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Austin Hormone Doctor Bioidentical Hormone Replacement Therapy

I would like to share my story of how taking bio-identical hormones and working with Dr. Edgertons group changed my life. I am still so thankful every day and take every opportunity I get to share my story in an effort to help others who may suffer from menopause.I started having, I would say, moderate symptoms that quickly became severe when I was 48 (almost 3 yrs. ago). When I say severe, I couldnt focus at work, I was always exhausted, I couldnt sleep at night, I had day & night sweats, I gained weightyou name it, I had it and felt it.Initially, I went to see my gynecologist who ran blood tests indicating my results were within normal ranges. We tried several different birth control medications (for the estrogen/progesterone) and none worked. When he indicated he didnt know what else to do, I felt so defeated but I wasnt giving up. I had been doing some research and wanted to try bio-identical therapy and coincidentally Dr. Edgerton was in the same practice as my gynecologist so he suggested that I see Dr. Edgerton.Once you finally have a doctor and staff that empathize with you and understand what you are going through, you immediately begin to feel that there is hope. I owe that to Dr. Edgerton and his staff. They explained that just because your hormone levels are within the standard ranges, it doesnt necessarily mean they are normal, especially if they are in the low end of the range. The idea is to get your hormones at the maximum level and this is exactly what they did with the bio-identical hormone therapy.I started seeing Dr. Edgerton in February, 2009 and after a few adjustments to the Bioidentical Hormones medications, by April, I was feeling so much better (no more sweating, focus was back, etc.) and the extra bonus was the weight really started coming off. Keep in mind, I had been going to the gym but I wasnt getting any resultsuntil my hormones were properly working. My weight was 143 in February and by July I was weighing 122 and I continued to lose. I couldnt believe it and still Im amazed which is why I want to help others. I have kept my weight at 115 for over a year now have never been more fit and healthy than I am now at 50almost 51.I want to thank Dr. Edgerton and his staff who helped me tell my storywith a happy ending.

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Austin Hormone Doctor Bioidentical Hormone Replacement Therapy

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Heart Failure Signs | Cardiac Stem Cell Therapies: Heart …

Human life is dependent upon the hearts ability to pump forcefully and frequently enough, but heart failure signs can disturb its normal function. Most humans cannot live more than four minutes without a heartbeat or continuous blood-flow. At that time, brain cells begin to die because they lack adequately oxygenated blood-flow.

The human adult body requires, on average, 5.0 liters of re-circulated blood per minute. In the cardiology field, this metric is called the Cardiac Output, which is calculated as Stroke Volume (SV) x Heart Rate (HR). Another key metric is a patients Ejection-Fraction (EF %). A patients EF tells a cardiologist and other physicians if his or her heart is functioning normally or low normally. It is a measurement of ones heart contraction, with a normal EF range being 55-70%.

This number can also be combined with a patients heart rate to provide physicians with a baseline of a patients cardiac status. A normal range for an adult is 60-100 beats per minute, and this can be significantly higher during a normal pregnancy.

In this article:

For a cardiologist, cardiac metrics indicate if their services are required and allowthem to sign-off on pre-operative cardiac clearances. For other physicians, it tells them if the organ which they specialize in is being perfused adequately (for example, a nephrologist would be interested to know kidney perfusion). It can also indicate the degree to which decreased heart function may affect the severity or spread of disease.

When the heart fails to contract forcefully enough and its performance decreases to the point where its ability to circulate blood adequately is compromised (the EF% falls below 40%), this is considered heart failure. The clinical parameters of heart failure are clearly defined by the New York Heart Association (NYHA), which places patients in NYHA Class III & IV into the heart failure category.

An echocardiogram (often called an Echo), as opposed to an Electrocardiogram (EKG or ECG), allows technicians and physicians to visualize the beating heart. Video clips of the heart contracting are digitally recorded, and a patients EF and Cardiac Output (CO) can be measured with several diagnostic tools (Fractional Shortening via 2D or M-Mode measurements and Simpsons Method via 2D and 3D Quantification) on a cardiovascular ultrasound system.

When an experienced echo tech or cardiologist views a failing heart, it is immediately apparent. Based on my experience reading echocardiograms, I can see that the heart walls or heart muscles (myocardium) are not contracting as vigorously as they should.

For patients with a 5% EF range, any physical movement is extremely strenuous, and they can go into cardiac arrest at any moment, which is why they are usually on cardiac telemetry in a hospital setting. Most likely, a patient with 5% EF range would be awaiting a heart transplant, unless there is a medical condition preventing them from being eligible.

Once a patient falls into the heart failure range, they will be lethargic and have severe limits on activities. Other clinical manifestations of heart failure can include peripheral edema (i.e. swelling in the feet, legs, ankles, or stomach), pulmonary edema, and shortness of breath. In many cases, this can lead to depression.

In evaluating the frequency of heart failure in the U.S, statistics from the U.S. Centers for Disease Control (CDC) find that approximately 5.7 million adults are afflicted with this condition. Additionally, care for congestive heart failure costs an estimated $30.7B per year. Furthermore, the mortality rates of patients suffering from heart failure indicate its clinical severity, with 1 in 5 patients with this condition dying within a year of receiving the diagnosis.

A patient experiencing severe heart failure has limited treatment options, which are expensive, complicated, and have major lifestyle implications.

These limited options include:

Consequently, physicians need more effective weapons for treating heart failure in order to improve patients lives and reduce healthcare-related costs. CHF patients have disproportionate hospital readmission rates when compared to other major diseases.

Enter in the growing field of cardiac stem cell treatments, which introduce fundamentally new treatment options for heart failure patients. In cardiac stem cell treatments, stem cells are taken from a patients bone marrow or fat tissue in a sterile surgical procedure and injected via a catheter-wire into infarcted or poorly contracting muscular segments of the hearts main pumping chamber, the left ventricle (LV).

Over the course of a few months, the stem cells impact myocardial cells and begin to improve the contractility of the affected segments, most likely through paracrine signaling mechanisms and impacting the local microenvironment. This can bring a patients EF to low-normal or even normal levels. As a result, a patient can live a more normal life and return to many activities.

A very early clinical trial aimed at evaluating the potential and effectiveness of cardiac stem cell therapy in humans was conducted in 2006 utilizing a commercial product, VesCellTM. The parameters and results of this trial were documented in the American Heart Associations Circulation, Abstract 3682: Treatment of Patients with Severe Angina Pectoris Using Intracoronarily Injected Autologous Blood-Borne Angiogenic Cell Precursors.The subjects of this trial received an intracoronary injection of VesCellTM, an Autologous Angiogenic Cell Precursor (ACP)-based product.

The authors drew their conclusion regarding this study. VesCell therapy for chronic stable angina seems to be safe and improves anginal symptoms at 3 and 6 months. Larger studies are being initiated to evaluate the benefit of VesCell for the treatment of this and additional severe heart diseases. (Source: Tresukosol et al. Abstract 3682: Treatment of Patients with Severe Angina Pectoris Using Intracoronarily Injected Autologous Blood-Borne Angiogenic Cell Precursors. Circulation. October 31, 2006. Vol. 114, Issue Suppl 18. Link: http://circ.ahajournals.org/content/114/Suppl_18/II_786.4 )

Another early cardiac stem cell clinical trial was performed in 2009 by a Cedars-Sinai team based on technologies and discoveries made by Eduardo Marban, MD, PhD, and led by Raj Makkar, MD. In this study, they explored the safety of harvesting, expanding, and administering a patients cardiac stem cells to repair heart tissue injured by myocardial infarction.

Recently, the American College of Cardiology (ACC) also announced results of a ground-breaking clinical study to evaluate the efficacy and effectiveness of cardiac stem cell treatment for heart failure patients. As stated by Timothy Henry, M.D., Director of Cardiology at Cedars-Sinai Heart Institute and one of the studys lead authors, This is the largest double-blind, placebo-controlled stem cell trial for treatment of heart failure to be presentedBased on these positive results, we are encouraged that this is an attractive potential therapy for patients with class III and class IV heart failure.

Additionally, Dr. Charles Goldthwaite, Jr, published a whitepaper titled, Mending a Broken Heart: Stem Cells and Cardiac Repair, in which he draws the conclusion, Given the worldwide prevalence of cardiac dysfunction and the limited availability of tissue for cardiac transplantation, stem cells could ultimately fulfill a large-scale unmet clinical need and improve the quality of life for millions of people with CVD. However, the use of these cells in this setting is currently in its infancymuch remains to be learned about the mechanisms by which stem cells repair and regenerate myocardium, the optimal cell types, and modes of their delivery, and the safety issues that will accompany their use.

Clearly, there is a trend toward acceptance of cardiac stem cell therapies as an emerging treatment option. Several world-renowned institutes are now conducting clinical studies involving cardiac stem cell treatment, as well as applying for intellectual property protection (patents) pertaining to the techniques required in administrating the therapies.

The key questions at this point in time appear to be:

An important whitepaper pertaining to cardiac stem cells is Ischemic Cardiomyopathy Patients Treated with Autologous Angiogenic and Cardio-Regenerative Progenitor Cells, written by Dr. Athina Kyritsis, et al. In it, the physicians describe their objective as investigating the feasibility, safety, and clinical outcome of patients with Ischemic Cardiomyopathy treated with Autologous Angiogenic and Cardio-Regenerative Progenitor cells (ACPs).

The researchers state: In numerous human trials there is evidence of improvement in the ejection fractions of Cardiomyopathy patients treated with ACPs. Animal experiments not only show improvement in cardiac function, but also engraftment and differentiation of ACPs into cardiomyocytes, as well as neo-vascularization in infarcted myocardium. In our clinical experience, the process has shown to be safe as well as effective.

The authors also found that patients treated with this approach gained increases in cardiac ejection fraction from their starting measurements, with improvements in their cardiac ejection fraction of 21 points (75% increase) at rest and 28.5 points (80% increase) at stress. As a result of these finding, the authors conclude, ACPs can improve the ejection fraction in patients with severely reduced cardiac function with benefits sustained to six months.

In the practice of medicine, the focus should be on delivering excellent care to patients. If there are cardiac stem cell treatments available, then regulatory obstacles should be removed when sufficient clinical trial evidence has been provided to indicate safety and efficacy.

Cardiologist Zannos Grekos, MD, a pioneer in cardiac stem cell therapy since 2006, points to the vastly untapped promise of related therapies, commenting Those of us that have been involved with cardiac stem cell treatment for the last 10-plus years can see the incredible potential this approach has.

As of 2017, the U.S. healthcare system is under enormous pressure to deliver affordable healthcareto a growing population of patients, especially those who are fully or partially covered under Medicare or Medicaid (many have secondary coverage). Although we are in the infancy of its development, cardiac stem cell treatments represent a potentially powerful treatment alternative to patients with heart failure symptoms.

To learn more, view the resources below.

1) Regenocyte http://www.regenocyte.com

2) Cleveland Clinic Stem Cell Therapy for Heart Disease my.clevelandclinic.org/health/articles/stem-cell-therapy-heart-disease

3) Harvard Stem Cell Institute (HSCI) hsci.harvard.edu/heart-disease-0

4) Cedars Sinai Cardiac Stem Cell Treatment http://www.cedars-sinai.edu/Patients/Programs-and-Services/Heart-Institute/Clinical-Trials/Cardiac-Stem-Cell-Research.aspx

5) Johns Hopkins Medicine Cardiac Stem Cell Treatments http://www.hopkinsmedicine.org/stem_cell_research/cell_therapy/a_new_path_for_cardiac_stem_cells.html

What do you think about heart failure signs and cardiac stem cell therapies? Share your thoughts in the comments section below.

Up Next:European Society of Cardiology (ESC) Congress Presentation Reveals Results From Pre-Clinical Study Using CardioCells Stem Cells for Acute Myocardial Infarction

Guest Post: This is a guest article by Clifford M. Thornton, a Certified Cardiovascular Technologist, experienced Echocardiographer Technician, and journalist in the cardiac and medical device fields. His articles have been published in Inventors Digest, Global Innovation Magazine, and Modern Health Talk. He is enthusiastic about progress with cardiac stem cell therapies and their role in heart failure treatment.He can be reached byphone at 267-524-7144 or by email at[emailprotected].

Editors Note This post was originally published on March 14, 2017, and has been updated for quality and relevancy.

Heart Failure Signs | Cardiac Stem Cell Therapies for Heart Failure Treatment

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Heart Failure Signs | Cardiac Stem Cell Therapies: Heart …

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AveXis Research & Development

The U.S. Food and Drug Administration (FDA) has granted AVXS-101 Orphan Drug Designation for the treatment of all types of SMA and Breakthrough Therapy Designation, as well as Fast Track Designation, for the treatment of SMA Type 1.

The European Medicines Agency (EMA) also granted AveXis access into its PRIority Medicines (PRIME) program for AVXS-101 for the treatment of SMA Type 1.

