Author Archives: admin


‘Pain is normal’: The frustrating present and possible future of … – Healio

February 27, 2023

13 min read

Source: Healio interviews

Disclosures: Gilbert reports no relevant financial disclosures. Lane reports consulting for Amgen, Genescense, GSK, Sanofi and Xalud. Neogi reports consulting for EMD-Merck Serono, Novartis, Pfizer/Lilly and Regeneron. Roos reports being the co-founder of Good Life with Osteoarthritis in Denmark (GLA:D), a not-for profit initiative hosted at University of Southern Denmark aimed at implementing clinical guidelines for osteoarthritis in clinical practice, as well as lecture fees, to herself or her institution, from TrustMe-ED and Learn.Physio. Skou reports being co-founder of GLA:D.

ADD TOPIC TO EMAIL ALERTS

Receive an email when new articles are posted on

Back to Healio

When it comes to patients with osteoarthritis adopting, much less maintaining, an exercise regimen to improve their condition, most rheumatologists have just one thought: Easier said than done.

However, because clinical trial programs for many potential OA drugs and surgical procedures have yielded mixed results, general wellness remains, at least for now, the optimal intervention for the condition.

Thus, the rheumatology community is tasked with a unique challenge: Encourage 32 million Americans, and 527 million individuals worldwide, to eat healthier and get moving.

Behavioral changes, including diet and exercise, continue to be first-line treatment for knee OA, Abigail Gilbert, MD, assistant professor of medicine in the division of rheumatology, allergy and immunology at the University of North Carolina Chapel Hill, told Healio Rheumatology.

It is not as though the pipeline for OA drugs has been lacking. Recent research has explored the Wnt pathway modulator lorecivivint (SM04690, Biosplice), as well as TPX-100 (OrthoTrophix), a 23-amino acid peptide that can be administered by intra-articular injection, among others.

In addition, an anabolic agent that appears to stimulate cartilage regeneration recombinant human fibroblast growth factor 18 (FGF18) will soon be studied in phase 3 clinical trials, Nancy E. Lane, MD, of the University of California, Davis Health System, told Healio Rheumatolgy, referring to the injectable drug sprifermin (TrialSpark).

However, while some OA trials have recently yielded successful or promising results, just as many, if not more, have historically produced frustrating dead ends.

There have been many studies investigating some of these treatment for osteoarthritis, Sren Thorgaard Skou, PT, PhD, MSc, head of research for PROgrez at Slagelse Hospital, and professor of exercise and human health, in the research unit for musculoskeletal function and physiotherapy at the University of Southern Denmark, said in an interview. But there is no magic cure for OA, and it is quite important to remember that.

As the various potential drugs and therapeutic mechanisms come and go, Ewa Roos, PT, PhD, professor in the faculty of health sciences, in the department of sports science and clinical biomechanics, and of the research unit of musculoskeletal function and physiotherapy, also at the University of Southern Denmark, suggested that it is just as important for providers to understand the whole of the OA patient experience.

There is the disease of OA, which includes structural changes and damage to tissue that we can see on imaging, Roos said. But then there is the illness of OA, which includes the patient experience of pain and loss of function.

It is the latter that drives patients to seek health care, according to Roos.

As of today, there are no effective treatments for the disease of OA, she said. So, what we are doing in clinical practice is treating the illness, or the patient experience.

The 2019 American College of Rheumatology recommendations for knee and hip OA, published by Kolasinski and colleagues in Arthritis & Rheumatology, are telling in this regard and underscore the points made by the above experts. Various types of exercise, from yoga to tai chi, received strong recommendations in the document.

Conversely, there are strong recommendations against novel and experimental approaches like transcutaneous electric nerve stimulation (TENS), chondroitin, platelet-rich plasma injections and stem cell injections. In addition, while there was significant concern surrounding opioid use, as expected, the voting panel even had reservations about pain management with acetaminophen.

Clinical trial programs for novel compounds and off-label medications will continue. Patients will undergo joint replacement procedures and other surgeries. However, until one or more therapies show the capacity to dramatically improve both the disease and the illness of OA, rheumatologists must manage pain as best they can and do whatever possible to encourage patients to be more active.

The benefits of physical exercise for a patient with OA are many, according to Tuhina Neogi, MD, PhD, chief of rheumatology and Alan S. Cohen professor of rheumatology at the Boston University School of Medicine and Boston Medical Center.

Importantly, exercise and physical therapy approaches are important to help address obesity, one of the most important risk factors for OA, she told Healio Rheumatology. By reducing the prevalence of obesity, an important reduction in OA prevalence and burden would be achieved.

For many experts, the issue of weight loss for OA is not one of if, but how.

While we frequently talk about weight loss as benefiting many diseases, including knee OA, we recognize this is often easier said than done, and we need more ways to support patients in being successful with weight loss, Gilbert said.

It is also important to remember that, in addition to obesity, patients with OA often have other chronic diseases like hypertension and diabetes that benefit from physical activity and weight loss, according to Gilbert.

Knee pain can certainly limit physical activity, so approaches to decrease pain can help patients be more successful at efforts to increase physical activity, she said.

Put simply, regardless of whether or not the patient demonstrates obesity or comorbid conditions, exercise is necessary. The question then becomes one of optimizing a regimen for each individual patient.

The COVID-19 pandemic, among its myriad impacts, brought a host of new technologies to the forefront in rheumatology, including a closer look at how wearable devices and other tech interventions can be wielded in patient care. At a time when many patients were confined indoors for months at a time, some of these interventions were used to remind patients to stand up, walk around or, if possible, run.

In a paper published in JMIR mHealth and uHealth by Bricca and colleagues, on which Skou was an investigator, 60 smartphone apps from the Apple App Store and Google Play underwent an analysis of their quality and potential for promoting behavior change, including exercise.

