview | VCA Animal Hospital

I. Background:

A. Information And Statistics About Diabetes

Diabetes mellitus is a disease of metabolism presenting as a complex group of syndromes that have in common elevated blood glucose levels. It occurs because the insulin produced by the beta cells of the pancreas is either absent, insufficient, or not used properly by target tissues. As a result, the body is unable to metabolize macronutrients in food in the normal way. Since the body cannot convert glucose into energy, high levels of glucose remain in the blood and spill into the urine, eventually resulting in micro-vascular complications (for example, kidney disease and eye disease) and macro-vascular complications (for example, stroke and ischemic heart disease).

There are two major types of diabetes that affect the Medicare population, Type 1 diabetes, previously called insulin dependent diabetes mellitus, and Type 2 diabetes, previously called non-insulin dependent diabetes mellitus.

The goals in the management of diabetes are to achieve normal metabolic control and reduce the risk of micro and macro-vascular complications. Numerous epidemiologic and interventional studies point to the necessity of maintaining good glycemic control to reduce the risk of the complications of diabetes.

Despite this knowledge, diabetes remains the leading cause of blindness, lower extremity amputations, and kidney disease requiring dialysis. Diabetes and its complications are primary or secondary factors in an estimated 9 percent of hospitalizations (Aubert, RE, et al., Diabetes-related hospitalizations and hospital utilization. In: Diabetes in America. 2nd ed. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Disease, NIH, Pub. No. 9514681995: 553570).

Overall, beneficiaries with diabetes are hospitalized 1.5 times more often than beneficiaries without diabetes. Ten percent of these hospitalizations are a direct result of uncontrolled diabetes, and more than half of these admissions occur in beneficiaries 65 and older (National Hospital Discharge Survey, U.S. National Center for Health Statistics, U.S. Department of Health and Human Services, 1990). In expanding the Medicare program to include outpatient diabetes self-management training services, the Congress intended to empower Medicare beneficiaries with diabetes to better manage and control their conditions. The Conference Report indicates that the conferees believed that this provision will provide significant Medicare savings over time due to reduced hospitalizations and complications arising from diabetes. (H.R. Conf. Rep. No. 105217, at 701 (1997)).

According to the National Health and Nutrition Examination Survey (NHANES):

(Diabetes Fact Sheet, The Centers for Disease Control & Prevention https://www.cdc.gov/diabetes/pubs/pdf/ndfs_2011.pdf)

According to the Department of Health and Human Services Centers for Disease Control and Prevention (CDC):

B. Statutory Authority for Diabetic Self-Management Training (DSMT)

Section 4105(a) of the Balanced Budget Act of 1997 (BBA) (pub. L. 105-33), enacted on August 5, 1997, provides for Medicare coverage for DSMT services provided by a certified provider. Section 4105 of the BBA amended section 1861 of the Social Security Act (The Act) by adding a new section (q)(q).

Section 1861(qq) of the Social Security Act (the Act) provides CMS with the statutory authority to regulate Medicare outpatient coverage of DSMT services.

Section 1861(q)(q)(2) provides that the Secretary may recognize a physician, individual, or entity that is recognized by an organization as meeting standards for furnishing these services as a certified DSMT provider. This statute also provides that a physician or other individual or entity shall be deemed to have met such standards if they meet applicable standards originally established by the National Diabetes Advisory Board.

Section 1861(q)(q)(2)(B) of the Act states that a physician, or such other individual or entity, meets the quality standards if the physician, or individual or entity, meets quality standards established by the Secretary, except that the physician or other individual or entity shall be deemed to have met such standards if the physician or other individual or entity meets applicable standards originally established by the National Diabetes Advisory Board and subsequently revised by organizations who participated in the establishment of standards by such Board, or is recognized by an organization that represents individuals (including individuals under this title) with diabetes as meeting standards for furnishing the services.''

Additionally, section 4105(c)(1) of the BBA requires the Secretary to establish outcome measurements for purposes of evaluating the improvement of the health status of Medicare beneficiaries with diabetes.

A final rule (65 FR 83130) was published in the Federal Register on December 29, 2000 which implemented the BBA provisions addressing the coverage, payment, quality standards, and accreditation requirements for DSMT. This final rule also implemented the DSMT regulations which are codified at Title 42 of the Code of Federal Regulation (CFR) sections 410.140 to 410.146.