The open-label, single-arm, single-dose, multi-center trial known as STR1VE is designed to evaluate the efficacy and safety of a one-time IV infusion of AVXS-101 in patients with SMA Type 1. The co-primary efficacy outcome measures of the trial include the achievement of independent sitting for at least 30 seconds at 18 months of age; and, event-free survival at 14 months of age. Co-secondary outcome measures include the ability to thrive, and the ability to remain independent of ventilatory support at 18 months of age.

The open-label, dose-comparison, multi-center Phase 1 trial known as STRONG is designed to evaluate the safety, optimal dosing, and proof of concept for efficacy of AVXS-101 in two distinct age groups of patients with SMA Type 2, utilizing a one-time IT route of administration. The primary outcome measure for patients less than 24 months of age at the time of dosing is the achievement of the ability to stand without support for at least three seconds. The primary outcome measure for patients between 24 months and 60 months of age at the time of dosing is the achievement of change in Hammersmith Functional Motor Scale Expanded from baseline. The secondary outcome measure for both age groups is the proportion of patients that achieve the ability to walk without assistance, defined as taking at least five steps independently while displaying coordination and balance. Developmental abilities, including motor function, will also be evaluated as exploratory objectives.

Learn more about clinical trials

We have exclusive worldwide license agreements to develop and commercialize gene therapy using the AAV9 vector to treat two rare neurological monogenic disorders: Rett syndrome (RTT) and a genetic form of amyotrophic lateral sclerosis (ALS) caused by mutations in the superoxide dismutase 1 (SOD1) gene.

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AveXis Research & Development

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Stem Cells Used in Anti-Aging Skin Care Radiant RG-Cell

Stem cells are biological cells that are able to stay dormant until triggered to reproduce into new tissue. Found in human embryos and in adult tissue, they can form into any cell type, and help repair organs and skin in the case of injury or other cause of damage.

So is it any surprise that their potential is also being trumpeted in the world of skin care? Cosmetic science has often taken inspiration from hard-core medical breakthroughs, and stem cells appear to possess the ideal skill set to throw the switch on a veritable fountain of youth.

While skin stem cells have found use in treating diseases, stem cells technology in skin care products have been largely based on hype rather than science, but in some cases like RG-CELL, it truly works magic.

The concept of topically applying stem cells, through cream, serum, mask, or facial procedure, with a promise to replenish dying cells and regenerate dying tissues has shown no real scientific evidence that it works.

If youre unfamiliar with the practice, you may question the validity of using live stem cells in anti-aging products when its already an enormous and time consuming challenge to use them in actual organ regenerating procedures.

Firstly, stem cells are highly unstable. They have little to no shelf life. Secondly, they will not enter the deep layers of the skin without an effective skin delivery system. And thirdly, stem cells need specific nutrition via a blood supply in the tissue to survive and function if they were layered onto intact skin the stem cells would just die.

It should be made abundantly clear that, no stem cell skin care products contain actual stem cells. Stem cell based products contain growth factors, along with enzymes and other nutrients, which help the cells grow. Other products dont contain any stem cell-related material at all.

[frame src=https://rg-cell.com/wp-content/uploads/2013/05/stem-cell-skin-care.jpg width=250 height=188 alt=Stem Cell Skin Care align=right]There are 2 ways in which stem cell technology is being used. Firstly, companies are creating products with specialized peptides and enzymes or plant growth factors which, when applied topically on the surface, help protect the human skin from damage and deterioration. Products claiming to contain plant stem cells dont contain human cytokines (or cell messengers), and in fact are really just ground up plant bits. In short, plant stem cell technology cannot effectively impact human stem cells. It can be useful as excellent antioxidants, but marketing has made the benefits bigger than reality.

Secondly, and bearing more scientific evidence, is an alternative application of skin care anti-aging products. These products utilize human stem cell technology, and your skin is the most active participant, NOT plant or apple stem cells. Using ingredients that promote the repair and rejuvenation of your skin by stimulating the activity of your own stem cells in the skin has proven to be safer, more ethical and far more scientifically proven than applying stem cells in a jar. This technology implies a superior product designed specifically to regenerate and rejuvenate your own skin cells.

These products contain epidermal growth factors (EGF) obtained by genetic engineering technology (microbial recombinant) totally identical to natural EGF, known as a BEAUTY FACTOR, boosts and regulates stem cell proliferation. When applied to the skin, stimulate collagen production, improve elasticity, firm sagging skin, improve tone and so much more.

[frame src=https://rg-cell.com/wp-content/uploads/2012/11/nano-encapsulation.jpg width=250 height=190 alt=Skin Delivery System align=right]EGF is a large molecule so it cannot penetrate the skin. In fact, it is too big to fit in between the spaces in cells of our skin. There is also speculation around the length of time, that it can remain stable in a formulation. Clinical studies and research are practically non-existent. Therefore, buyer beware: If you opt for using a product that contains EGF consider whether or not the mechanism of action employed to deliver the ingredient to the dermal layers, will actually work.

Only special technology, can deliver EGF into the skin deeper layers. One of the biggest advances is the use of a patented nano-particulate lipid bi-layer delivery system that allows the products to be delivered deep into the skin where the stem cells live.

RG-Cell uses a unique patented nano-encapsulation technology as its delivery system. This improves the permeation and penetration efficiency of the active ingredients. Owing to this fact, RG-CELL can make valid claims about the efficiency in it is delivery of EGF where it is needed the most. This technology also stabilizes the EGF thereby prolonging its shelf life in the actual product.

Thus we can see that there are already many choices in skin care products with specialized peptides and enzymes or EGFs which, when applied topically stimulate the skins own stem cells. But, only one uses the most advanced technology to deliver nutrients into the skin. Expect many more good choices to be developed in the years to come!

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Stem Cells Used in Anti-Aging Skin Care Radiant RG-Cell

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CRISPR – Simple English Wikipedia, the free encyclopedia

CRISPR is a term used in microbiology. It stands for Clustered Regularly-Interspaced Short Palindromic Repeats. These are a natural segment of the genetic code found in prokaryotes: most bacteria and archaea have it.[1]

CRISPR has a lot of short repeated sequences. These sequences are part of an adaptive immune system for prokaryotes. It allows them to remember and counter other organisms that prey on them, such as bacteriophages.

They have the potential to modify the genes of almost any organism. They are part of a tool that allows precisely targeted cutting and insertion of genes in genetic modification (GM). Work is under way to find how they can be used to attack virus diseases in humans.[2]

Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid.[2] CRISPR spacers recognize and cut up the foreign genetic elements in a manner like RNA interference in eukaryotic organisms.

In effect, the spacers are fragments of DNA from viruses that have previously tried to attack the cell line. The foreign source of the spacers was a sign to researchers that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[3]

The actual cutting is done by a nuclease called Cas9. Cas9 has two active cutting sites, one for each strand of the DNA’s double helix. Cas9 does this by unwinding foreign DNA and checking whether it is complementary to the 20 basepair spacer region of the guide RNA (the spacer region RNA). If it is, the foreign DNA gets chopped up.

The technology has been used to switch off genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuel and to genetically modify crop strains.[4]

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CRISPR – Simple English Wikipedia, the free encyclopedia

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CRISPR NIH Director’s Blog

Posted on September 11th, 2018 by Dr. Francis Collins

Caption: A CRISPR/cas9 gene editing-based treatment restored production of dystrophin proteins (green) in the diaphragm muscles of dogs with Duchenne muscular dystrophy.Credit: UT Southwestern

CRISPR and other gene editing tools hold great promise for curing a wide range of devastating conditions caused by misspellings in DNA. Among the many looking to gene editing with hope are kids with Duchenne muscular dystrophy (DMD), an uncommon and tragically fatal genetic disease in which their musclesincluding skeletal muscles, the heart, and the main muscle used for breathinggradually become too weak to function. Such hopes were recently buoyed by a new study that showed infusion of the CRISPR/Cas9 gene editing system could halt disease progression in a dog model of DMD.

As seen in the micrographs above, NIH-funded researchers were able to use the CRISPR/Cas9 editing system to restore production of a critical protein, called dystrophin, by up to 92 percent in the muscle tissue of affected dogs. While more study is needed before clinical trials could begin in humans, this is very exciting news, especially when one considers that boosting dystrophin levels by as little as 15 percent may be enough to provide significant benefit for kids with DMD.

Posted In: News

Tags: animal models, beagles, Cavalier King Charles Spaniel, CRISPR, CRISPR/Cas9, diaphragm muscle, DMD, dogs, Duchenne muscular dystrophy, dystrophin, gene editing, genetic diseases, heart, muscle, muscular dystrophy, rare diseases, Somatic Cell Genome Editing

Posted on October 10th, 2017 by Dr. Francis Collins

About a month ago, I had the pleasure of welcoming the Juip (pronounced Yipe) family from Michigan to NIH. Although youd never guess it from this photo, two of the Juips five children9-year-old Claire and 11-year-old Jake (both to my left)have a rare genetic disease called Friedreichs ataxia (FA). This inherited condition causes progressive damage to their nervous systems and their hearts. No treatment currently exists for kids like Claire and Jake, yet this remarkable family has turned this serious health challenge into an opportunity to raise awareness about the need for biomedical research.

One thing that helps keep the Juips optimistic is the therapeutic potential of CRISPR/Cas9, an innovative gene editing systemthat may someday make it possible to correct the genetic mutations responsible for FA and many other conditions. So, Im sure the Juips were among those encouraged by the recent news that NIH-funded researchers have developed a highly versatile approach to CRISPR/Cas9-based therapies. Instead of relying on viruses to carry the gene-editing system into cells, the new approach uses tiny particles of gold as the delivery system!

Posted In: Health, Science, technology, Uncategorized

Tags: CRISPR, CRISPR-Gold, CRISPR/Cas9, DMD, Duchenne muscular dystrophy, dystrophin, FA, Friedreichs ataxia, gene editing, Juip, rare diseases, stem cells

Posted on July 18th, 2017 by Dr. Francis Collins

Credit: Seth Shipman, Harvard Medical School, Boston

Theres a reason why our cells store all of their genetic information as DNA. This remarkable molecule is unsurpassed for storing lots of data in an exceedingly small space. In fact, some have speculated that, if encoded in DNA, all of the data ever generated by humans could fit in a room about the size of a two-car garage and, if that room happens to be climate controlled, the data would remain intact for hundreds of thousands of years! [1]

Scientists have already explored whether synthetic DNA molecules on a chip might prove useful for archiving vast amounts of digital information. Now, an NIH-funded team of researchers is taking DNAs information storage capabilities in another intriguing direction. Theyve devised their own code to record information not on a DNA chip, but in the DNA of living cells. Already, the team has used bacterial cells to store the data needed to outline the shape of a human hand, as well the data necessary to reproduce five frames from a famous vintage film of a horse galloping (see above).

But the researchers ultimate goal isnt to make drawings or movies. They envision one day using DNA as a type of molecular recorder that will continuously monitor events taking place within a cell, providing potentially unprecedented looks at how cells function in both health and disease.

Posted In: Health, Science, Video

Tags: biosensor, biotechnology, Cas1, Cas2, CRISPR, CRISPR-Cas, DNA, DNA movie, DNA storage, E. coli, film, gene editing, genomics, Human and Animal Locomotion, imaging, information storage, molecular recorder, movie, spacers

Posted on May 4th, 2017 by Dr. Francis Collins

Jesse Dixon

As a kid, Jesse Dixon often listened to his parents at the dinner table discussing how to run experiments and their own research laboratories. His father Jack is an internationally renowned biochemist and the former vice president and chief scientific officer of the Howard Hughes Medical Institute. His mother Claudia Kent Dixon, now retired, did groundbreaking work in the study of lipid molecules that serve as the building blocks of cell membranes.

So, when Jesse Dixon set out to pursue a career, he followed in his parents footsteps and chose science. But Dixon, a researcher at the Salk Institute, La Jolla, CA, has charted a different research path by studying genomics, with a focus on understanding chromosomal structure. Dixon has now received a 2016 NIH Directors Early Independence Award to study the three-dimensional organization of the genome, and how changes in its structure might contribute to diseases such as cancer or even to physical differences among people.

Posted In: Health, Science

Tags: 2016 NIH Directors Early Independence Award, 3D genome structure, chromatin, chromatin structure, CRISPR, CRISPR/Cas9, DNA, DNA packaging, ENCODE, Encyclopedia of DNA Elements, enhancer, gene editing, genome, genomics, histones, TAD, topologically associated domains

Posted on January 24th, 2017 by Dr. Francis Collins

Caption: This image represents an infection-fighting cell called a neutrophil. In this artists rendering, the cells DNA is being edited to help restore its ability to fight bacterial invaders.Credit: NIAID, NIH

For gene therapy research, the perennial challenge has been devising a reliable way to insert safely a working copy of a gene into relevant cells that can take over for a faulty one. But with the recent discovery of powerful gene editing tools, the landscape of opportunity is starting to change. Instead of threading the needle through the cell membrane with a bulky gene, researchers are starting to design ways to apply these tools in the nucleusto edit out the disease-causing error in a gene and allow it to work correctly.