Apps for patients with a chronic condition or multimorbidity appear to be of acceptable quality but have low to moderate potential for promoting behavior change, the researchers wrote.

According to Skou, the use of technology in OA care is promising but needs further development. The main problem, he said, is in the maintenance phase.

It is easier to get patients to start exercising, but much more difficult to get them to maintain it, Skou said.

Until technology improves, Roos continues to rely on education as a cornerstone of managing weight and exercise habits in her patients.

The primary aim of education is to address common beliefs that exist, she said. For example, it is a common myth that exercise will be bad for your joints and cartilage. We have shown repeatedly in animals and humans that exercise therapy is not bad for the cartilage.

Another myth is that patients should not exercise if they are in pain.

It is OK to exercise if you are in pain, Roos said.

In fact, a certain amount of muscle fatigue and pain is expected, particularly for patients who have not exercised much previously, she added.

You are using your body in a way that maybe you have not used it before, Roos said. It will hurt. Pain is normal.

It is important in these situations to communicate to patients that muscle pain will decrease after the initial flare and, ideally, disappear with regular exercise.

The final myth described by Roos was that patients with radiographic changes, or who have undergone joint replacement, should not exercise.

I encourage all of my patients to exercise, she said. You can expect similar pain relief regardless of severity of radiographic changes.

Because it can be so difficult to get patients to lose weight and be more active even with consistent education or regular reminders from a phone or watch it is unavoidable that many patients progress to a point where further intervention is necessary. For those individuals, surgical options remain viable.

Joint replacement continues to be life-changing for some patients with advanced arthritis, Gilbert said. Some patients have been able to return to prior activities that they had set aside due to pain and have significantly improved quality of life.

Of course, as with any major surgery, there is potential for complications and adverse outcomes. The risks and benefits need to be carefully discussed and considered, Gilbert added.

It is definitely not for everyone, and not the first option to try, Roos said.

Findings from a randomized, controlled trial published by Skou and colleagues in The New England Journal of Medicine underscore this point. They randomly assigned 100 patients with moderate-to-severe knee OA to total knee replacement (TKR) followed by 12 weeks of non-surgical treatment, or only 12 weeks of non-surgical treatment. Change from baseline to 12 months in four Knee Injury and Osteoarthritis Outcome Score (KOOS4) subscales assessing pain, function and quality of life served as the primary outcome measure.

According to the researchers, the surgery group experienced a 32.5-point improvement on the KOOS4 scale compared with a 16-point improvement for the non-surgery group (95% CI, 10-21.5). However, surgery also yielded significantly more serious adverse events (P = .005).

For these reasons, TKR is often a complicated and personal choice, according to Gilbert.

Many patients want to exhaust conservative management before undergoing the knife, she said.

The good news is that the technology involved in joint surgery continues to improve, according to Lane.

The materials used for the implants and the ability to 3D print implants, to customize them to patients, is impressive, she said. In addition, the lifespan of joint replacements has increased to the point that a 60-year-old patient may only need one joint replacement in their lifetime.

Shoulder joint replacements have also improved, Lane added.

Patients report significant improvements in pain and function after the surgery, she said.

Additionally, although Skou agrees that it is possible to come a long way without surgery, he still sees joint replacement as a desirable and effective option for patients who do not improve from non-surgical care, albeit with one caveat.

If you have an unsuccessful surgery, the chances of success for a second or third surgery are lower, he said.

With exercise and weight loss presenting adherence challenges and joint replacement subject to pitfalls regarding patient choice and selection, a robust armamentarium of therapeutic options would be hugely beneficial for OA populations. Unfortunately, that is not what rheumatologists have to work with.

Lorecivivint and sprifermin were top of mind for Neogi when discussing potential new therapeutic agents.

Phase 2b data published by Yazici and colleagues in Osteoarthritis and Cartilage showed that lorecivivint yielded improvements over placebo, in terms of patient-reported outcomes like WOMAC pain and function, in a cohort of nearly 700 patients. A dosage of 0.07 mg yielded the best responses and may be optimal for future studies, according to the researchers.

However, in a review paper published in Deutsche Medizinische Wochenschrift, Krasselt and Baerwald described phase 2 results for lorecivivint as barely encouraging.

Regarding sprifermin, Zeng and colleagues published a meta-analysis of studies focusing on the drug in Arthritis Research & Therapy. They suggested that sprifermin had no adverse effects but did not likely have any positive effect on symptom alleviation.

Lorecivivint and sprifermin are the two agents that have had the most data available recently regarding potential disease-modifying effects, Neogi said. Both programs have highlighted the need for phenotyping to identify the right target population, particularly to demonstrate symptom modification.

Matching the right patient to the right intervention is no easy task for any rheumatologist. Often, the clinical community must look in unlikely places for answers of how to accomplish that goal.

Meanwhile, in a post-hoc analysis of a large cardiovascular trial of the interleukin (IL)-1 inhibitor canakinumab (Ilaris, Novartis) published in the Annals of Internal Medicine, Scheiker and colleagues reported a lower risk for joint replacement in the treatment arms compared with placebo.

This raises the possibility that IL-1 inhibition may have a symptom- and/or perhaps structure-modifying effect, Neogi said. This analysis also illustrated that promising signals may be missed in smaller phase 1/2 trials that are likely to have enrolled participants that are too heterogeneous to pick up an efficacy signal, whereas the much larger canakinumab trial was able to pick up a signal despite the noise due to sheer sample size.

Turning to more experimental interventions like platelet-rich plasma and stem cell therapies, Neogi called on the research community for well-designed and powered RCTs before recommendations can be made.

Despite the current lack of high-powered trials, Lane expressed optimism regarding these potential therapies.

Please note that while small clinical studies have shown that platelet-rich plasma, hyaluronic acid and mesenchymal stem cell injections are effective in some phase 2 and 3 clinical trials, Lane said.