The CMS regulations at 42 CFR 410.144 provide the authority for the CMS to require the DSMT AOs to use one of the following types of accreditation standards: (1) the accreditation standards set forth at 410.144(a); (2) the accreditation standards issued by the National Standards for Diabetes Self-Management Education Support (NSDSMES) (410.144(b)); or (3) other accreditation standards, so long as they have been submitted to CMS and approved as meeting or exceeding the CMS quality standards described at 410.144(a).

The American Diabetes Association (ADA) and the American Association of Diabetic Educators (AADE) are the two national DSMT AOs approved by CMS to accredit entities that furnish DSMT services. These DSMT AOs are approved by CMS for six-year terms. Section 410.143(a) sets forth the ongoing responsibilities of the DSMT AOs. The requirement at section 410.143(b) sets forth the oversight activities that CMS, or its agent, will perform to ensure that a CMS approved DSMT AO and the entities the organization accredits continue to meet a set of quality standards described at 410.144.

Section 410.145 of the regulations specifies requirements that DSMT entities must meet. Section 410.146 requires that approved entity must collect and record in an organized systematic manner, patient assessment information at least on a quarterly basis for a beneficiary who receives DSMT training.

II. Information about the Diabetic Self-Management Training Accrediting Organizations

A. General Information

B. Information About The ADA Education Recognition Program (ERP)

C. Information About the AADE

III. Contact Information for CMS-designated DSMT Accrediting Organizations

There are two accrediting organizations approved by CMS to accredit DSMT entities. The contact information for these DSMT AO is listed below.

American Diabetes Association (ADA)

2451 Crystal City Drive

Suite 800

Arlington, VA 22202

703-549-1500

Phone: 800-342-2383

Website: http://www.diabetes.org

American Association of Diabetic Educators (AADE)

200 W. Madison

Suite 800

Chicago, IL 60606

Phone: 800-338-3633

Website: https://www.diabeteseducator.org

IV. Oversight and Validation Process for DSMT Accrediting Organizations Accreditation Processes

V. Where to Submit Questions Related to the DSMT Accreditation Program

Questions about the DSMT Accreditation Program may be submitted to the DSMT Accreditation email box at DSMTAccreditation@cms.hhs.gov.

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Process of producing genetically identical individuals of an organism

Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cloning is the process of creating cloned organisms (copies) of cells and of DNA fragments (molecular cloning).

Coined by Herbert J. Webber, the term clone derives from the Ancient Greek word (kln), twig, which is the process whereby a new plant is created from a twig. In botany, the term lusus was used.[1] In horticulture, the spelling clon was used until the early twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o".[2][3] Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.

Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years. It is a reproduction method used by plants, fungi, and bacteria, and is also the way that clonal colonies reproduce themselves.[4][5] Examples of these organisms include blueberry plants, Hazel trees, the Pando trees,[6][7] the Kentucky coffeetree, Myrica, and the American sweetgum.

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed, and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein production, affinity tagging, single-stranded RNA or DNA production and a host of other molecular biology tools.

Cloning of any DNA fragment essentially involves four steps[8]

Although these steps are invariable among cloning procedures a number of alternative routes can be selected; these are summarized as a cloning strategy.

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation, the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitisation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders).[9] In this technique a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies, each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few cells, sterile polystyrene rings (cloning rings), which have been dipped in grease, are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

Somatic-cell nuclear transfer, popularly known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning". The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.[10]

Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer's. The process begins by removing the nucleus (containing the DNA) from an egg cell and inserting a nucleus from the adult cell to be cloned.[11] In the case of someone with Alzheimer's disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient.[11] The embryo will then form a blastocyst which has the potential to form/become any cell in the body.[12]

The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the donor's somatic cell nucleus is inserted into the oocyte.[13] The oocyte will react to the somatic cell nucleus, the same way it would to a sperm cell's nucleus.[13]

The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use.[13] The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm.[13] This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current.[13] This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals.[13]

SCNT is seen as a good method for producing agriculture animals for food consumption. It successfully cloned sheep, cattle, goats, and pigs. Another benefit is SCNT is seen as a solution to clone endangered species that are on the verge of going extinct.[13] However, stresses placed on both the egg cell and the introduced nucleus can be enormous, which led to a high loss in resulting cells in early research. For example, the cloned sheep Dolly was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood.[14] As the procedure could not be automated, and had to be performed manually under a microscope, SCNT was very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg was also far from being well understood. However, by 2014 researchers were reporting cloning success rates of seven to eight out of ten[15] and in 2016, a Korean Company Sooam Biotech was reported to be producing 500 cloned embryos per day.[16]

In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus.

Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants and some insects. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation,[17] has been common practice in the horticultural world for hundreds of years.

The term clone is used in horticulture to refer to descendants of a single plant which were produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction.[18] As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana.[19]

Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.

Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies naturally. Parts of an individual plant may become detached by fragmentation and grow on to become separate clonal individuals. A common example is in the vegetative reproduction of moss and liverwort gametophyte clones by means of gemmae. Some vascular plants e.g. dandelion and certain viviparous grasses also form seeds asexually, termed apomixis, resulting in clonal populations of genetically identical individuals.

Clonal derivation exists in nature in some animal species and is referred to as parthenogenesis (reproduction of an organism by itself without a mate). This is an asexual form of reproduction that is only found in females of some insects, crustaceans, nematodes,[20] fish (for example the hammerhead shark[21]), and lizards including the Komodo dragon[21] and several whiptails. The growth and development occurs without fertilization by a male. In plants, parthenogenesis means the development of an embryo from an unfertilized egg cell, and is a component process of apomixis. In species that use the XY sex-determination system, the offspring will always be female. An example is the little fire ant (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.

Artificial cloning of organisms may also be called reproductive cloning.

Hans Spemann, a German embryologist was awarded a Nobel Prize in Physiology or Medicine in 1935 for his discovery of the effect now known as embryonic induction, exercised by various parts of the embryo, that directs the development of groups of cells into particular tissues and organs. In 1924 he and his student, Hilde Mangold, were the first to perform somatic-cell nuclear transfer using amphibian embryos one of the first steps towards cloning.[22]

Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg from which the nucleus has been removed, or to a cell from a blastocyst from which the nucleus has been removed.[23] If the egg begins to divide normally it is transferred into the uterus of the surrogate mother. Such clones are not strictly identical since the somatic cells may contain mutations in their nuclear DNA. Additionally, the mitochondria in the cytoplasm also contains DNA and during SCNT this mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to death.

Artificial embryo splitting or embryo twinning, a technique that creates monozygotic twins from a single embryo, is not considered in the same fashion as other methods of cloning. During that procedure, a donor embryo is split in two distinct embryos, that can then be transferred via embryo transfer. It is optimally performed at the 6- to 8-cell stage, where it can be used as an expansion of IVF to increase the number of available embryos.[24] If both embryos are successful, it gives rise to monozygotic (identical) twins.

Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned from an adult somatic cell. Dolly was formed by taking a cell from the udder of her 6-year-old biological mother.[25] Dolly's embryo was created by taking the cell and inserting it into a sheep ovum. It took 435 attempts before an embryo was successful.[26] The embryo was then placed inside a female sheep that went through a normal pregnancy.[27] She was cloned at the Roslin Institute in Scotland by British scientists Sir Ian Wilmut and Keith Campbell and lived there from her birth in 1996 until her death in 2003 when she was six. She was born on 5 July 1996 but not announced to the world until 22 February 1997.[28] Her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.[29]

Dolly was publicly significant because the effort showed that genetic material from a specific adult cell, designed to express only a distinct subset of its genes, can be redesigned to grow an entirely new organism. Before this demonstration, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg.[30] However, this concept was not yet demonstrated in a mammalian system.

The first mammalian cloning (resulting in Dolly) had a success rate of 29 embryos per 277 fertilized eggs, which produced three lambs at birth, one of which lived. In a bovine experiment involving 70 cloned calves, one-third of the calves died quite young. The first successfully cloned horse, Prometea, took 814 attempts. Notably, although the first[clarification needed] clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.

There were early claims that Dolly had pathologies resembling accelerated aging. Scientists speculated that Dolly's death in 2003 was related to the shortening of telomeres, DNA-protein complexes that protect the end of linear chromosomes. However, other researchers, including Ian Wilmut who led the team that successfully cloned Dolly, argue that Dolly's early death due to respiratory infection was unrelated to problems with the cloning process. This idea that the nuclei have not irreversibly aged was shown in 2013 to be true for mice.[31]

Dolly was named after performer Dolly Parton because the cells cloned to make her were from a mammary gland cell, and Parton is known for her ample cleavage.[32]

The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Notable experiments include:

Human cloning is the creation of a genetically identical copy of a human. The term is generally used to refer to artificial human cloning, which is the reproduction of human cells and tissues. It does not refer to the natural conception and delivery of identical twins. The possibility of human cloning has raised controversies. These ethical concerns have prompted several nations to pass legislation regarding human cloning and its legality. As of right now, scientists have no intention of trying to clone people and they believe their results should spark a wider discussion about the laws and regulations the world needs to regulate cloning.[68]

Two commonly discussed types of theoretical human cloning are therapeutic cloning and reproductive cloning. Therapeutic cloning would involve cloning cells from a human for use in medicine and transplants, and is an active area of research, but is not in medical practice anywhere in the world, as of 2021[update]. Two common methods of therapeutic cloning that are being researched are somatic-cell nuclear transfer and, more recently, pluripotent stem cell induction. Reproductive cloning would involve making an entire cloned human, instead of just specific cells or tissues.[69]

There are a variety of ethical positions regarding the possibilities of cloning, especially human cloning. While many of these views are religious in origin, the questions raised by cloning are faced by secular perspectives as well. Perspectives on human cloning are theoretical, as human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and in livestock production.

Advocates support development of therapeutic cloning to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants,[70] to avoid the need for immunosuppressive drugs,[69] and to stave off the effects of aging.[71] Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology.[72]

Opponents of cloning have concerns that technology is not yet developed enough to be safe[73] and that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested),[74][75] as well as concerns about how cloned individuals could integrate with families and with society at large.[76][77]

Religious groups are divided, with some opposing the technology as usurping "God's place" and, to the extent embryos are used, destroying a human life; others support therapeutic cloning's potential life-saving benefits.[78][79]

Cloning of animals is opposed by animal-groups due to the number of cloned animals that suffer from malformations before they die, and while food from cloned animals has been approved by the US FDA,[80][81] its use is opposed by groups concerned about food safety.[82][83]

Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream. Possible implications of this were dramatized in the 1984 novel Carnosaur and the 1990 novel Jurassic Park.[84][85] The best current cloning techniques have an average success rate of 9.4 percent[86] (and as high as 25 percent[31]) when working with familiar species such as mice,[note 1] while cloning wild animals is usually less than 1 percent successful.[89]

Several tissue banks have come into existence, including the "Frozen zoo" at the San Diego Zoo, to store frozen tissue from the world's rarest and most endangered species.[84][90][91] This is also referred to as "Conservation cloning".[92][93]

In 2001, a cow named Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after two days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean ibex) were frozen in liquid nitrogen immediately after it died in 2000. Researchers are also considering cloning endangered species such as the Giant panda and Cheetah.[94][95][96][97]

In 2002, geneticists at the Australian Museum announced that they had replicated DNA of the thylacine (Tasmanian tiger), at the time extinct for about 65 years, using polymerase chain reaction.[98] However, on 15 February 2005 the museum announced that it was stopping the project after tests showed the specimens' DNA had been too badly degraded by the (ethanol) preservative. On 15 May 2005 it was announced that the thylacine project would be revived, with new participation from researchers in New South Wales and Victoria.[99]

In 2003, for the first time, an extinct animal, the Pyrenean ibex mentioned above was cloned, at the Centre of Food Technology and Research of Aragon, using the preserved frozen cell nucleus of the skin samples from 2001 and domestic goat egg-cells. The ibex died shortly after birth due to physical defects in its lungs.[100]

One of the most anticipated targets for cloning was once the woolly mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint Russo-Japanese team is currently working toward this goal.[when?] In January 2011, it was reported by Yomiuri Shimbun that a team of scientists headed by Akira Iritani of Kyoto University had built upon research by Dr. Wakayama, saying that they will extract DNA from a mammoth carcass that had been preserved in a Russian laboratory and insert it into the egg cells of an Asian elephant in hopes of producing a mammoth embryo. The researchers said they hoped to produce a baby mammoth within six years.[101][102] It was noted, however that the result, if possible, would be an elephant-mammoth hybrid rather than a true mammoth.[103] Another problem is the survival of the reconstructed mammoth: ruminants rely on a symbiosis with specific microbiota in their stomachs for digestion.[103]

Scientists at the University of Newcastle and University of New South Wales announced in March 2013 that the very recently extinct gastric-brooding frog would be the subject of a cloning attempt to resurrect the species.[104]

Many such "De-extinction" projects are described in the Long Now Foundation's Revive and Restore Project.[105]