While the research is just getting under way, progress is already being made for a rare inherited immunodeficiency called chronic granulomatous disease (CGD). As published recently in Science Translational Medicine, a team of NIH researchers has shown with the help of the latest CRISPR/Cas9 gene-editing tools, they can correct a mutation in human blood-forming adult stem cells that triggers a common form of CGD. Whats more, they can do it without introducing any new and potentially disease-causing errors to the surrounding DNA sequence [1].

When those edited human cells were transplanted into mice, the cells correctly took up residence in the bone marrow and began producing fully functional white blood cells. The corrected cells persisted in the animals bone marrow and bloodstream for up to five months, providing proof of principle that this lifelong genetic condition and others like it could one day be cured without the risks and limitations of our current treatments.

Posted In: Health, Science

Tags: adult stem cells, bacteria, CGD, chronic granulomatous disease, clinical trials, CRISPR, CRISPR-Cas, CRISPR/Cas9, DNA editing, fungi, gene therapy, genetics, hematopoietic stem cells, immunodeficiency, immunology, infectious disease, inherited immuodeficiency, neutrophil, rare disease, translational medicine, X chromosome, X-linked chronic granulomatous disease

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CRISPR NIH Director’s Blog

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Cell and Gene Therapy | Alliance for Cancer Gene Therapy …

What is Cell and Gene Therapy for Cancer?Gene therapy is a technique that uses genes to treat or prevent disease such as cancer by inserting a gene into a patients cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including: replacing a mutated or abnormal gene that causes disease with a healthy copy of the gene; inactivating, or knocking out, a mutated gene that is functioning improperly; and introducing a new gene into the body to help fight a disease.

Cell Therapy is the infusion or transplantation of whole cells into a patient for treatment of an inherited or acquired disease like cancer.

Primary Forms of Cell and Gene Therapies for Cancer Treatment

The long-term goal of cancer cell and gene therapy is to develop treatments that attack only cancer cells, eliminating adverse effects on the body. Furthermore, these therapies have potential for treating other diseases such as cardiovascular, disorders, cystic fibrosis, hemophilia, sickle-cell anemia, muscular dystrophy, diabetes, and Parkinsons. All research in this area, therefore, makes a difference.

About Molecular Medicine

Molecular medicine uses the bodys own cells and genes as both the source and medicine for diseases of all types the basis for all cell and gene therapies.

Molecular medicine began with the identification of DNA in the early 1900s. Progress was slow until the mapping of the human genome in the new millennium and the rapid technological advances that made it possible to isolate and target specific cells and genes.

This field of study explains the fundamental genetic errors that cause diseases like cancer and helps establish a blueprint for good health.

Molecular medicine and advanced technology make it possible to target cancers directly without damage to other parts of the body.

Molecular medicine is also referred to as genetic medicine, gene therapy, targeted therapeutics, genetic epidemiology or individualized medicine.

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Cell and Gene Therapy | Alliance for Cancer Gene Therapy …

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Gene Therapy Market Share Insights – Grand View Research

Industry Outlook

The global gene therapy market sizewas valued at USD 7.6 million in 2017. It is estimated to expand at a CAGR of over 19.0% during the forecast period. Increasing number of molecules in the development phase is expected to stoke market growth. It was projected that in 2016, more than 900 molecules are in the development phase that can prove to be an effective treatment for several incurable diseases, which are generally caused by an error in a single gene.

Increasing gene therapy innovations for cardiovascular and rare diseases treatment is one of the key trends driving the market. Rising focus on development of gene therapy treatment for rare diseases is a result of intensifying competition among market players to consolidate their position in the industry.

Gene therapy involves incorporation of an artificial or a modified gene using modified viral vectors that help deliver the gene at intended site of action or even kill the cell that may cause the disease. This treatment is mostly a one-time treatment or requires very few doses of medication to completely cure the disease.

The method of treatment, which was once considered impossible, has now become a trend among big and small companies. A consequence of this has been an upsurge in the number of successful startups, backed by investors in line with big companies. The trends is poised to continue and boost the growth of the market during the forecast period.

Some novel molecules to be used for gene therapy are set to reach the commercialization stage. The growth of the market is largely dependent on key decisions made by manufacturers such as pricing, regulations, and reimbursement for treatment along with payers who help cover treatment costs. However, concerns regarding unethical use of the therapy can hamper growth prospects, especially in developing countries.

The gene therapy market is witnessing an upswing in innovations in various therapeutic fields of medicine. However, oncology is at the forefront in terms of innovation. On the basis of indication, the cancer segment accounted for the leading share of the overall market revenue in 2017. This is due to the high number of pipeline molecules that were registered over the last three years. Rising prevalence of cancer caused due to genetic mutations is also contributing to the growth of the segment. The genetic disorders segment, however, is anticipated to register the highest CAGR during the forecast period.

There are very few drugs in the market that have been approved by various regulatory bodies across the globe. These drugs are considered to change the treatment methods and regimen for rare and orphan diseases, however, their sky high pricing is limiting their commercial success.

For instance, Glybera, the first drug approved for the treatment of LPLD, was a breakthrough in the medical history. However, the drug couldnt be a commercial success due to high pricing at 1.6 million USD at the time of launch and very low prevalence of the disease (1-2 per a million of population).

In 2016, GlaxoSmithKline got another drug Strimvelis approved by European drug regulatory authority for the treatment of ADA-SCID. Other techniques in R&D are likely to witness a significant growth rate in the forecast years due to proven success of approved drugs.

The key element of gene therapy lies in the delivery of modified gene or functioning gene. Delivery systems used should be able to deliver functioning gene to intended cell target through modified viruses, which are considered the best vectors by scientists as viruses are highly evolved in delivering nucleic acid, bypassing the immune system of the host.

Several viruses such as Adeno-associated virus, retrovirus, lentivirus, and herpes simplex virus are modified in labs and are used as carriers for gene therapy drugs. Adenovirus is the most used viral vector followed by Retrovirus due to their reduced immunogenicity. Each of the viruses has its own disadvantage such as toxicity, limited DNA carrying capacity, etc.

Non-Viral vectors are being developed lately that can reduce or eliminate viral toxicity completely, however, none of the non-viral vectors possess ideal vector properties as of now.

Europehas seen two efficient gene therapy molecules after 2010, one was released in 2012 by UniQure N.V and other by GlaxoSmithKline, which were groundbreaking and were approved by the European regulatory body.

The U.S. is estimated to become a leader in terms of revenue as the country more than 64.0% of clinical trials by various big and small companies in the overall clinial trials. Among emerging economies, Russia and China are expected to be at the forefront of the market by a significant margin as they have two approved drugs in the market that can be used for cancer treatment.

Some of the key players are UniQure N.V, Spark Therapeutics LLC, Bluebird Bio, Juno Therapeutics, GlaxoSmithKline, Celgene Corporation, Shire Plc, Sangamo Biosciences, Dimension Therapeutics, Voyager Therapeutics, Human Stem Cell Institute, Bristol Myers Squibb, and Chiesi Farmaceutici S.p.A.

Due to a large number of pipeline molecules in development and intense competition among companies to augment their revenue growth, the market is projected to tread along a healthy growth track. Most of the startups are attracting capital investments to support their research for new molecules and initiate new product development.

Attribute

Details

Base year for estimation

2016

Actual estimates/Historical data

2014 – 2016

Forecast period

2017 – 2025

Market representation

Revenue in USD Million and CAGR from 2017 to 2025

Regional scope

North America, Europe, Asia Pacific, Latin America, Middle East & Africa

Country scope

U.S., Canada, Germany, U.K., China, Japan, Brazil, South Africa

Report coverage

Revenue forecast, company share, competitive landscape, growth factors and trends

15% free customization scope (equivalent to 5 analyst working days)

If you need specific information, which is not currently within the scope of the report, we will provide it to you as a part of customization

This report forecasts revenue growth and provides an analysis of themarket trends in each of the sub-markets from 2014 to 2026. For the purpose of this report, Grand View Research has segmented the global gene therapy market report on the basis of indication, vector type, and region:

Indication Outlook (Revenue, USD Million, 2014 – 2026)

Cancer

Cardio Vascular Diseases

Infectious Diseases

Genetic Disorders

Neuro Disorders

Others

Vector Type Outlook (Revenue, USD Million, 2014 – 2026)

Viral Vectors

Retrovirus

Adenovirus

Adeno-associated virus

Vaccinia virus

Herpes simplex virus

Others

Non-Viral Vectors

Injection of Naked DNA

Lipofection

Others

Regional Outlook (Revenue, USD Million, 2014 – 2026)

North America

Europe

Asia Pacific

Latin America

MEA

See more here:
Gene Therapy Market Share Insights – Grand View Research

Recommendation and review posted by Bethany Smith

Stem Cell Skin Care – anti-aging cream and hydration Serum

SC21 BioTech: Stem Cell Skin Care Set

SC21 nowoffers a rejuvenating stem cell skin careset that is available to help restore aging skin. At SC21, we have been able to combine human mesenchymal stem cell growth factors, polypeptide complexes, and cytokines, with our day time anti-aging cream & evening hydration serum.

Our SC21 biotechnology scientists have developed a process to isolate potent rejuvenating factors from human stem cells. By resupplying the skin with these powerful missing factors, SC21 Day & Night Stem Cell Skin Care promotes cell renewal, boosts the production of collagen and elastin, restores aging cells, and, ultimately, provides you with more youthful looking skin.

It is important to note that as we age, the stem cell population that is vital in providing healing signals to the skin dramatically diminishes. As a result of this, the rejuvenating components the skin needs to maintain its appearance lessen. By replenishing lost peptides, cytokines & growth factors with the use of a topical product on the skin, we, through the day &night skin care set, are able to effectively re-engage the skins healing process.

The SC21 day & night stem cell skin care rejuvenation set also has a complete solution for restoring aging skin. We have, through the day anti-aging cream & night hydration serum been able to use: human mesenchymal stem cell growth factors, to regenerate human tissues; polypeptide complexes, (which penetrate the epidermis, outer layer of our skin) to send signals to the skin cells and cytokines proteins to send signals between the skin cells.

Focus Ingredient of Growth Factor Skin Care:

Mesenchymal Stem Cell (MSC) Peptide Complex = 15% (cytokines, growth factors, peptide complex)

Other Key Ingredients:

Focus Ingredient of Growth Factor Skin Care:

Mesenchymal Stem Cell (MSC) Peptide Complex = 20%(cytokines, growth factors, peptide complex)

Other Key Ingredients:

Apply 2-3 pumps to clean & dry skin.

Peptides are easier explained as signaling molecules produced by cells to instruct other cells.

As cellular messengers, cytokines influence and control our biological processes from start to finish. There are hundreds of unique cytokines in the human body. Cells talk with cytokines to repair injury, repel microbes, fight infections, and develop immunity.

Growth factors, are, on the other hand, diffusible signaling proteins that stimulate the growth of specific tissues and play a crucial role in promoting cell differentiation and division.

Many modern medical advances, including stem cell breakthroughs, are made possible due to our growing understanding of cytokines & growth factors. We use modern culture techniques (the same type used to produce human insulin and other naturally occurring substances) to grow human stem cells in the laboratory to harvest their regenerative cytokines and growth factors.

Mesenchymal stem cells (MSCs), which are traditionally found in the bone marrow, are used to improve function upon integration because they are self-renewing cells that have the capacity to differentiate, and are capable of replacing and repairing damaged tissues.

MSCs can consequently during culture, produce the following:

Our skin cells are biologically designed to continuously renew themselves, but, starting from our mid 20s, the skin cell renewal process slows down and our skin becomes thinner. This thinning causes us to be more prone to skin damage from external elements.

However, there are other factors that can contribute to our aging process, and in other cases even cause premature aging. Some of these factors include:

More:
Stem Cell Skin Care – anti-aging cream and hydration Serum

Recommendation and review posted by Bethany Smith

Which spare body parts will stem cells deliver first? | Cosmos

On 6 November 1998, the world woke to news of an astonishing discovery. James Thomson and his colleagues at the University of Wisconsin-Madison had generated stem cells from human embryos. Unlike other types of stem cells, these were pluripotent meaning they had the potential to generate any type of body tissue if given the right signals.

For many this news, and the accompanying claims that embryonic stem (ES) cells could revolutionise medicine, appeared to come out of the blue. However, for those of us already working in the stem cell space it was the vital next step in exploring the potential of stem cell science.

Back in 1998, I was a keen PhD student, part of the stem cell research effort at Monash University. I was trying to create pluripotent stem cells from the skin cells of a mouse. The idea was to first clone a mouse embryo from its skin cell and harvest the ES cells. In the lab next door, Ben Reubinoff had been working with Alan Trounson and Martin Pera for several years to see if they could make embryonic stem cells from donated human embryos effectively in parallel to their colleagues in Wisconsin.