However, she ultimately agreed with Neogis assessment that more data are necessary.

Additional study is needed to determine what patients would benefit from these therapies, Lane added.

According to Neogi, the biggest story of the past year, in terms of pain management, was that the anti-NGF tanezumab (Pfizer and Eli Lilly & Co.) program was discontinued. Meanwhile, another major anti-NGF program, fasinumab (Regeneron), was also recently discontinued, she added.

We will have to wait and see whether any future programs emerge for this target, Neogi said.

For Lane, the list of exciting new therapeutics in the OA space includes the use of adenovirus technology to introduce inhibitors of inflammatory cytokines like IL-1R and anti-inflammatory molecules like IL-10, which are entering, or have entered, phase 2 studies.

Another approach highlighted by Lane, and currently heading into phase 2 trials, involves targeting senolytic cells.

It will be important to know if reducing the burden of senolytic cells within the knee joint will reduce both symptoms and reduce structural deterioration in our OA patients, she said.

Looking deeper into the pipeline, research into cartilage transplants and bioengineered cartilage is also underway, according to Lane.

However, for the most part, the studies currently are preclinical, she said.

Despite the excitement surrounding these interventions, and the potential they hold, it remains unlikely that any of them will emerge as a cure-all for OA any time soon, according to Neogi. Instead, rheumatologists should focus their energies on using a multimodal approach to managing OA, she said. This involves treating patients based on their individual symptoms, needs and goals.

There is no longer a pyramid approach or hierarchy of therapies to consider in order, Neogi said. A multimodal approach should be considered, individualizing management plans by matching therapies to the patients symptoms, impairments, goals of care, acceptability, safety and feasibility. In addition, therapies may be revisited at multiple times over the course of someones OA journey.

To that point, patients may also look beyond traditional pharmacotherapies to relieve pain and reduce inflammation, among other outcomes.

Neogi suggested that turmeric and krill oil may have benefits in improving knee OA symptoms.

These provide additional modalities people may wish to try that are likely to have minimal side effects, she said.

Other non-pharmacotherapeutic interventions, according to Neogi, include radiofrequency ablation and genicular artery embolization. However, they come with some important caveats.

These procedures have shown some short-term symptom relief, but longer-term safety data are needed before they can be recommended given the theoretical concerns regarding long-term sequelae of disruption to sensation in joints, she said.

There may be some evidence that muscle mass and quality may be associated with knee OA, according to Lane.

Studies are underway to perform deep phenotyping of the muscle in knee OA subjects, she said, noting that her group is looking into this issue.

Meanwhile, although there is consensus among experts that biomarkers like type II collagen, PRO-C2, PRO-C2, C2M, CTX-II and T2CM could hold many clues as to OA disease pathogenesis and progression, there is just as much agreement that the data produced by research into these biomarkers are lacking.

When asked about new or promising biomarkers that are going to fundamentally alter the nature of OA management, Lane stated that none so far are ready for actual use in practice.

While there are some novel biomarkers from large longitudinal studies that have shown associations with development and progression of knee OA, these markers are not yet ready to be incorporated into OA management, she said. However, studies are closely evaluating serum for both proteomics and metabolics, and in the next few years we may know more.

Regarding injectable approaches, TPX-100 is a novel, 23-amino-acid peptide derived from matrix extracellular phosphoglycoprotein (MEPE), a 525-amino-acid protein that occurs naturally in humans and is known to be involved in the regulation of hard tissue and phosphate metabolism, according to Lane.

MEPE is a sibling protein made by osteocytes and osteoblasts, and its actions are often related to mineralization of musculoskeletal tissues, she said. Its mechanism related to reducing pain in knee OA is not clear. However, the phase 2 study does have some interesting results.

The findings for TPX-100 were presented by McGuire at the 2022 OARSI World Congress on Osteoarthritis. According to the presenter, the intervention yielded strong improvements in bone shape change and function that may ultimately minimize the need for knee replacement.

However, whether these results will lead to clinical use remains to be seen, according to Lane.

It is too soon to know if the results of this study a reduction in femoral bone shape change over 5 years translates to overall reduction in disease activity and change in joint degeneration, she said.

Further findings presented by McGuire showed that the symptoms of knee pain and knee function were different from placebo at the 2-year endpoint.

The results of this long-term extension study are intriguing, and hopefully will be followed by a phase 3 study that will incorporate both joint structure and symptoms as endpoints, Lane said.

Skou was more pointed in his assessment.

The results for TPX-100 look interesting, but we need more evidence before we can recommend it to patients, he said.

As the rheumatology community awaits results for this and other interventions, it may be useful to get back to basics, according to Roos.

Go here to read the rest:
'Pain is normal': The frustrating present and possible future of ... - Healio

11 Reasons You May Be Seeing Excessive Hair Loss, And What To … – Glam

Most of us are guilty of putting our hair through the wringer, so to speak. Thanks to all the amazing treatments out there that can alter the appearance of our locks, like perms and color treatments, most of us like to treat ourselves to a brand-new look every once in a while. And while our new hairdo might make us feel extra amazing, our hair usually does not have the same experience.

Whether you frequently color, perm, straighten, or relax your hair, you're introducing harmful chemicals to your hair follicles, and they can only put up with it for so long if you don't give your hair breaks. "After repeated insults, the hair follicles just won't grow back," Bethanee Schlosser, M.D. tells Self. Your hair will start to thin out, and you might start to notice that your scalp is more visible than it used to be eek!

If you suspect your hair loss is due to excessive chemical treatments, it's time to slow it way down and give your natural hair a chance to thrive. Holding off on any immediate chemical treatments can help you prevent the existing damage from getting worse. Growing back the hair you lost, however, isn't as simple, and it's a good idea to enlist the help of a dermatologist.