After an eight-year project involving the use of a pioneering cloning technique, Japanese researchers created 25 generations of healthy cloned mice with normal lifespans, demonstrating that clones are not intrinsically shorter-lived than naturally born animals.[31][106] Other sources have noted that the offspring of clones tend to be healthier than the original clones and indistinguishable from animals produced naturally.[107]

Some posited that Dolly the sheep may have aged more quickly than naturally born animals, as she died relatively early for a sheep at the age of six. Ultimately, her death was attributed to a respiratory illness, and the "advanced aging" theory is disputed.[108][dubious discuss]

A detailed study released in 2016 and less detailed studies by others suggest that once cloned animals get past the first month or two of life they are generally healthy. However, early pregnancy loss and neonatal losses are still greater with cloning than natural conception or assisted reproduction (IVF). Current research is attempting to overcome these problems.[32]

Discussion of cloning in the popular media often presents the subject negatively. In an article in the 8 November 1993 article of Time, cloning was portrayed in a negative way, modifying Michelangelo's Creation of Adam to depict Adam with five identical hands.[109] Newsweek's 10 March 1997 issue also critiqued the ethics of human cloning, and included a graphic depicting identical babies in beakers.[110]

The concept of cloning, particularly human cloning, has featured a wide variety of science fiction works. An early fictional depiction of cloning is Bokanovsky's Process which features in Aldous Huxley's 1931 dystopian novel Brave New World. The process is applied to fertilized human eggs in vitro, causing them to split into identical genetic copies of the original.[111][112] Following renewed interest in cloning in the 1950s, the subject was explored further in works such as Poul Anderson's 1953 story UN-Man, which describes a technology called "exogenesis", and Gordon Rattray Taylor's book The Biological Time Bomb, which popularised the term "cloning" in 1963.[113]

Cloning is a recurring theme in a number of contemporary science fiction films, ranging from action films such as Anna to the Infinite Power, The Boys from Brazil, Jurassic Park (1993), Alien Resurrection (1997), The 6th Day (2000), Resident Evil (2002), Star Wars: Episode II Attack of the Clones (2002), The Island (2005) and Moon (2009) to comedies such as Woody Allen's 1973 film Sleeper.[114]

The process of cloning is represented variously in fiction. Many works depict the artificial creation of humans by a method of growing cells from a tissue or DNA sample; the replication may be instantaneous, or take place through slow growth of human embryos in artificial wombs. In the long-running British television series Doctor Who, the Fourth Doctor and his companion Leela were cloned in a matter of seconds from DNA samples ("The Invisible Enemy", 1977) and then in an apparent homage to the 1966 film Fantastic Voyage shrunk to microscopic size to enter the Doctor's body to combat an alien virus. The clones in this story are short-lived, and can only survive a matter of minutes before they expire.[115] Science fiction films such as The Matrix and Star Wars: Episode II Attack of the Clones have featured scenes of human foetuses being cultured on an industrial scale in mechanical tanks.[116]

Cloning humans from body parts is also a common theme in science fiction. Cloning features strongly among the science fiction conventions parodied in Woody Allen's Sleeper, the plot of which centres around an attempt to clone an assassinated dictator from his disembodied nose.[117] In the 2008 Doctor Who story "Journey's End", a duplicate version of the Tenth Doctor spontaneously grows from his severed hand, which had been cut off in a sword fight during an earlier episode.[118]

The 2021 film, Girl Next in which a woman is adducted, drugged and brainwashed into becoming obedient, living sex doll. Later proving that she is a clone of a clone designed to assassin the traffickers.

After the death of her beloved 14-year-old Coton de Tulear named Samantha in late 2017, Barbra Streisand announced that she had cloned the dog, and was now "waiting for [the two cloned pups] to get older so [she] can see if they have [Samantha's] brown eyes and her seriousness".[119] The operation cost $50,000 through the pet cloning company ViaGen.[120]

Science fiction has used cloning, most commonly and specifically human cloning, to raise the controversial questions of identity.[121][122] A Number is a 2002 play by English playwright Caryl Churchill which addresses the subject of human cloning and identity, especially nature and nurture. The story, set in the near future, is structured around the conflict between a father (Salter) and his sons (Bernard 1, Bernard 2, and Michael Black) two of whom are clones of the first one. A Number was adapted by Caryl Churchill for television, in a co-production between the BBC and HBO Films.[123]