There was a lot of excitement about how we might one day be able to use these cells to make replacement body tissues effectively on demand and alleviate suffering for many patients. Although we all recognised this was going to take an enormous amount of effort and time to deliver.

Outside the lab if I mentioned that I worked in stem cell research, I was met with overwhelming curiosity. But people also wondered why we couldnt just use adult stem cells which are found in some of our organs. Many people I spoke to already knew somebody who had been helped by a stem cell transplant using bone marrow or cord blood. Why did we need to use human embryos and ES cells at all?

The reason was, and still is, that adult stem cells are not able to generate any type of tissue because they are not pluripotent. Bone marrow stem cells, for instance, can regenerate an immune system but they cannot regenerate the pancreas or brain tissue. The only source of pluripotent cells was surplus human embryos originally created in an IVF clinic and then donated to research.

In 2007, Japanese scientists made a landmark discovery that side-stepped the need to use embryos. They were able to manipulate ordinary human skin cells to make them pluripotent (a much more elegant and effective approach than my attempts with mice skin cells during my PhD). Dubbed induced pluripotent stem cells or iPSC, these cells share the same desirable features as ES cells. They can be grown in the lab and coaxed to form specific types of body cells.

But both sources of pluripotent stem cells also carry the risk that they could form a tumour if we dont fully direct their developmental fate. Any clinical application must meticulously weed out the stem cells as part of the laboratory recipe used to make the replacement cells. For me, the crucial challenge is how to harness the potential of stem cells to develop safe and effective treatments.

These days, as the head of the outreach and policy program for Stem Cells Australia, a nationwide consortium of Australian stem cell scientists, I spend a lot of my time talking to the public. To some extent Ive become a race caller frequently asked to predict what new treatments are likely to come galloping down the track. Sometimes Im asked to offer an opinion on stem cell treatments that are not on the track at all. Promoted as a sure thing and available now for a price, these interventions lack credible evidence that they work or are even safe. Providers are effectively peddling hope and should be viewed with caution.

Fortunately, we do have providers committed to responsibly advancing the field with lots of bona fide contenders in clinical trials. So with my binoculars firmly in place, here is my reading of whats coming down the track.

Jeffrey Phillips

Leading the charge towards the clinic is a possible treatment for the most common cause of age-related vision loss: macular degeneration. In Australia about one in seven people over the age of 50 have some evidence of this disease. In this condition, damage to the cells at the back of the eye the macula affects central vision and the ability to read, drive and recognise faces. The actual seeing cells in the macula are intact but sight is lost because a tiny underlying patch of darkly pigmented cells are damaged. Known as retinal pigmented epithelial cells or RPE cells, they act like a pit stop team, feeding and clearing away waste for the highly active cells of the retina.

Because the number of RPE cells needed is very small and pluripotent stem cells readily develop into this exact tissue (you can easily spot a patch of darkly pigmented cells in the dish), macular degeneration has long been a favourite. Clinical trials are now underway in the United States, United Kingdom and Japan to determine whether replacing faulty RPE cells with those made in the lab from either human embryonic stem cells or induced pluripotent stem cells could help.

At this early stage, safety is a key concern. The surgical technique to deliver the cells carries the risk of detaching the retina and causing further vision loss. In May 2018, the London Project to Cure Blindness announced that two patients with macular degeneration specifically whats called the wet form due to extensive blood vessel growth under the retina had improved their vision with no significant side-effects after participating in a clinical trial.

Another early entrant in the race to the clinic is type 1 diabetes. Its a disease caused by friendly fire: the immune system seeks and destroys the beta cells of the pancreas. These remarkable cells can both sense rising blood sugar levels and release the exact amount of insulin needed to lower glucose levels to normal. When these cells are destroyed, which often occurs in childhood, the person is no longer able to control their blood sugar levels.

More than 120,000 Australians manage the disease with regular injections of insulin. But they cant regulate their blood sugar levels as precisely as beta cells do. And there are consequences: high blood sugar levels can damage the blood vessels in the heart, eyes and kidneys, while low levels can be fatal. Some patients have been lucky enough to receive a whole pancreas transplant or tissues containing beta cells from cadavers. But there are two problems. First, transplant donors are in short supply. Second, the donated tissue will likely suffer the fate of the original: attack by the immune system.

Enter pluripotent stem cells. Supply is no longer a problem. After two decades of trying, scientists are now able to make large quantities of fully functional beta cells in the lab. And as far as keeping the immune system at bay, several start-up companies have come up with the tea-bag approach. They encase the beta cells in a porous capsule. Like tea leaves, the beta cells are netted in but soluble factors easily move in and out across the net, including insulin and blood-borne glucose as well as other nutrients. Crucially, the net also stops marauding immune cells from getting to the beta cells.

The Californian company, Viacyte, is trialling a teabag about the size and shape of a credit card. Made of surgical-grade polymer, the capsule encases immature beta cells (theyre more robust if they mature inside the body), and is inserted just under the patients skin.

The key challenge, so far, is providing intimate contact with surrounding blood vessels so that the transplanted cells increase in number and survive. In June this year, the company reported its results at a meeting of the American Diabetes Association. Overall, they said there was a low rate of survival, but when cells did survive they produced insulin.

The company is now evaluating a second device that allows the patients blood vessels to grow through the walls of the capsule.

Jeffrey Phillips

A strong stayer in the race to the clinic is Parkinsons disease (PD). Predominantly a disease of ageing, around 1% of people over the age of 60 suffer from it.

The disease results from the death of brain neurons that release the neurotransmitter dopamine. Like a conductor, dopamine ensures different parts of the brain act in synchrony to execute routine movements. Without dopamine, patients have trouble controlling their walking and experience tremors in their hands and other parts of their bodies. Could replacing the faulty dopamine-producing neurons with healthy ones provide a way to combat PD?

More than 20 years ago, a few different research groups around the world gave it a try. Using human foetal tissue, they dissected out the dopamine-producing cells, and surgically implanted these into the brains of patients, specifically in a region called the striatum.

Some patients improved, but others reported significant side effects, particularly uncontrollable jerky movements known as dyskinesia. Questions were asked about whether the correct types of cells were being transferred to the correct part of the brain and further experiments were put on hold. A key question was whether pluripotent stem cells could offer a more precise and reliable source of dopamine-producing cells.

Jump forward to 2018 and several groups are on the cusp of testing new types of replacement cells for PD in a series of clinical trials. Years of research has shown that ES cells and iPS cells can be directed to develop into the correct type of neurons and that sufficiently large numbers can be generated.

When tested in animals, the dopamine-producing cells corrected movement disorders and did not form tumours.

This time around, rather than working in silos, different groups of researchers in Japan, Sweden, UK and US have banded together in a coalition called G-Force PD. Although each group is using a slightly different approach for their clinical trial, by sharing their results and expertise they hope to bring a cell-based therapy for PD closer to reality.

Jeffrey Phillips

Skin stem cells have long been solid performers for growing skin grafts to treat severe burns. But in November 2017, headlines ran hot with a report that a seven-year-old refugee Syrian boy, on the verge of death from a genetic skin condition, had been saved by a graft of skin stem cells corrected by gene therapy.

Hassan, now living with his family in Germany, suffered from a severe form of Epidermolysis Bullosa (EB). Its been referred to as the worst disease youve never heard of. It affects about 500,000 people worldwide, and can be caused by mutations to 18 different genes. In each case, the mutation disrupts the anchoring of the skins upper layer, the epidermis, to the underlying dermis. The result is skin that tears as easily as a butterflys wing. The only treatment is painful bandaging and re-bandaging.

Hassans skin had started blistering from birth but by the time he was seven, a bacterial infection had robbed him of 80% of his skin cover. In a last ditch effort to save his life, his German doctors contacted veteran stem cell researcher Michele De Luca at the University of Modena and Reggio Emilia in Italy. In 2006, De Luca had used skin grafts corrected by gene therapy to treat a leg wound of a woman who suffered from the same form of EB that Hassan suffered from. It was caused by a mutation to a gene called LAMB3.

De Lucas team took a tiny patch of skin containing stem cells from Hassans groin. They also spliced a copy of the LAMB3 gene into a benign virus. Then they infected the skin cells with the virus which ferried the LAMB3 gene into their DNA. The genetically corrected skin grew into a sheet which was grafted onto Hassans body. Five months after the first graft, Hassan was discharged. A month later he was back at school and playing soccer. Thanks to the genetically corrected stem cells, his grafted skin no longer blisters or shreds. The executive director of the Dystrophic Epidermolysis Bullosa Research Association of America dubbed Hassans treatment a sea change to the world of EB. Besides de Lucas group, Peter Marinkovich and Jean Tang at Stanford University School of Medicine, United States, are also trialling genetically-corrected skin grafts for a different type of EB.

Jeffrey Phillips

One of the front runners at the start of the stem cell race was spinal cord injury. Perhaps you remember the actor Christopher Reeve, aka Superman? Following a horse riding accident that left him a quadriplegic, he campaigned tirelessly for researchers to be allowed to use human embryonic stem cells to treat spinal cord injury which claims about 180,000 new cases each year. Perhaps thanks to his efforts in 2010, the world saw the first clinical trial using cells made from human ES cells.

Conducted by the California based biotech company Geron, the researchers had directed ES cells to develop into precursors of oligodendrocytes. These octopus-like cells wind their arms around neurons in the spinal cord to provide electrical insulation as well as nurturing factors. With a spinal cord injury, these important support cells can be lost. Four patients were injected with stem cell-derived oligodendrocyte precursors soon after their injury.

Controversially, Geron discontinued the study in 2011 to refocus their business. Asterias Biotherapeutics picked up the baton and last July, in a company press release, reported the results of an early clinical trial on 25 additional patients who were all injected with oligodendrocyte precursors three to six weeks post-injury. They reported no serious adverse events and that four patients recovered a degree of motor function that may increase their ability to lead an independent life. However, we have to wait to see the peer reviewed published results before we can assess the state of progress.

Beyond replacing oligodendrocytes made from ES cells, other clinical trials are testing different types of cells ranging from neurons obtained from donated foetal tissue to using the patients own cells obtained from the back of the nose where they play an important role in supporting the regeneration of the olfactory neurons. Some types of transplanted cells may act as paramedics, helping damaged motor neurons to recover. Others are designed to directly replace spinal cord neurons.

It remains too early to tell which approach will result in long-term improvements. While many with spinal cord injury are eager for even small improvements such as bladder or bowel control, patients should be careful about trying marketed experimental procedures outside well-conducted clinical trials as they may cause further harm. In a chilling example, one young woman who sought treatment using olfactory cells developed a large, painful mucus-secreting tumour in her spine and no improvement of her paraplegia. Unfortunately, many stem cell cures promoted online, especially for spinal cord injury, lack credibility.

Seeking advice from your medical specialist is the best way to find out more. If they dont know about a trial or claimed treatment, it is probably a mirage.

Jeffrey Phillips

Marked as a long shot for many years, stem cell research is starting to pay dividends for kidney disease. Though its not ready to provide transplants, it is already helping to discover new treatments.

Kidneys are the bodys vital cleansing and balancing system. They filter waste products and toxins from our blood into urine, maintain the bodys water balance and also make hormones important for regulating blood pressure and the production of red blood cells.

Kidney disease, which affects one in 10 Australians, damages the filtration units called nephrons. The major causes are diabetes and high blood pressure. Once gone, the nephrons cannot regenerate. But waiting for a donated kidney can take years; close to 1,000 Australians are currently on the waiting list for a transplant. This health crisis has catapulted researchers into trying to recreate kidney tissue from pluripotent stem cells an immense challenge as these are complex biological machines composed of many interacting parts.

Melissa Littles group, based at the Murdoch Childrens Research Institute in Melbourne, have pioneered this research. In 2015, they successfully grew tiny kidney-like structures that were showcased on the cover of Nature with the headline: Kidney in a dish. While their mini-kidneys possess many of the working parts of a mature kidney, theres a long way to go before they can be used as transplants. The plumbing for example bringing blood in and taking waste out is not yet functional. Also they are tiny, smaller than the tip of your finger.

Nevertheless, these mini-kidneys are already making a difference to our understanding of how kidneys develop and what goes awry in kidney disease, especially the hereditary form. For example, researchers were recently able to make mini-kidneys from a child suffering from a rare genetic condition that can cause end-stage kidney disease. They did it by first generating iPS cells from the childs skin. In the lab they were able to observe structural abnormalities in the childs cells and also showed that when the genetic mutation was corrected, the structural defect was corrected. This provides a new insight into inherited kidney disease where previously we knew very little about how these conditions develop.

Jeffrey Phillips

This article appeared in Cosmos 80 – Spring 2018 under the headline “The stem cell race”

More:
Which spare body parts will stem cells deliver first? | Cosmos

Recommendation and review posted by Bethany Smith

Male Hypogonadism | Endocrinology | Dartmouth-Hitchcock

Alternative names: Gonadal Deficiency, Testosterone Deficiency

What is male hypogonadism? What are the signs of male hypogonadism? What causes male hypogonadism? How does my doctor tell if I have male hypogonadism? How is male hypogonadism treated?