See the original post here:
11 Reasons You May Be Seeing Excessive Hair Loss, And What To ... - Glam

5 Things to Know About Plantar Fasciitis – Right as Rain by UW Medicine

Plantar fasciitis is a common cause of foot pain. But did you know it might be more common in women? And that most of us will experience it at some point?

Here's how to manage it and prevent it from becoming a long-term problem.

More than 2 million people are treated for plantar fasciitis each year, according to the American Academy of Orthopaedic Surgeons. Its an equal-opportunity foot problem that doesnt care how old you are, how active you are or what type of feet you have.

Usually we can figure out why people get other foot conditions, but with plantar fasciitis, there isnt always a pattern, says Dr. Edward Blahous, a podiatrist and podiatric surgeon who sees patients at the UW Medicine Sports Medicine Clinic at Ballard.

It may be more common in women, who make up most of Blahous patients (though it could also be that women are more likely to go to the doctor for it).

People who have what Blahous calls a tight Achilles tendon may be more likely to get plantar fasciitis, as well as people who have flat feet, high arches or are obese.

A lot of people who get it also have something wrong with their big toe joint, so there seems to be some correlation, though its not proven, Blahous adds.

Read more:
5 Things to Know About Plantar Fasciitis - Right as Rain by UW Medicine

How Injectable Fillers Became the Biggest Thing in Penis Enlargement – GQ

Six years ago, New York plastic surgeonDavid Shafer was performing filler injections on a female patients face as her boyfriend looked on. Its a routine cosmetic service, as these things go: filler is a gel-like substance designed to be shot below the skin for aesthetic tweaks. Half-jokingly, the man asked the doctor if he could do that same procedure to his penis. Rather than roll his eyes, Shafer told me that a light went off in his head. Not onlycould he do it, but the boyfriend could be his guinea pig. Theres no polite way to say this: Two days later the man returned to the office to have his dick pumped up with dermal filler.

View more

He called me a week later, Shafer told me recently. And said, Doc, I love it. Can I come back and get more?

Now plastic surgeons around the country are offering penis enlargement with the same tech used to plump lips anddefine jawlines. Shafer's version, which hes named the Shafer Width and Girth procedure (or, uh,SWAG), is a hit. Its become a huge part of our business, he said. When I first started it was one or two a week, then one a day, then two a day. Now its four or five a day. The procedure has gained so much momentum that in January the clinic opened up an entire floor dedicated exclusively to below-the-belt injections.

By all accounts, its a quick and easy procedure. Patients are first numbed with a shot of anesthetics at the base of their shaft before its then shot up with syringes of hyaluronic acid-based filler. Shafer uses Voluma or the newer Volux brand, which are more typically used for firmer areas of the face (the cheeks and chin) as opposed to areas that require softer consistencies, like the lips. Depending on the patient's preference, this can require 10 to 20 syringes of filler, setting a patient back anywhere between $11,000 to $20,000. While the results begin instantly, the full effects won't be seen (and felt) for about two weeks, and intercourse is discouraged for 48 hours after the surgery.

It looks like your penis gained weight, Shafer said. If you have a thin arm and then you gain weight and your arm gets thicker? It's like that. And while it technically doesnt add inches, Shafer does say that the added weight can often create an elongated appearance.

A patient who Ill call Jason said he stumbled on Dr. Shafer when scouring the Internet for penile enhancement products. I never had any complaints about my size, and had what I considered a healthy sex life, he said. But I was always looking for somethingmore. I think its beneficial to try to optimize ourselves, and whether its in the gym, or how we eat, or, trying a procedure like this, I feel its a way to enhance what we have, and just another process to become the best version of ourselves.

Jason told me he was nervous on his first visit, but still dropped $12,000 on injections. Im generally a reserved person, so you could imagine discussing private parts is difficult enough, he said. But pulling down your pants and saying, Hey, I would like to try to make this better can truly be a daunting thing.

He said the result speaks for itself: Not only does sex feel physically better, but the excitement around it is heightened due to the confidence attained. It appears that you can, in fact, buy big dick energy: Its walking around with this new, heavy dick that truly feels amazing.

One might guess that penile injections are for single men only, but William (not his real name) got injections during a short-term separation from his wife and, now that they've reconciled, has gone back for more. While he may have gotten them during a low period, he discovered that they enhance both his and his partners intercourse experience. More girth, of course, means more friction, and he said that the sensation for him is superior that the filler feels like his penis is wrapped in a jelly-like substance. Its hard to explain, he told me. It just feels better.

Down in Fort Lauderdale, Florida, dermatologistDr. Matthew Zarraga has cooked up a similar procedure, calledZ Roc Hard. Its a more comprehensive penile treatment that, in addition to dermal fillers, includes double chin treatment Kybella to break-up suprapubic fat over the genitals (which can give the penis a small appearance); platelet rich plasma injections into the shaft which can increase vascularity and sensitivity; andbotox into the testicles for a smoother, larger appearance. While the whole shebang cost up to $18,000, Zarraga still has guys flying in from all over the country for appointments

He recalls one man who had been considering the procedure for some time before his wife encouraged him to bite the bullet. She told him that, while she was perfectly satisfied, she had gotten a boob job. Along those lines, if he wanted to get penile injections to make himself feel better, he should do it. I injected him with six syringes, Zarraga said, and he comes back a month later and says I havent had this much sex with my wife in years. she loved the outcome so much that she wants me to get another six.

But it goes without saying that the benefits are often more for the recipients state of mind. Its telling that Shafer has said that hes seen plenty of guys who are quite well-endowed come in wanting to beef up.

Look, I have porn stars that come in who are like a horse, he said. And even they want more.

See original here:
How Injectable Fillers Became the Biggest Thing in Penis Enlargement - GQ

Popular injectables: Erection specialists set up shop – Townsville Bulletin

To use this website, cookies must be enabled in your browser. To enable cookies, follow the instructions for your browser below.