In 2012, a Japanese television series named "Bunshin" was created. The story's main character, Mariko, is a woman studying child welfare in Hokkaido. She grew up always doubtful about the love from her mother, who looked nothing like her and who died nine years before. One day, she finds some of her mother's belongings at a relative's house, and heads to Tokyo to seek out the truth behind her birth. She later discovered that she was a clone.[124]

In the 2013 television series Orphan Black, cloning is used as a scientific study on the behavioral adaptation of the clones.[125] In a similar vein, the book The Double by Nobel Prize winner Jos Saramago explores the emotional experience of a man who discovers that he is a clone.[126]

Cloning has been used in fiction as a way of recreating historical figures. In the 1976 Ira Levin novel The Boys from Brazil and its 1978 film adaptation, Josef Mengele uses cloning to create copies of Adolf Hitler.[127]

In Michael Crichton's 1990 novel Jurassic Park, which spawned a series of Jurassic Park feature films, the bioengineering company InGen develops a technique to resurrect extinct species of dinosaurs by creating cloned creatures using DNA extracted from fossils. The cloned dinosaurs are used to populate the Jurassic Park wildlife park for the entertainment of visitors. The scheme goes disastrously wrong when the dinosaurs escape their enclosures. Despite being selectively cloned as females to prevent them from breeding, the dinosaurs develop the ability to reproduce through parthenogenesis.[128]

The use of cloning for military purposes has also been explored in several fictional works. In Doctor Who, an alien race of armour-clad, warlike beings called Sontarans was introduced in the 1973 serial "The Time Warrior". Sontarans are depicted as squat, bald creatures who have been genetically engineered for combat. Their weak spot is a "probic vent", a small socket at the back of their neck which is associated with the cloning process.[129] The concept of cloned soldiers being bred for combat was revisited in "The Doctor's Daughter" (2008), when the Doctor's DNA is used to create a female warrior called Jenny.[130]

The 1977 film Star Wars was set against the backdrop of a historical conflict called the Clone Wars. The events of this war were not fully explored until the prequel films Attack of the Clones (2002) and Revenge of the Sith (2005), which depict a space war waged by a massive army of heavily armoured clone troopers that leads to the foundation of the Galactic Empire. Cloned soldiers are "manufactured" on an industrial scale, genetically conditioned for obedience and combat effectiveness. It is also revealed that the popular character Boba Fett originated as a clone of Jango Fett, a mercenary who served as the genetic template for the clone troopers.[131][132]

A recurring sub-theme of cloning fiction is the use of clones as a supply of organs for transplantation. The 2005 Kazuo Ishiguro novel Never Let Me Go and the 2010 film adaption[133] are set in an alternate history in which cloned humans are created for the sole purpose of providing organ donations to naturally born humans, despite the fact that they are fully sentient and self-aware. The 2005 film The Island[134] revolves around a similar plot, with the exception that the clones are unaware of the reason for their existence.

The exploitation of human clones for dangerous and undesirable work was examined in the 2009 British science fiction film Moon.[135] In the futuristic novel Cloud Atlas and subsequent film, one of the story lines focuses on a genetically-engineered fabricant clone named Sonmi~451, one of millions raised in an artificial "wombtank", destined to serve from birth. She is one of thousands created for manual and emotional labor; Sonmi herself works as a server in a restaurant. She later discovers that the sole source of food for clones, called 'Soap', is manufactured from the clones themselves.[136]

In the film Us, at some point prior to the 1980s, the US Government creates clones of every citizen of the United States with the intention of using them to control their original counterparts, akin to voodoo dolls. This fails, as they were able to copy bodies, but unable to copy the souls of those they cloned. The project is abandoned and the clones are trapped exactly mirroring their above-ground counterparts' actions for generations. In the present day, the clones launch a surprise attack and manage to complete a mass-genocide of their unaware counterparts.[137][138]

In the series of A Certain Magical Index and A Certain Scientific Railgun, one of the espers, Mikoto Misaka, had DNA was harvested unknowingly, creating 20,000 exact but not equally powerful clones for an experiment. They were used as target practice by Accelerator, just to level up, as killing the original multiple times is impossible. The experiment ended when Toma Kamijo saved and foiled the experiment. The remaining clones have been dispersed everywhere in the world to conduct further experiments to expand their lifespans, save for at least 10 who remained in Academy City, and the last clone, who was not fully developed when the experiment stopped.[citation needed]

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Cloning - Wikipedia