Male hypogonadism is caused by a man’s testes failing to produce normal levels of the male sex hormone, testosterone. Some men are born with hypogonadism, while others may develop the condition later in life.

There are two kinds of male hypogonadism:

Male hypogonadism at puberty can slow a boy’s growth, and affect the development of normal male sexual characteristics. He may not undergo the normal changes a boy has during puberty, such as a deepening voice, body and facial hair, and increased muscle mass.

Male hypogonadism in adults can cause:

Primary hypogonadism, in which the testes do not work properly, can be caused by many conditions, including:

Secondary hypogonadism, in which the endocrine glands do not stimulate the testes to produce hormones, can be caused by:

Your doctor may check for low levels of testosterone (male sex hormone) by performing a blood test. He or she may also use blood tests to check the levels of the pituitary hormones (FSH and LH) that stimulate the testes to produce their hormones.

Other laboratory tests can help your doctor tell if hypogonadism is being caused by a problem with the testes, or with the pituitary gland. Such tests include:

If male hypogonadism is caused by a pituitary or other tumor, treatment is aimed at removing the tumor, or reducing its effects. This can include medication, surgery, and/or radiation therapy.

Male hormone replacement therapy has been used successfully for years to treat male hypogonadism. This involves a man taking testosterone by injection, transdermal system (patch), or gel.

Information on the Dartmouth-Hitchcockwebsite:

Our goals are to provide people with meaningful information to make informed decisions about their health and health care.

Dartmouth-Hitchcock and its affiliated component organizations aspire to deliver consistent high quality medical care to all patients and to continually improve its quality of care as evolving technology and medical knowledge permits.

Please call 911 in the case of any medical emergency.

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Male Hypogonadism | Endocrinology | Dartmouth-Hitchcock

Recommendation and review posted by Bethany Smith

What is CRISPR?

In this video Paul Andersen explains how the CRISPR/Cas immune system was identified in bacteria and how the CRISPR/Cas9 system was developed to edit genomes.

Do you speak another language? Help me translate my videos:http://www.bozemanscience.com/transla…

Music Attribution

Intro Title: I4dsong_loop_main.wavArtist: CosmicDLink to sound: http://www.freesound.org/people/Cosmi…Creative Commons Atribution License

OutroTitle: String TheoryArtist: Herman Jollyhttp://sunsetvalley.bandcamp.com/trac…

All of the images are licensed under creative commons and public domain licensing:Adenosine. (2009). English: Artistic rendering of a T4 bacteriophage. The colours grey and orange do not signify anything, they are just used to illustrate structure. Created for Wikipedia. Retrieved from https://commons.wikimedia.org/wiki/Fi…E. coli Bacteria. (n.d.). Retrieved February 17, 2016, from https://www.flickr.com/photos/niaid/1…Fioretti, B. F. Hallbauer &. (2015). English: Director, Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology. Visiting professor The Laboratory for Molecular Infection Medicine Sweden MIMS; http://www.mpiib-berlin.mpg.de/resear…. Retrieved from https://commons.wikimedia.org/wiki/Fi…Foresman, P. S. ([object HTMLTableCellElement]). English: Line art drawing of a chimera. Retrieved from https://commons.wikimedia.org/wiki/Fi…Magladem96. (2014). English: Picture of DNA Base Flipping. Retrieved from https://commons.wikimedia.org/wiki/Fi…project, C. wiki. (2014). English: Crystal Structure of Cas9 bound to DNA based on the Anders et al 2014 Nature paper. Rendition was performed using UCSFs chimera software. Retrieved from https://commons.wikimedia.org/wiki/Fi…Providers, P. C. (1979). English: Photomicrograph of Streptococcus pyogenes bacteria, 900x Mag. A pus specimen, viewed using Pappenheims stain. Last century, infections by S. pyogenes claimed many lives especially since the organism was the most important cause of puerperal fever and scarlet fever. Streptococci. Retrieved from https://commons.wikimedia.org/wiki/Fi…RRZEicons. (2010). English: zipper, open, close. Retrieved from https://commons.wikimedia.org/wiki/Fi…UC Berkeley. (n.d.). Gene editing with CRISPR-Cas9. Retrieved from https://www.youtube.com/watch?v=avM1Y…

Read more from the original source:
What is CRISPR?

Recommendation and review posted by Bethany Smith

NOVA – Official Website | Cryonics

Major funding for “Making Stuff” is provided by the National Science Foundation.

This material is based upon work supported by the National Science Foundation under Grant No. DRL-1222986. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Additional funding is provided by the U.S. Department of Energy’s Office of Science.

This material is based upon work supported by the Department of Energy under Award Number(s) DE-SC0008715. This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

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NOVA – Official Website | Cryonics

Recommendation and review posted by Bethany Smith

Cryonics | Fallout Wiki | FANDOM powered by Wikia

Cryonics is the low-level temperature preservation of humans and animals in a suspended animation by slowing their vital functions, for the purposes of preserving them and keeping them alive for periods of time ranging from decades or even centuries until they are needed. Other purposes include safekeeping, or for keeping them alive for as long as possible while their brains are connected to an external expression device.

Before the Great War, Robert House and his company, as well as the Big Mountain Research Facility, are known to have produced technology within the cryogenic and cryonic field. Robert House extensively researched extending the human life, and created his own cryonic chamber that allowed the user to connect their brain pattern and consciousness to an external interface. Big Mountain produced hibernation chambers with similar purposes, however, the nature of their research into the field is unknown.[1] The United States Armed Forces experimented with cryonic technology as well, making the systems at the Raven Rock facility, and using cryonic chambers in the Sierra Army Depot.

The Sierra Army Depot dealt in the field of cryonics, with one example being their cryonic submergence of Dobbs in bio med gel.

The Environmental Protection Agency made use of hibernation chambers, holding three subjects within each. These editions were large transparent tanks filled with ice cold water, that is filled and drained through a long serpentine pipe system.

The Enclave’s scientific personnel at the Raven Rock base in the Capital Wasteland have several rooms dedicated to stasis, and their prisoner restraints use the same systems. There are two different versions of the Raven Rock stasis chambers: a green chamber holding the subject in the center, and a transparent blue holographic/light-based/photonic resonance chamber. The systems in Raven Rock appear to be the most advanced systems of their kind currently known on Earth. These chambers have been used to preserve yao guai, deathclaws, super mutants, feral ghouls, and human prisoners. Whether these stasis chambers are built for long-term preservation on the scale of years is unknown. The same blue-light stasis technology is used in Fort Constantine’s T-51b power armor storage room. Although unseen in-game, Vault 87 was meant to have been equipped with four stasis-chambers.[2]

Its history unknown, a liquid nitrogen-based weapon commonly known as a Cryolator could be easily constructed.

The alien civilization aboard Mothership Zeta made cryonics a core aspect of their invasion and their research. The longest known preservation of a subject aboard the ship was over 600 years. They worked by entirely sealing the subject and the chamber from the outside, and used freezing cold air to suspend the subject. When opening, the chamber would decompress and stabilize with outside air, and force the subject to drop to the floor. Furthermore, there was a second method of cryonics used by the aliens. In their dedicated cryo lab, during the Zeta Uprising, the Lone Wanderer could have frozen alien soldiers with the loose systems, thawing them into an ice block for a mere few seconds. Elliott Tercorien could have harnessed the energy to create cryo grenades and cryo mines, which exact the same effect on targets within the blast radius.

The only instance of cryonic technology in the Mojave Wasteland is Robert House’s cryonic preservation chamber in the Lucky 38. This is one of the only known instances of a cryonic chamber that links the subject’s brain and consciousness with an external interface. It is an advanced piece of technology, linking Robert House to an external interface, from which he has access to large amounts of data from the Lucky 38’s mainframe, as well as the ability to control his Securitrons. Opening the chamber, even for a second, will doom the subject to having only little more than a year left at life due to exposure to outside contaminants. House hopes that with the Courier’s help, he will be able to make the same cryonic technology he uses available to other high-value individuals in the future. The only other instance of cryonic technology is at the hazmat testing ground in the Big Empty, for storing the hazmat suit

Vault 111, located in Boston, was built to observe the effects of suspended animation on unsuspecting test subjects for 180 days, and holds multiple cryosleep pods. They are first shown to the Sole Survivor pre-War, disguised as “decontamination” pods. The Sole Survivor was preserved for exactly 210 years, with their spouse and son being taken in the year 2227, 150 years in. All the other residents of the Vault perished by 2227, due to Conrad Kellogg not reactivating their life support, with Shaun being abducted as an infant by him for the Institute.

Green stasis tanks are also seen holding super mutants in the FEV Lab in the Institute’s Bioscience sector.

Vault 0 kept pre-War geniuses in cryogenic stasis, by extracting their brains from their body and freezing them. They were then hooked up to the Calculator supercomputer, melding all of the identities of each connected brain into one.

The Boulder Dome in Denver was equipped with prototype military medical cryo tanks that had a very high malfunction rate, and before the Great War, scientists were frozen in sleeper tanks. Victor Presper continued to use the equipment during his time spent there.

Cryonic technology appears in Fallout 2, Fallout 3, its add-on Mothership Zeta, Fallout: New Vegas and its add-on Old World Blues, Fallout 4, and Fallout Tactics. It is also mentioned in the Fallout: New Vegas add-on Dead Money. Cryonic technology was also meant to appear in the canceled Van Buren.

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Cryonics | Fallout Wiki | FANDOM powered by Wikia

Recommendation and review posted by Bethany Smith

Female genetics – Women and Alzheimer’s

Several genes are known to be associated with higher risk of Alzheimers disease (conversely, in some cases these genes have been found to protect against the disease). One of these genes is known as APOE.

APOE has three common forms:

APOE2, the least common gene, is believed to play a role in reducing the risk of Alzheimers diseaseAPOE4, more common than APOE2, is believed to increase the risk of Alzheimers diseaseAPOE3, the most common gene, has not been found to increase or decrease the risk of developing Alzheimers disease

Inheriting one of the APOE4 gene variants found in about 20% of the populationmay increase the chance of developing Alzheimers disease by a factor of four. Inheriting two of APOE4 variants (one from each parent) will increase the chance by a factor of 10. Additionally, a 2014 study found that women with the APOE4 gene were twice as likely to get Alzheimers disease than women who do not carry the gene; for men with the APOE4 gene the risk factor does not increase.

Known genetic factors such as this account for a small percentage of all Alzheimers disease cases, but current research supported by Cure Alzheimers Fund and others indicates that genetics have a far greater influence than was previously thought. Additional candidate genes are being discovered and studied to determine their possible role in the disease. The genes we do know about account for a large percentage of early-onset cases: The rare Presenilin 1 and Presenilin 2 genes, for example, virtually guarantee development of Early Onset Alzheimers disease.

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Female genetics – Women and Alzheimer’s

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Membership | Cryonics Institute

Cryonics is a fascinating concept that has inspired the imagination and dreams of thousands of people worldwide. If you’re someone interested in the theory, remarkable potential and practical applications of cryonics, joining CI is a great way to learn more and get involved in the cryonics movement. Our members come from all walks of life and from all around the world, united by our interest in cryonics and the potential benefits it holds for ourselves and for all mankind.

As a member, you will be one of the owners and operators of the Cryonics Institute, as we are wholly owned and operated by our membership. CI is run by a Board of Directors elected exclusively from our members, by our members – so we have no outside investors, managers or other parties dictating our operations. Our responsibilities are to our patients, our membership, and to advancing our founder Robert Ettinger’s vision. CI membership is an excellent starting point for anyone interested in cryonics to learn more and be part of an exciting, potentially world-changing community of forward-thinking people. There are no specific duties or formal responsibilities required for membership, apart from applying and paying the membership fees. However a large number of our members take a more active role in the organization either as officially elected Officers or as volunteers. How active you choose to be is completely up to your own discretion.

Please note, Cryonics Institute Membership is required if you are actively planning cryonic suspension services for yourself or a loved one through CI. Our “Members-Only” policy for cryonic services helps ensure the quality of our suspensions, and maintains the integrity of our organization and operations. CI is our organization and as member-owners it’s clearly in our own best interests to manage it efficiently and especially to insure the highest standards for our suspension arrangements.

There are two classes of CI Membership. A Lifetime Member pays a one-time fee of $1,250 and can arrange for cryopreservation at CI for $28,000, usually by making CI the beneficiary of a life insurance policy. Other close family members can join for an additional $625 (there is no charge for minor children). An Annual (or Yearly) Member pays a $75 initiation fee plus $120 yearly (or $35 quarterly) and can arrange for cryopreservation at CI for $35,000. Every Yearly Membership family member must pay the same price. Neither of these fees include the cost of preparation or shipment by a local funeral director, which must be arranged separately (often with a Local Help Rider). To join, simply fill out a membership form for the type of membership you desire, Annual ($120/year recurring) or Lifetime ($1,250 one time.) The forms are available below or can be mailed on request.