Facebook App: Open links in External BrowserThere is a specific issue with the Facebook in-app browser intermittently making requests to websites without cookies that had previously been set. This appears to be a defect in the browser which should be addressed soon. The simplest approach to avoid this problem is to continue to use the Facebook app but not use the in-app browser.

This can be done through the following steps:1. Open the settings menu by clicking the hamburger menu in the top right2. Choose App Settings from the menu3. Turn on the option Links Open Externally (This will use the devices default browser)

Enabling Cookies in Internet Explorer 7, 8 & 91. Open the Internet Browser2. Click Tools > Internet Options > Privacy > Advanced3. Check Override automatic cookie handling4. For First-party Cookies and Third-party Cookies click Accept5. Click OK and OK

Enabling Cookies in Firefox1. Open the Firefox browser2. Click Tools > Options > Privacy > Use custom settings for history3. Check Accept cookies from sites4. Check Accept third party cookies5. Select Keep until: they expire6. Click OK

Enabling Cookies in Google Chrome1. Open the Google Chrome browser2. Click Tools > Options > Privacy Options > Under the Hood > Content Settings3. Check Allow local data to be set4. Uncheck Block third-party cookies from being set5. Uncheck Clear cookies6. Close all

Enabling Cookies in Mobile Safari (iPhone, iPad)1. Go to the Home screen by pressing the Home button or by unlocking your phone/iPad2. Select the Settings icon.3. Select Safari from the settings menu.4. Select accept cookies from the safari menu.5. Select from visited from the accept cookies menu.6. Press the home button to return the the iPhone home screen.7. Select the Safari icon to return to Safari.8. Before the cookie settings change will take effect, Safari must restart. To restart Safari press and hold the Home button (for around five seconds) until the iPhone/iPad display goes blank and the home screen appears.9. Select the Safari icon to return to Safari.

Excerpt from:
Popular injectables: Erection specialists set up shop - Townsville Bulletin

UW sports medicine helps one athlete make her comeback – University of Washington Magazine

For six weeks following surgery, she was on crutches and in a brace. Stueckle, the teams athletic trainer, developed a program of non-weight-bearing exercises to keep Powells upper body in shape and build a foundation for the rehabilitation of her kneeincluding stretching her leg muscles and increasing her knees range of motion, and setting a volleyball while sitting on a box. And then, when Powell was able to start putting weight on her leg, Stueckle introduced new, challenging exercises, like standing on an unstable surface with her left leg and working on her sets.

I had lost a ton of muscle in my quad, calf and hamstring. So the silliest little exercises like leg raises would absolutely gas me. It was a lot of lonely work, Powell says. But she wasnt truly alone. Every day before practice, and often multiple times a day, she would meet with Stueckle for her ever-evolving rehabilitation. She also had periodic check-ins with OKane and Hagen, who confirmed her knee was improving as expected.

When a resurgence of COVID-19 pushed the 2020 season to January 2021 and the NCAA granted athletes an extra year of eligibility, Powell had renewed hope and a clear recovery target. Still, she endured her share of low moments, including recurring pain in her knee due to a suture. Hagen had OKane evaluate the area using ultrasound and inject it with a steroid to decrease inflammation, which solved the problem. Later, Powell went through a two-week slump in which she didnt feel like she was making any progress, but Stueckle continued to reassure her that she was on the right path. A big aspect of rehab is educating and collaborating with my athletes, Stueckle says. A lot of it is, first and foremost, having that relationship with athletes, building that trust.

Jenn has been there with me since day one, says Powell, who, like the rest of the team, has seen Stueckle in the training room for preventive maintenance and minor injuries since she was a freshman. She did such a great job handling both the physical and emotional side of my recovery.

When Powell returned home to Arkansas for winter break in 2020, Stueckle provided her and her personal trainer with a rehab program. And in January, when Powell was back in Seattle, Hagen had her run through a litany of tests and a thorough knee examand told her she was physically ready to play. It was just up to Powell to decide if she was mentally ready.

For athletes, the mental hurdle in recovering from an injury is often the hardest, says Hagen. UW Medicine doctors sometimes refer athletes to team psychologists, who help them process everything from the academic and personal struggles to intense pressure and career-ending injuries. That holistic care wasnt always the case, says OKane, speaking of sports medicine practices in general, not specifically at the UW. It used to be a race to see how fast surgeons could get athletes back out there after injuries like ACL tears.

In the early 2000s, UW Athletics formalized a partnership with UW Medicine to provide team physician services. To avoid any potential conflict of interest, says OKane, the doctors have the final say in whether an athlete is ready to play: Its written into our contract. Today, this type of autonomous health care is a guiding NCAA principle, and while some institutions have found it challenging to achieve, its fundamental to the relationship between UW Athletics and UW Medicine.

In her final season, Powell reached 162 career aces, setting a school record and prompting the Seattle Times to call her the Queen of Aces.

Coaches and players are often the gas, and were the brakes, OKane says. Theyre the ones who are pushing a little bit, and were the ones who are holding back a little bit. Its a really tight connection at the UW, but the final say about play, not play, is with the physician.

Fortunately for Powell, her recovery had gone smoothly, and Hagen and OKanes reassurance was all she needed mentally. We dont see a reason to hold you back, Hagen told Powell in that final appointment. Powell didnt either.

Over the next few months, she helped lead the Huskies to a conference championship and to the Final Four for the first time since 2013. Ella May went from sitting on a box in the fall to first-team All-American in the spring, Stueckle says.

But Powell wasnt done. Just months later, she and the Huskies were back again, winning their second-straight Pac-12 title and advancing to the Sweet Sixteen. Powell was named Pac-12 setter of the year both seasons, cementing her spot as a Husky great.

While OKane and his team see more than 700 student-athletes for their primary-care needs, and Hagens team treats them for musculoskeletal injuries, thats just part of their caseloads. The rest are patients from the community. (Other specialists at the center, including physiatrists and physical therapists, also treat UW athletes and community patients alike. And a number of UW sports medicine clinics in the greater Seattle area extends that community reach even further.)

UW Medicine as a research powerhouse applies just as much to someone like Powell as it does to anyone else. Theres nothing about Ella May that resembles most of the other people in the world, says OKane. But the way you approach her is carried over to the way that you approach other folks.

Those other folks could include a young soccer player or a senior who hikes. And regardless of who the patient is, the same tools are available, from technologically advanced surgery to nonsurgical treatments like ultrasound-guided procedures, nerve pain reduction techniques and platelet-rich plasma injections to help inflamed tendons and joints. Whether the treatments and technologies are new or have been used for decades, UW Medicine relies on its wealth of research and expertise to ensure they remain safe and effective.

In 2015, Dr. John Drezner, a UW Athletics team physician and director of the UW Medicine Center for Sports Cardiology, was a leader in a Seattle meeting of international experts who used new research to standardize the interpretation of electrocardiograms in athletes. Known as the Seattle criteria, it is today used by doctors around the world to improve the screening of amateur and professional athletes and identify potentially life-threatening cardiovascular abnormalities before they strike on the court or on the field. There is a lot of community trickle-down from UW Medicines research, says OKane.

Dr. John OKane examines a student-athletes knee.

* * *

Continue reading here:
UW sports medicine helps one athlete make her comeback - University of Washington Magazine

This stem cell startup is designing a therapy to restore and boost … – The Boston Globe

That bold vision has now attracted personal investments from several local life science leaders, including George Church, the Harvard University geneticist who has founded and advised dozens of biotech companies. Church told Wang a former postdoctoral researcher in his lab that his thymus cell therapy was one of the most exciting ideas hed come across, since it has the potential to impact almost every person on the planet.

Wangs startup recently raised $7 million in seed financing, bringing the total to $13 million, he said. Thymmunes investors include the biotech venture capital firm Pillar VC and NYBC Ventures, the investment arm of the New York Blood Center. Biotech entrepreneurs and investors Mark Bamforth, James Fordyce, John Maraganore, Judy Pagliuca, Philip Reilly, and Mark de Souza pitched in, too.

Maraganore, the former founding chief executive of Alnylam Pharmaceuticals, a genetic medicines company in Cambridge worth more than $23 billion, said he was fascinated by both the near-term and long-term goals of Wangs vision for what thymus cell therapies might do for rare and common conditions alike.

If successful, it could be pretty transformative, Maraganore said. At the end of the day, we all age and die due to our immune system falling apart, and if theres a way to reconstitute it, that would be pretty cool.

Physicians once thought that the thymus, a small gland situated behind the breastbone, between the lungs, and above the heart, was a dispensable organ. But it plays a vital role in developing immunity.

Its job is to basically be the schoolhouse for T cells, those critical cells in your immune system that help you fight everything from pathogens to cancer, Wang said. From a young age, the thymus also teaches T cells about the inventory of molecules normally found in people so that they dont attack their own body and cause an autoimmune disease.

The thymus is perhaps the most important organ youve never heard of. Many folks dont even know it exists, said Thomas de Vlaam, an investor at Pillar VC. If it works well, its unnoticeable, but once it starts failing for whatever reason, the effects are detrimental.

Thymmunes ambitions span the gamut of thymus biology, from replacing missing thymuses to bolstering shrinking ones, and its technology is largely based on work from a group of scientists at the University of California, San Francisco. In 2010, Audrey Parent, a postdoctoral researcher working with UCSF professors Matthias Hebrok and Mark Anderson, was trying to figure out how to make thymus cells in the lab for the first time.

Parent, now an assistant professor at UCSF, said the project involved a lot of trial and error to find a molecular recipe that could turn a stem cell into a thymus cell. There was no recipe to do that at the time, Parent said. We looked at how the embryo does it, we tried to replicate what nature has been doing really successfully, and then transferred that into a recipe that you can do in a dish.

Their results, published in 2013, used human embryonic stem cells to make thymus cells that were transplanted into mice that lacked a thymus. Crucially, the implants allowed the mice to make their own T cells. Thymmune has licensed patents from UCSF, and like many new startups in the stem cell field, it is forgoing difficult-to-source and ethically fraught embryonic stem cells in favor of induced pluripotent stem cells, or iPSCs, which can be made from adult skin cells.

Sometime in the next few years, Wang plans to start a clinical trial in children who are born without a thymus. Its a ticking time bomb where these kids usually dont survive past one or two years, Wang said.

The Food and Drug Administration approved the first therapy for the condition, called congenital athymia, in 2021. Slices of thymus obtained from organ donations, cultured in a lab and surgically implanted into an infants thigh, improved the chances of surviving the otherwise fatal condition to 76 percent after two years. The results there have been fantastic, Wang said. He hopes to replicate them and make an off-the-shelf product that doesnt require organ donations.

If that approach is successful, Wang wants to use his companys thymus cells as a therapy that helps people getting bone marrow or organ transplants recover more quickly and trains their immune systems to not reject the transplant. He also has plans to develop engineered thymus cells that can quell autoimmune diseases by retraining haywire immune cells to stand down and stop attacking the body.

Wangs ultimate vision, and the one thats especially invigorated investors like Church and Maraganore, is to inject thymus cells into aging people to bolster their immune response. To be clear, theres a lot more we need to do before getting to that point, Wang said. But that is the level of aspiration were aiming for. ... We want to provide everyone the opportunity for healthier aging.

Ryan Cross can be reached at ryan.cross@globe.com. Follow him on Twitter @RLCscienceboss.