To learn more about membership options and details, please see our Frequently Asked Questions. We also provide a special Membership Outreach program that gives you the opportunity to speak one-on-one with a current CI member who will help answer your questions via phone or email.

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Membership | Cryonics Institute

Recommendation and review posted by Bethany Smith

Bioidentical Hormone Doctors

NOTE: Forever Health provides a directory where people like you can connect with BHRT and other physicians and innovative health practitioners. We do not provide medical advice or services directly. While BHRT and prevention are important to the physicians and health practitioners listed, each has his or her own approach to practicing medicine. So when scheduling your appointment, be sure to clarify the reason for your visit, as well as your goals for seeking out such treatment.

NOTE on Insurance: Innovative practitioners and insurance companies have long debated the importance of preventive medicine and services such as BHRT. Due to variations in coverage for novel treatment options, many practitioners will resolve this issue by providing their services on a cash-only basis. Forever Health encourages you to contact the practitioner to determine if any alternative payment options or post-visit reimbursements exist.

DISCLAIMER: Inclusion in this directory is free to practitioners and does not constitute endorsement by Forever Health. All health practitioners who appear on this list do so on the sole basis of their own expression of interest in the fields of BHRT or other integrative medicine. Forever Health does not verify the competence, professional credentials, business practices or validity of the expressed interests of these health practitioners. Forever Health makes no recommendation of any health practitioner on this list and makes no suggestion that any such health practitioner will cure, treat, or prevent any disease or condition.

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Bioidentical Hormone Doctors

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Cellular Therapy – fujifilmcdi.com

iPSCells Represent a Superior Approach

iPS cell-derived cardiomyocyte patch demonstrates spontaneous and synchronized contractions after 4 days in culture.

One of the greatest promises of human stem cells is to transform these early-stage cells into treatments for devastating diseases. Stem cells can potentially be used to repair damaged human tissues and to bioengineer transplantable human organs using various technologies, such as 3D printing. Using stem cells derived from another person (allogeneic transplantation) or from the patient (autologous transplantation), research efforts are underway to develop new therapies for historically difficult to treat conditions. In the past, adult stem and progenitor cells were used, but the differentiation of these cell types has proven to be difficult to control. Initial clinical trials using induced pluripotent stem (iPS) cells indicate that they are far superior for cellular therapy applications because they are better suited to scientific manipulation.

CDIs iPS cell-derived iCell and MyCell products are integral to the development of a range ofcell therapyapplications. A study using iCell Cardiomyocytesas part of a cardiac patch designed to treat heart failure is now underway. This tissue-engineered implantable patch mayemerge as apotential myocardial regeneration treatment.

Another study done with iPS cell-derived cells and kidney structures has marked an important first step towards regenerating, and eventually transplanting, a functioning human organ. In this work, iCell Endothelial Cellswere used to help to recapitulatethe blood supply of a laboratory-generated kidney scaffold. This type of outcome will be crucial for circulation and nutrient distribution in any rebuilt organ.

iCell Endothelial Cells revascularize kidney tissue. (Data courtesy of Dr. Jason Wertheim, Northwestern University)

CDI and its partners are leveraging iPS cell-derived human retinal pigment epithelial (RPE) cells to develop and manufacture autologous treatments for dry age-related macular degeneration (AMD). The mature RPE cells will be derivedfrom the patients own blood cells using CDIs MyCell process. Ifapproved by the FDA, this autologous cellular therapy wouldbe one of the first of its kind in the U.S.

Learn more about the technologybehind the development of these iPScell-derived cellular therapies.

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Cellular Therapy – fujifilmcdi.com

Recommendation and review posted by Bethany Smith

What is gene therapy? – Genetics Home Reference – NIH

Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patients cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including:

Replacing a mutated gene that causes disease with a healthy copy of the gene.

Inactivating, or knocking out, a mutated gene that is functioning improperly.

Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently being tested only for diseases that have no other cures.

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What is gene therapy? – Genetics Home Reference – NIH

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Storing Stem Cells For Life – Smart Cells

One of the bravest moves in that direction has come from stem cell research and therapy. Stem cell therapy is currently being used to successfully treat more than 80 diseases, but the field is rapidly evolving backed by prestigious research and clinical trials.

Smart Cells is the first private UK stem cell storage company to have released stored stem cell units for use in the treatment of children with life-threatening illnesses. We have released the greatest number of samples for use in transplants from the UK.

We believe with the development of technology in the future we will be able to treat even more illnesses.

We believe our customers deserve the best service available and we run our state of the art facility with leading professionals in the field.

We believe that storing your childs stem cells at birth can be a crucial part of treating or curing an unexpected illness.

We believe that in the future this service should be available to every parent, child and family.

We are a company that is for life.

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Storing Stem Cells For Life – Smart Cells

Recommendation and review posted by sam

Bio-Identical Hormone Replacement Therapy – Hormone Clinic

Youve been using Natural Hormones all your life, why change now?

The term Bio-identical Hormone Replacement Therapy is interchangable with Natural Hormone Replacement Therapy.

Health is a holistic goal, and hormonal health put simply, is a large significant factor of that big picture. To control your hormonal health you need towork with an understanding doctorwho believes in the value of hormonal testing to use as a tool in the treatment of your health.

Combining your symptoms with your hormonal profile, looking at you individually, and considering your own, and your family history a picture can be brought into focus. Once obtained, this picture of your individuality can be used to give you a predictable and active future so that age is not a barrier to wellness.

When hormone supplementation is warranted, there arefour major points to consider. These stem from the belief that if we are going to relieve or reverse the ageing process, it should be done in such a way as to mimic the bodys system as it was created. In that endeavour, only hormones that are the exact molecules made by the human endocrine system should be used. The human race would not have survived this long if these hormones were dangerous and subjected us to fatal diseases.

Secondly, those hormones used should be introduced into the bloodstream in a way that emulates the glands as closely as possible, that is, avoiding the digestive tract and liver and minimising first-pass effects.

Thirdlya broader spectrum of hormones, at lower doses, offers a more complete, physiological balance.

Finallythe dose must be individualised to the patients needs and goals which in the end will result in fewer side effects if any, along with reducing or eliminating other medical problems.

Bio-identical Hormone Replacement Therapyproducts are compounded by a compounding pharmacist, which offers you and your physician a choice for individualised hormone replacement.

Naturalrefers to thestructureof the hormone (i.e. they are bio-identical in structure and function to the hormones naturally produced in our bodies). The hormones start from wild yam and soybeans which are rich in precursor molecules. These are easily converted by biochemists into other molecules that areidentical in all aspectsto our own naturally occurring hormones.

It should be emphasised that although these precursor molecules can be synthesised into natural bio-identical hormones in a test tube, our bodies are incapable of doing so. Therefore although wild yam creams are good moisturisers they do nothing to increase our hormonal levels.

Insulin used by diabetics was obtained in the past from pigs, which saved many lives, yet caused a significant amount of side effects and problems. Pig insulin is no longer used by diabetics as a Natural identical human insulin has been established, overriding any reason for using pig insulin. In this same light, there is now NO cause for women to be using oestrogens and progesterones produced from the urine of pregnant mares, aswe now have natural bio-identical human hormones available.

Bio-identical hormone replacement therapy is administered generally, in the form of atroche(lozenge) or as a transdermal cream which areabsorbed into the blood streamwithout having to go through the digestive system creating stress on the liver. We find Troches are the most suited form for Natural Hormone Replacement Therapy mainly because of the ease in which the ingredients in each prescription can be altered, and because of their superior transbuccal absorption.

Compare this to the mass produced formulas available commercially where titrating a dose is almost impossible and one must fit in with what is available.

Results have been good to excellent and in many cases extraordinary. The natural or bio-identical hormones used in the treatment of menopause are any combination of natural Progesterone, Oestrogen, Testosterone, at levels determined specifically for each patient.

Trochescan be mixed so that each individual troche contains a combination of various natural bio-identical hormones in small doses. For example, a troche can be made containing a mixture of any of the hormones, eg: oestrogen, progesterone, testosterone in any possible doses according to your needs. This combination is used to treat the symptoms of menopause.

Small doses oftestosteroneare useful inmenopausefor depression, to enhance energy levels, mood and libido without causing side effects.

Troches containing natural progesterone alone are helpful for premenstrual syndrome and peri menopause and can be taken during the latter half of the menstrual cycle to alleviate depression, migraine, nausea and basically all those PMT symptoms that occur premenstrually.

Menopausal women who have had ahysterectomycan benefit from troches containingnatural bio-identical estrogens,progesteroneandtestosterone. Even though the uterus may not be present, in the case of hysterectomy,progesterone is still essentialto balance the oestrogen component and stop oestrogenic side effects. Your requirements should be determined with a blood test to measure your sex hormones we recommend tests for blood oestrogen, progesterone and testosterone.

Troches can be made palatable, by making them in different flavours. Each troche is made up on an individual basis and the whole process takes a few days.

Once deciding to embark on taking hormone replacement one is usually forced to grapple with the risk/benefits factor. It must be stressed that although we believe Bio-identical hormones used in troches may be safer than Synthetic hormones,there are still risksrelating to the use of natural hormones. These risks are mostly related to the uses of oestrogen and the increase risk of breast cancer. For medicolegal purposes we have to assume the use of bio-identical hormones carries the same risk as synthetic hormones. The current thinking suggests that if we need to use any form of oestrogen to treat menopause we should attempt to stop this within 2 yrs of commencement.

Tailor made troches allow slow reduction of oestrogen and in my experience it is most definitely easier to slowly wean a patient off Oestrogen due to ease of dose adjustment. It would appear that continuation on progesterone and testosterone over a longer period is a safer option.

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Bio-Identical Hormone Replacement Therapy – Hormone Clinic

Recommendation and review posted by Bethany Smith

Low Testosterone (Male Hypogonadism) | Cleveland Clinic

What is low testosterone (male hypogonadism)?

Low testosterone (male hypogonadism) is a condition in which the testes (testicles, the male reproductive glands) do not produce enough testosterone (a male sex hormone).

In men, testosterone helps maintain and develop:

Low testosterone affects almost 40% of men aged 45 and older. It is difficult to define normal testosterone levels, because levels vary throughout the day and are affected by body mass index (BMI), nutrition, alcohol consumption, certain medications, age and illness.

As a man ages, the amount of testosterone in his body gradually drops. This natural decline starts after age 30 and continues (about 1% per year) throughout his life.

There are many other potential causes of low testosterone, including the following:

Symptoms of low testosterone depend on the age of person, and include the following:

Other changes that occur with low testosterone include:

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Low Testosterone (Male Hypogonadism) | Cleveland Clinic

Recommendation and review posted by simmons

Hypogonadism? | Go Ask Alice!

Dear Just Wondering,

Hypogonadism and hypergonadism are syndromes that result from abnormal levels of testosterone and estrogen. They affect the reproductive systems in both sexes, permanantly causing the testes and ovaries to not function properly. Hypogonadism acts by lowering the production and quality of testosterone and sperm in men and estrogen and eggs in women. This imbalance in the body’s chemistry can result in a lowered sex drive in both men and women. Hypogonadism can also cause infertility.

Hypogonadism can appear either before or after puberty. If it occurs before puberty, the symptoms can include:

Hypogonadism after puberty can cause:

Treatment of hypogonadism usually comes through hormone replacement therapy. In men, testosterone is replaced, and in women, estradiol (a precursor to estrogen) and progesterone. While this therapy has improved the chances of many couples trying to have children, it does not help everybody.

On the other end of the spectrum is hypergonadism. As you most likely guessed, those with hypergonadism have higher levels of testosterone and estrogen in their systems. While this may sound great for the dating scene, the extra hormones are not as fun as they may seem.

Hypergonadism is rarer than hypogondism. But, like hypogonadism it can appear either before or after puberty.

Hypergonadism occuring before puberty actually prods puberty into action. After puberty those diagnosed exhibit the same affects as the prepubescent. Hypergonadism causes the same changes in both men and women, including:

Like hypogonadism, hormonal treatments are needed to correct hypergonadism. But since there are higher levels of estrogen and testosterone coursing through the body, a delicately balanced hormonal cocktail is needed. Treating hypergonadism is much more difficult because it is tougher to lower an excess of hormones than it is to add them to the body.

The latest research points towards many different sources as the cause of hypogonadism and hypergonadism in both males and females including:

It appears that the best course of action for treating both hypogonadism and hypergonadism is hormonal therapy. An endocrinologist, a medical provider specializing in the body’s hormones, can make certain that hormonal balance is achieved in the safest possible way.

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Hypogonadism? | Go Ask Alice!