More:
This stem cell startup is designing a therapy to restore and boost ... - The Boston Globe

How to generate new neurons in the brain – Science Daily

Some areas of the adult brain contain quiescent, or dormant, neural stem cells that can potentially be reactivated to form new neurons. However, the transition from quiescence to proliferation is still poorly understood. A team led by scientists from the Universities of Geneva (UNIGE) and Lausanne (UNIL) has discovered the importance of cell metabolism in this process and identified how to wake up these neural stem cells and reactivate them. Biologists succeeded in increasing the number of new neurons in the brain of adult and even elderly mice. These results, promising for the treatment of neurodegenerative diseases, are to be discovered in the journal Science Advances.

Stem cells have the unique ability to continuously produce copies of themselves and give rise to differentiated cells with more specialized functions. Neural stem cells (NSCs) are responsible for building the brain during embryonic development, generating all the cells of the central nervous system, including neurons.

Neurogenesis capacity decreases with age

Surprisingly, NSCs persist in certain brain regions even after the brain is fully formed and can make new neurons throughout life. This biological phenomenon, called adult neurogenesis, is important for specific functions such as learning and memory processes. However, in the adult brain, these stem cells become more silent or ''dormant'' and reduce their capacity for renewal and differentiation. As a result, neurogenesis decreases significantly with age.The laboratories of Jean-Claude Martinou, Emeritus Professor in the Department of Molecular and Cellular Biology at the UNIGE Faculty of Science, and Marlen Knobloch, Associate Professor in the Department of Biomedical Sciences at the UNIL Faculty of Biology and Medicine, have uncovered a metabolic mechanism by which adult NSCs can emerge from their dormant state and become active.

''We found that mitochondria, the energy-producing organelles within cells, are involved in regulating the level of activation of adult NSCs,'' explains Francesco Petrelli, research fellow at UNIL and co-first author of the study with Valentina Scandella. The mitochondrial pyruvate transporter (MPC), a protein complex discovered eleven years ago in Professor Martinou's group, plays a particular role in this regulation. Its activity influences the metabolic options a cell can use. By knowing the metabolic pathways that distinguish active cells from dormant cells, scientists can wake up dormant cells by modifying their mitochondrial metabolism.

New perspectives

Biologists have blocked MPC activity by using chemical inhibitors or by generating mutant mice for the Mpc1gene. Using these pharmacological and genetic approaches, the scientists were able to activate dormant NSCs and thus generate new neurons in the brains of adult and even aged mice. ''With this work, we show that redirection of metabolic pathways can directly influence the activity state of adult NSCs and consequently the number of new neurons generated,'' summarizes Professor Knobloch, co-lead author of the study. ''These results shed new light on the role of cell metabolism in the regulation of neurogenesis. In the long term, these results could lead to potential treatments for conditions such as depression or neurodegenerative diseases'', concludes Jean-Claude Martinou, co-lead author of the study.

See original here:
How to generate new neurons in the brain - Science Daily

Enhanced mitochondrial biogenesis promotes neuroprotection in … – Nature.com

Reagents and resources

All the reagents including qPCR primers, antibodies, and software used are listed in Supplementary Table1

Human embryonic stem cell (H7-hESCs; WiCell, Madison, WI, https://www.wicell.org) reporter line with CRISPR-engineered multicistronic BRN3B-P2A-tdTomato-P2A-Thy1.2 construct into the endogenous RGC specific BRN3B locus was used for isogenic control20. CRISPR mutated H7-hESC reporter with OPTNE50K-homozygous mutation (H7-E50K) was done as explained previously25. Patient-derived induced pluripotent stem cells (iPSCs) with E50K mutation24 (iPSC-E50K), E50K mutation corrected to WT by CRISPR in the patient-derived iPSC (iPSC-E50Kcorr)25 with BRN3B::tdTomato reporter were obtained from the Jason Meyer lab. All the above cell lines were grown in mTeSR1 media (mT) at 37C in 5% CO2 incubator on matrigel (MG) coated plates. These cells were maintained by clump passaging using Gentle Cell Dissociation Reagent (GD) after 7080% confluency. Media was aspirated and GD was added to cells followed by incubation at 37C in 5% CO2 incubator for 5min. Next, mT was used to break up the colonies into small clumps by repeated pipetting and then seeded onto new MG plates.

For differentiation, stem cells were dissociated to single cells using accutase for 10min and then quenched with twice the volume of mT with 5M blebbistatin (blebb). The cells were centrifuged at 150xg for 6minutes and resuspended in mT with 5M blebb, then 100,000 cells were seeded per well of a 24-well MG coated plate. The next day, media was replaced with mT without blebb. After 24h, media was replaced with differentiation media (iNS) and further small molecule-based differentiation was carried out with iNS media as elucidated previously23. Differentiation of hRGCswas monitored by tdTomato expression and cells were purified during days 4555 using THY1.2 microbeads and magnetic activated cell sorting system (MACS, Miltenyi Biotec) as explained before20,23. Next, hRGCs were resuspended in iNS media, counted using a hemocytometer and seeded on MG-coated plates, coverslips, or MatTek dishes for experiments.

Purified hRGCs were seeded at 30,000 cells per well of a 96-well MG-coated plate and maintained for 3 days. For measuring mitochondrial mass, hRGCs were labeled with mitochondria-specific MTDR dye. To measure mitochondrial degradation, hRGCs were labeled with 10nM MTDR dye for 1h, washed, and then treated with 10M CCCP for a time course. To measure mitochondrial biogenesis, hRGCs were labeled with MTDR first, treated with 10M CCCP or equal amount of DMSO for 3h, then washed and incubated with fresh media with MTDR for the time course. After treatments, hRGCs were dissociated to single-cell suspension using accutase and analyzed using Attune NxT flow cytometer (Thermo Fisher).