Recommendation and review posted by Bethany Smith

Causes of Hair Loss in Women | Bernstein Medical

Common baldness in women, also called female pattern alopecia, is genetically inherited and can come from either the mothers or fathers side of the family. Female alopecia most commonly presents in a diffuse pattern, where hair loss occurs over the entire scalp. Less commonly, women exhibit a patterned distribution where most of the thinning occurs on the front and top of the scalp with relative sparing of the back and sides.

The type of hair loss, diffuse or patterned, has important implications for treatment. Women with diffuse hair loss are generally best treated medically, whereas women with patterned hair loss may be good candidates for hair transplant surgery. Interestingly, patterned hair loss is the most common type seen in men and accounts for why a greater proportion of men are candidates for surgery compared to women.

In women who are genetically predisposed to hair loss, both diffuse and patterned distributions are caused by the actions of two enzymes: aromatase (which is found predominantly in women) and 5-a reductase (which is found in both women and men). Diffuse hair loss is most often hereditary, but it can also be caused by underlying medical conditions, medications, and other factors; therefore, a thorough medical evaluation is an important part of the management.

In the next sections, we will take a closer look at both the mechanisms of genetically induced female hair loss as well as the medical conditions and drugs that can cause diffuse hair loss in women.

As with hair loss in men, female genetic hair loss largely stems from a complex stew of genes, hormones, and age. However, in women, there are even more players. In addition to 5-a reductase, testosterone, and dihydrotestosterone (DHT); which are also found in mens hair loss; also present in women are the enzyme aromatase and the female hormones estrone and estradiol. So lets break down the process that leads to common hair loss in women.

In both men and women, 5-a reductase reacts with testosterone to produce DHT, the hormone responsible for the miniaturization (shrinking) and the gradual disappearance of affected hair follicles. This explains why both men and women lose their hair. But one of the reasons why women seldom have the conspicuous bald areas that men do is because women naturally have only half the amount of 5-a reductase compared to men.

Adding to this complexity, in women, the enzyme aromatase is responsible for the formation of the female hormones, estrone, and estradiol, counteract the action of DHT. Women have higher levels of aromatase than men, especially at the frontal hairline. It is this presence of aromatase which may help explain why hair loss in women looks so different than in men, particularly with respect to the preservation of the frontal hairline. It may also explain why women have a poor response to the drug finasteride (Propecia), a medication widely used to treat hair loss in men that works by blocking the formation of DHT.

The following is a schematic chart of how the female hormones estrone and estradiol are produced and their relationship to DHT:

Womens hair seems to be particularly sensitive to underlying medical conditions. Since systemic medical conditions often cause a diffuse type of hair loss pattern that can be confused with genetic balding, it is important that women with undiagnosed alopecia be properly evaluated by a doctor specializing in hair loss (i.e., a dermatologist).

Below is a list of medical conditions that can lead to a diffuse pattern of hair loss:

A relatively large number of drugs can cause telogen effluvium, a condition where hair is shifted into a resting stage and then several months later shed. Fortunately, this shedding is reversible if the medication is stopped, but the reaction can be confused with genetic female hair loss if not properly diagnosed. Chemotherapy and radiotherapy can cause a diffuse type of hair loss called anagen effluvium that can be very extensive. This hair loss is also reversible when the therapy is over, but the hair does not always return to its pre-treatment thickness.

Causes of Telogen Effluvium

Causes of Anagen Effluvium

A host of dermatologic conditions can cause localized hair loss in women. The pattern that they produce is usually quite different from the diffuse pattern of female genetic hair loss and is easily differentiated from it by an experienced dermatologist. Occasionally, the diagnosis is difficult to make and tests, such as a scalp biopsy are necessary.

Localized hair loss in women may be sub-divided into scarring and non-scarring types.

Non-Scarring Alopecias

Alopecia Areata is a genetic, auto-immune disease that typifies the non-scarring type. It manifests with the sudden onset of discrete, round patches of hair loss associated with normal underlying skin. It usually responds quite well to local injections of corticosteroids.

Localized hair loss can be also be caused by constant pulling on scalp hair, either through braiding, tight clips or hair systems. Traction alopecia, the medical term for this condition, often causes reversible thinning but, if the tugging on the follicles persists for an extended period of time, the hair loss can be permanent. The most common presentation is thinning, or complete hair loss, at the frontal hairline and in the temples of women who wear their hair pulled tightly back. Early traction alopecia can reverse itself by simply wearing the hair loose. A hair transplant may be needed to restore the hair that is permanently lost from sustained traction.

Scarring Alopecias

Scarring hair loss can be caused by a variety of medical or dermatologic conditions such as Discoid Lupus, Lichen Planus, and infections. It can also be caused by thermal burns or local radiation therapy. Face-lift surgery may result in permanent localized hair loss that can be particularly bothersome if it occurs at the frontal hairline or around the temples. Fortunately, localized hair loss from injury or from medical problems are often amenable to hair transplantation.

Many of the factors that cause the rate of loss to speed up or slow down are unknown, but we do know that with age, a persons total hair volume will decrease. This is referred to as senile alopecia. Even when there is no predisposition to genetic balding, hair across the entire scalp will thin over time resulting in the appearance of less density. The age at which these effects finally manifest themselves varies from one individual to another and is mainly related to a persons genetic makeup.

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Causes of Hair Loss in Women | Bernstein Medical

Recommendation and review posted by simmons

Addgene: CRISPR Guide

Generating a Knockout Using CRISPR

You can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cpf1 and a gRNA specific to the gene to be targeted. The genomic target can be any 20 nucleotide DNA sequence, provided it meets two conditions:

The PAM sequence is essential for target binding, but the exact sequence depends on which Cas protein you use. We’ll use the popular S. pyogenes Cas9 (SpCas9) as an example, but check out our list of additional Cas proteins and PAM sequences. Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation. Importantly, the spacer region of the gRNA remains free to interact with target DNA.

Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3 end of the gRNA targeting sequence) will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3 to 5 direction. The zipper-like annealing mechanics of Cas9 may explain why mismatches between the target sequence in the 3 seed sequence completely abolish target cleavage, whereas mismatches toward the 5 end distal to the PAM often still permit target cleavage.

The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (3-4 nucleotides upstream of the PAM sequence).

The resulting DSB is then repaired by one of two general repair pathways:

The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations (for more information, jump to Plan Your Experiment.) In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. However, the strength of the knockout phenotype for a given mutant cell must be validated experimentally. Learn more about non-homologous end joining (NHEJ).

Browse Plasmids: Double-Strand Break (Cut)

CRISPR specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing a gRNA for your experiment (see the Plan Your Experiment section below).

In addition to optimizing gRNA design, CRISPR specificity can also be increased through modifications to Cas9. As discussed previously, Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB.

Thus, two nickases targeting opposite DNA strands are required to generate a DSB within the target DNA (often referred to as a double nick or dual nickase CRISPR system). This requirement dramatically increases target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. Therefore, if high specificity is crucial to your experiment, you might consider using the dual nickase approach to create a double nick-induced DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene edits.

In 2015, researchers used rational mutagenesis to develop two high fidelity Cas9s: eSpCas9(1.1) and SpCas9-HF1. eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9’s interactions with the DNA phosphate backbone. HypaCas9, developed in 2017, contains mutations in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wildtype Cas9.

Browse Plasmids: Single-Strand Break (Nick)

While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag.

In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence must be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms.) The length of each homology arm is dependent on the size of the change being introduced, with larger insertions requiring longer homology arms.

Depending on the application, the repair template may be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double-stranded DNA plasmid. When designing the repair template, do not include the PAM sequence present in the genomic DNA. This step prevents the repair template from being a suitable target for Cas9 cleavage. For example, you could alter the DNA sequence of the PAM with a silent mutation that does not change the amino acid sequence.

The efficiency of HDR is generally low (

Since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a large portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally and to isolate clones containing the desired edit (see the validation section in Plan Your Experiment). Learn more about homology directed repair (HDR).

Browse Plasmids: Endogenous Tagging

As discussed above, the efficiency of HDR is very low due to the number of DSBs repaired by NHEJ. To make point mutations without using HDR, researchers have developed CRISPR base editors that fuse Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC1. Base editors are targeted to a specific locus by a gRNA, and they can convert cytidine to uridine within a small editing window near the PAM site. Uridine is subsequently converted to thymidine through base excision repair, creating a C->T change (or G->A on the opposite strand.) This class of base editors is available with multiple Cas9 variants and using high fidelity Cas9s. In addition, new base editors have been engineered to convert adenosine to inosine, which is treated like guanosine by the cell, creating an A->G (or T->C) change. Learn more about CRISPR DNA base editors.

Browse Plasmids: Base Edit

Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing an ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA base editor that converts adenosine to inosine in RNA (termed REPAIR.) Since inosine is functionally equivalent to guanosine, the result is an A->G change in RNA. dPspCas13b does not appear to require a specific sequence adjacent to the RNA target, making this a very flexible editing system. Editors based on ADAR2(E488Q/T375G) display improved specificity, and editors carrying the delta-984-1090 ADAR truncation retain RNA editing capabilities and are small enough to be packaged in AAV particles.

Browse Plasmids: RNA Editing

CRISPR is a remarkably flexible tool for genome manipulation, as Cas enzymes bind target DNA independently of their ability to cleave target DNA. Specifically, both RuvC and HNH nuclease domains can be rendered inactive by point mutations (D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the gRNA targeting sequence.

Early experiments demonstrated that targeting dCas9 to transcription start sites was sufficient to repress transcription by blocking initiation. dCas9 can also be tagged with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcriptional repression or activation of downstream target genes. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator, A (e.g. VP64) or transcriptional repressor, R (e.g. KRAB; see panel A to the right).

Additionally, more elaborate activation strategies have been developed for more potent activation of target genes in mammalian cells. These include: co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, panel B), dCas9 fused to several different activation domains in series (e.g. dCas9-VPR, panel C) or co-expression of dCas9-VP64 with a modified scaffold gRNA and additional RNA-binding helper activators (e.g. SAM activators, panel D). Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.

Browse Plasmids: Activate, Repress/Interfere

Cas enzymes can be fused to epigenetic modifiers like p300 and TET1 to create programmable epigenome-engineering tools. Like CRISPR activators and repressors, these tools alter gene expression without inducing double-strand breaks. However, they are much more specific for particular chromatin and DNA modifications, allowing researchers to isolate the effects of a single epigenetic mark.

Another potential advantage of CRISPR epigenetic tools is their persistence and inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated/removed from the system. In contrast, epigenetic marks left by targeted epigenetic modifiers may be more frequently inherited by daughter cells. In certain cases, epigenetic modifiers may work better than activators/repressors in modulating transcription. However, since the effects of these tools are likely cell type- and context-dependent, it might be beneficial to try multiple CRISPR strategies when setting up your experimental system.

Browse Plasmids: Epigenetics

Expressing several gRNAs from the same plasmid ensures that each cell containing the plasmid will express all of the desired gRNAs, thus increasing the likelihood that all desired genomic edits will be carried out by Cas9. Such multiplex CRISPR applications include:

Current multiplex CRISPR systems enable researchers to target anywhere from 2 to 7 genetic loci by cloning multiple gRNAs into a single plasmid. These multiplex gRNA vectors can conceivably be combined with any of the aforementioned CRISPR derivatives to not only knock out target genes, but activate or repress target genes as well. Read more about Cas9 multiplexing and Cpf1 multiplexing.

Browse Plasmids: Multiplex gRNA vectors

The ease of gRNA design and synthesis, as well as the ability to target almost any genomic locus, make CRISPR the ideal genome engineering system for large-scale forward genetic screening. Forward genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known. In general, the goal of a genetic screen is to generate a large population of cells with mutations in a wide variety of genes and then use these mutant cells to identify the genetic perturbations that result in a desired phenotype.

Before CRISPR, genetic screens relied heavily on shRNA technology, which is prone to off-target effects and false negatives due to incomplete knockdown of target genes. In contrast, CRISPR is capable of making highly specific, permanent genetic modifications that are more likely to ablate target gene function. CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis. Currently, the most popular method for conducting genome-wide screens using CRISPR involves the use of pooled lentiviral CRISPR libraries.

Pooled lentiviral CRISPR libraries (often referred to as CRISPR libraries) are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.

Guide RNAs are designed in silico and synthesized (see panel A below), then cloned in a pooled format into lentiviral transfer vectors (panel B). CRISPR libraries have been designed for common CRISPR applications including genetic knockout, activation, and repression for human and mouse genes.

Each CRISPR library is different, as libraries can target anywhere from a single class of genes up to every gene in the genome. However, there are several features that are common across most CRISPR libraries. First, each library typically contains 3-6 gRNAs per gene to ensure modification of every target gene, so CRISPR libraries contain thousands of unique gRNAs targeting a wide variety of genes. Guide RNA design for CRISPR libraries is usually optimized to select for guide RNAs with high on-target activity and low off-target activity.

Keep in mind that the exact region of the gene to be targeted varies depending on the specific application. For example, knockout libraries often target 5 constitutively expressed exons, but activation and repression libraries will target promoter or enhancer regions. Be sure to check the library page/original publication to see if a library is suitable for your experiment. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.

CRISPR libraries from Addgene are available in two formats: as DNA, or in select cases, as pre-made lentivirus.

In the case of DNA libraries, the CRISPR library will be shipped at a concentration that is too low to be used in experiments. Thus, the first step in using your library is to amplify the library (panel C) to increase the total amount of DNA. When amplifying the library, it is important to maintain good representation of gRNAs so that the composition of your amplified library matches that of the original library. You’ll use next-generation sequencing (NGS) to verify that this is the case. Learn more about library verification.

Once the library has been amplified/verified, the next step is to generate lentivirus containing the entire CRISPR library (panel D). Then, you will transduce cells with the lentiviral library (panel E). Remember – if you are using a 2-vector system, you will transduce cells that are already expressing Cas9. After applying your screening conditions, you will look for relevant genes (hits) using NGS technology. For more detail on using CRISPR for both positive and negative screens, see our pooled library guide.

As noted above, forward genetic screens are most useful for situations in which the physiology or cell biology behind a particular phenotype or disease is well understood, but the underlying genetic causes are unknown. Therefore, genome-wide screens using CRISPR libraries are a great way to gather unbiased information regarding which genes play a causal role in a given phenotype. With any experiment, it is important to verify that the hits you identify are actually important for your phenotype of interest. You can individually test the gRNAs identified in your screen to ensure that they reproduce the phenotype of interest.

Find more information on factors to consider before starting your pooled library experiment in Practical Considerations for Using Pooled Lentiviral CRISPR Libraries (McDade et al., 2016).

Browse Libraries: CRISPR Pooled Libraries

Using catalytically inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable DNA labeler compatible with fluorescence microscopy in living cells. Alternatively, gRNAs can be fused to protein-interacting RNA aptamers, which recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize targeted genomic loci.

CRISPR imaging has numerous advantages over other imaging techniques, including that it is easy to implement due to the simplicity of gRNA design, programmable for different genomic loci, capable of detecting multiple genomic loci, and compatible with live cell imaging. Compared to techniques like fluorescence in situ hybridization (FISH), CRISPR imaging offers a unique method for detecting the chromatin dynamics in living cells.

Multicolor CRISPR imaging allows for simultaneous tracking of multiple genomic loci in living cells. One method uses orthogonal dCas9s (e.g., S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent proteins. Alternatively, one can fuse gRNAs to orthogonal protein-interacting RNA aptamers, which recruit specific orthogonal RNA-binding proteins (RBPs) tagged with different fluorescent proteins, as seen in the popular CRISPRainbow kit.

The fluorescent CRISPR system has been used for dynamic tracking of repetitive and non-repetitive genomic loci, as well as chromosome painting in living cells. Visualizing a specific genomic locus requires recruitment of many copies of labeled proteins to the given region. For example, chromosome-specific repetitive loci can be efficiently visualized in living cells using a single gRNA that has multiple targeted sequences in close proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus. Chromosome painting requires delivery of hundreds of gRNAs with target sites throughout the chromosome.

Browse Plasmids: Label

Identifying molecules associated with a genomic region of interest in vivo is essential to understanding locus function. Using CRISPR, researchers have expanded chromatin immunoprecipitation (ChIP) to allow purification of any genomic sequence specified by a particular gRNA.

In the enChIP (engineered DNA-binding molecule-mediated ChIP) system, catalytically inactive dCas9 is used to purify genomic DNA bound by the gRNA. An epitope tag(s) can be fused to dCas9 or gRNA for efficient purification. Various epitope tags including 3xFLAG-tag, PA, and biotin tags, can be used for enChIP, as well as an anti-Cas9 antibody. Biotin tagging of dCas9 can be achieved by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin ligase, as seen in the CAPTURE system. The locus is subsequently isolated by affinity purification against the epitope tag.

After purification of the locus, molecules associated with the locus can be identified by mass spectrometry (proteins), RNA-sequencing (RNAs), and next-generation sequencing (NGS) (other genomic regions). Compared to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward and enable direct identification of molecules associated with a genomic region of interest in vivo.

Browse Plasmids: Purify

In bacteria, type VI CRISPR systems recognize ssRNA rather than dsDNA. Many type VI enzymes also have the ability to process crRNA precursors to mature crRNAs. Upon ssRNA recognition by the crRNA, the target RNA is degraded. In bacteria, Cas13 enzymes can also cleave RNAs nonspecifically after the initial crRNA-guided cleavage. This promiscuous cleavage activity slows bacterial cell growth and may further protect bacteria from viral pathogens. Non-specific cleavage does not occur in mammalian cells. Similar to Cas9 and Cpf1, Cas13 can be converted to an RNA-binding protein through mutation of its catalytic domain. Learn more about Cas13a.

Browse Plasmids: RNA Targeting

While S. pyogenes Cas9 (SpCas9) is certainly the most commonly used CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every application. For example, the PAM sequence for SpCas9 (5 NGG 3) is abundant throughout the human genome, but an NGG sequence may not be positioned correctly to target your desired genes for modification. This limitation is of particular concern when trying to edit a gene using homology directed repair (HDR), which requires PAM sequences in very close proximity to the region to be edited. Kleinstiver et al. generated synthetic SpCas9-derived variants with non-NGG PAM sequences. Gao et al. subsequently engineered Cpf1 PAM variants. The inclusion of these variants into the CRISPR arsenal effectively doubles the targeting range of CRISPR in the human genome. Read more about Cas9 variants.

Additional Cas9 orthologs from various species bind a variety of PAM sequences. These enzymes may have other characteristics that make them more useful than SpCas9 for specific applications. For example, the relatively large size of SpCas9 (4kb coding sequence) means that plasmids carrying the SpCas9 cDNA cannot be efficiently packaged into adeno-associated virus (AAV). Since the coding sequence for Staphylococcus aureus Cas9 (SaCas9) is 1 kilobase shorter than SpCas9, SaCas9 can be efficiently packaged into AAV. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo.

Another limitation of SpCas9 is the low efficiency of making specific genetic edits via HDR. For specific point edits, CRISPR base editing is a useful alternative to HDR. For larger edits, Cpf1, first described by Zetsche et al., may be a better option. Unlike Cas9 nucleases, which create blunt DSBs, Cpf1-mediated DNA cleavage creates DSBs with a short 3 overhang. Cpf1s staggered cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 also expands the range of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.

CRISPR is a powerful system that enables researchers to manipulate the genome like never before. This section will provide a general framework to get you started using CRISPR in your research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR in other organisms. First, consider the genetic manipulation that is necessary to model your specific disease or process of interest. Do you want to:

Once you have a clear understanding of your experimental goal, you are ready to start navigating the different reagents that are available for your particular experiment.

Different genetic manipulations require different CRISPR components. Selecting a specific genetic manipulation can be a good way to narrow down which reagents are appropriate for a given experiment. Make sure to check whether reagents are available to carry out your experiment in your particular model organism. There may not be a perfect plasmid for your specific application, and in such cases, it may be necessary to customize an existing reagent to suit your needs.

To use CRISPR, you will need both Cas9 and a gRNA expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may be sufficient to express the CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may be more appropriate. In cases where off-target editing is a major concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression.

The table below summarizes the major expression systems and variables for using CRISPR in mammalian cells. Some of the variables include:

Once you have selected your CRISPR components and method of delivery, you are ready to select a target sequence and design your gRNA.

When possible, you should sequence the region you are planning to modify prior to designing your gRNA, as sequence variation between your gRNA targeting sequence and target DNA may result in reduced cleavage. The number of alleles for each gene may vary depending on the specific cell line or organism, which may affect the observed efficiency of CRISPR knockout or knockin.

In order to manipulate a given gene using CRISPR, you will have to identify the genomic sequence for the gene you are trying to target. However, the exact region of the gene you target will depend on your specific application. For example:

A PAM sequence is absolutely necessary for Cas9 to bind target DNA. As such, one can start by identifying all PAM sequences within the genetic region to be targeted. If there are no PAM sequences for your chosen enzyme within your desired sequence, you may want to consider alternative Cas enzymes (see Cas9 variants and PAM sequences). Once possible PAM sequences and putative target sites have been identified, it is time to choose which site is likely to result in the most efficient on-target cleavage.

The gRNA target sequence needs to match the target locus, but it is also critical to ensure that the gRNA target sequence does NOT match additional sites within the genome. In a perfect world, your gRNA target sequence would have perfect homology to your target with no homology elsewhere in the genome. Realistically, a given gRNA target sequence will have partial homology to additional sites throughout the genome. These sites are called off-targets and should be examined during gRNA design. In general, off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. To increase specificity, you can also consider using a high-fidelity Cas enzyme.

In addition to off-target activity, it is also important to consider factors that maximize cleavage of the desired target sequence or on-target activity. Two gRNA targeting sequences with 100% homology to their DNA targets may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. For example, gRNA targeting sequences containing a G nucleotide at position 20 (1 bp upstream of the PAM) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the target sequence.

Many gRNA design programs can locate potential PAM and target sequences and rank the associated gRNAs based on their predicted on-target and off-target activity (see gRNA design software). Additionally, many plasmids containing validated gRNAs are now available through Addgene. These plasmids contain gRNAs that have been used successfully in genome engineering experiments. Using validated gRNAs can save your lab valuable time and resources when carrying out CRISPR experiments. Read more about how to design your gRNA.

Browse Plasmids: Validated gRNAs

Once you have selected your target sequences it is time to design your gRNA oligos and clone these oligos into your desired vector. In many cases, targeting oligos are synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors).

Choose a delivery method that is compatible with your experimental system. CRISPR efficiency will vary based on the method of delivery and the cell type. Before proceeding with your experiment, it may be necessary to optimize your delivery conditions. Learn more about CRISPR delivery in mammalian systems.

Once you have successfully delivered the gRNA and Cas enzyme to your target cells, it is time to validate your genome edit. CRISPR editing produces several possible genotypes within the resulting cell population. Some cells may be wild-type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA.

Edited cells may be homozygous or heterozygous for edits at your target locus. Furthermore, in cells containing two mutated alleles, each mutated allele may be different owing to the error-prone nature of NHEJ. In HDR gene editing experiments, most mutated alleles will not contain the desired edit, as a large percentage of DSBs are still repaired by NHEJ.

How do you determine that your desired edit has occurred? The exact method necessary to validate your edit will depend upon your specific application. However, there are several common ways to verify that your cells contain your desired edit, including but not limited to:

More information on each of these techniques can be found in our blog post CRISPR 101: Validating your Genome Edit.

The majority of the CRISPR plasmids in Addgenes collection are from S. pyogenes unless otherwise noted.

Engineered Cpf1 variants with altered PAM specificities. 2017. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F. Nat Biotechnol. 35(8):789-792. PMID: 28581492

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. 2017. Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR. Sci Adv. 3(8):eaao4774. PMID: 28875174

Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. 2017. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR. Nat Commun. 8:15790. PMID: 28585549

Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. 2017. Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Nat Biotechnol. 35(4):371-376. PMID: 28191901

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. 2017. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F. Nat Biotechnol. 35(1):31-34. PMID: 27918548

Nucleic acid detection with CRISPR-Cas13a/C2c2. 2017. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science. 356(6336):438-442. PMID: 28408723

Programmable base editing of AT to GC in genomic DNA without DNA cleavage. 2017. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Nature. 551(7681):464-471. PMID: 29160308

RNA editing with CRISPR-Cas13. 2017. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. Science. pii: eaaq0180. PMID: 29070703

RNA targeting with CRISPR-Cas13. 2017. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. . 550(7675):280-284. PMID: 28976959

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. 2016. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. Nature. 529(7587):490-5. PMID: 26735016

Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. 2016. Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T. Nat Biotechnol. . PMID: 27088723

Naturally occurring off-switches for CRISPR-Cas9. 2016. Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR. Cell. 167(7):1829-1838. PMID: 27984730

Practical Considerations for Using Pooled Lentiviral CRISPR Libraries. 2016. McDade JR, Waxmonsky NC, Swanson LE, Fan M. Curr Protoc Mol Biol. 115:31.5.1-31.5.13. PMID: 27366891

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. 2016. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Nature. 533(7603):420-4. PMID: 27096365

Rationally engineered Cas9 nucleases with improved specificity. 2016. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Science. 351(6268):84-8. PMID: 26628643

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells. 2015. Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Ct J, Doyon Y. Cell Rep. 13(3):621-33. PMID: 26456817

An updated evolutionary classification of CRISPR-Cas systems. 2015. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. Nat Rev Microbiol. 13(11):722-36. PMID: 26411297

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. 2015. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. Nat Biotechnol. 33(1):73-80. PMID: 25357182

CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins. 2015. Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts BS, Mackiewicz M, Mendenhall EM, Myers RM. Genome Res. 25(10):1581-9. PMID: 26355004

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