Purified hRGCs were seeded at 500,000 cells per well of 24-well MG-coated plates and maintained for 3 days. Cells were then treated with indicated molecules and time points. DMSO was used as the control as the small molecules were dissolved in DMSO. The cells were lysed and collected in 100l of M-PER extraction buffer with 5mM EDTA and protease inhibitors. Protein quantification was done using a BSA standard following the DC Protein Assay Kit II (Bio-Rad) and measured on microplate reader. Loading samples were prepared with heat denaturation at 95C for 5minutes with laemmli sample buffer (1X). 1020g protein per sample were run on Bio-Rad Mini-PROTEAN TGX precast gels, in running buffer (Tris-Glycine-SDS buffer; Bio-Rad) at 100V until the dye front reached to the bottom. The transfer sandwich was made using PVDF membrane (activated in methanol) in Tris-Glycine transfer buffer (Bio-Rad) with 20% methanol and transferred for 2h at 30V, 4C. For the visualization of proteins, the membranes were blocked in 5% skim milk in TBST (TBS buffer with 20% Tween20) for 2h at room temperature and incubated overnight at 4C in 1:1000 dilution of primary antibodies for PGC1 (Abcam), Phospho-PGC1Ser571 (R&D Systems), PGC1 (Abcam), AMPK (Cell Signaling Technologies, CST), Phospho-AMPKThr172 (CST), LC3B (Sigma), GAPDH (CST), or ACTIN (CST). Membranes were then washed three times for 5minutes each in TBST, followed by 2h of incubation in 5% milk in TBST containing anti-rabbit HRP linked secondary antibody (CST) at 1:10,000 dilution. The membranes were again washed three times with TBST and then placed in Clarity Max Western ECL (Bio-Rad) substrate for 5min. The membranes were imaged in a Bio-Rad ChemiDoc Gel Imager, and the raw integrated density for each band was measured and normalized with respect to the corresponding GAPDH or actin loading control using Image J. Treatment conditions were further normalized to the corresponding DMSO control for each experiment. Complete blots of the representative western blot images, with protein molecular weight marker (Thermo Scientific), are provided in Supplementary Fig.6. Protein bands corresponding to the appropriate size were quantified following product datasheets and published literature.

Purified hRGCs were seeded on MG-coated coverslips (1.5 thickness) at a density of 30,00040,000 cells per coverslip and grown for 3 days. Next, hRGCs were treated with indicated molecules and time points. After treatment the media was aspirated, the cells were washed with 1 PBS, and then fixed with 4% Paraformaldehyde for 15min at 37C. Fixed cells were permeabilized with 0.5% Triton-X100 in PBS for 5min and then washed with washing buffer (1% donkey serum, 0.05% Triton-X100 in PBS) three times for 5min each. Cells were blocked with blocking buffer (5% donkey serum, 0.2% Triton-X100 in PBS) for 1h. After blocking, antibodies against TOM20 (Mouse, Santa Cruz), Tubulin 3 (mouse, Biolegend), RBPMS (rabbit, GeneTex) and Optineurin (Rabbit, Cayman Chemicals) were added (1:200 in blocking buffer) and the coverslips were incubated overnight at 4C. Next, coverslips were washed with washing buffer three times for 5min each and incubated for 2h at room temperature in the dark with fluorophore conjugated anti mouse or rabbit secondary antibodies (1:500). The coverslips were washed with washing buffer three times for 5min each, with 1.43M DAPI added to the second wash. The coverslips were then mounted onto slides with DAKO mounting medium. Visualization of above proteins and nucleus was done by confocal immunofluorescence microscopy using Zeiss LSM700 with 63x/1.4 oil objective. Analysis was carried out using ImageJ with maximum projections of DAPI channel (number of nuclei) and sum projections of TOM20 and OPTN channels for the corresponding confocal z-stacks. For OPTN aggregate size, we analyzed particles from 0.02 a.u. to infinity to account for the small and big aggregates.

Purified hRGCs were seeded at 300,000 cells per well of 24-well MG-coated plates and maintained for 3 days. Cells were then treated with indicated molecules and durations. Media was aspirated and cells were incubated in 200l accutase for 10min and then quenched with 400l iNS media. Cells were centrifuged at 150xg for 6min, media aspirated, and the cell pellets were stored at 20C. RNA was extracted from cell pellets following the kit protocol (Qiagen 74104). The RNA concentration was measured using Nanodrop 2000c (Thermo) and 6l of RNA was used to prepare cDNA following the kit protocol (Abm #G492). Primers were designed as detailed in TableS1 and qPCR were performed using BlasTaq qPCR MasterMix with 100ng total cDNA in a 20l reaction mixture using QuantStudio6 Flex RT PCR system (Applied Biosystems). GAPDH or actin was used as a housekeeping gene in every plate to calculate the Ct values. The Ct was calculated with respect to (w.r.t) the average Ct of the control sample. All conditions were measured by averaging three technical repeats for each biological repeat with total three biological replicates.

Purified hRGCs were seeded at 50,00075,000 cells per well of 96-well MG-coated plates and maintained for 3 days. Cells were then treated with indicated molecules for the designated durations. Media was aspirated and cells were incubated in 30l accutase for 10min and then quenched with 100l iNS media. Cells were centrifuged at 150xg for 6min, media aspirated, and the cell pellets were stored at 20C. DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen) and eluted with 30l elution buffer. DNA concentration was measured using Nanodrop 2000c (Thermo). 10ng of DNA was used to measure both mitochondrial ND1 gene and internal control, human nuclear RNase P gene copy numbers, as done previously23.

hRGCs were seeded on MG-coated glass bottom (1.5 thickness)MatTek dishes at 40,000 cells per dish and maintained for 3 days. For the experiments in Fig.2ac, cells were incubated with 250l of JC1 media (1:100 in iNS) for 30min in the incubator, then an additional 1.75ml of dilute JC1 (1:1000 in iNS) was added to the dish. The dish was then transferred to the live cell chamber (5% CO2, 37C, Tokai Hit) and confocal z-stacks were acquired prior to (before) and 10minutes after CCCP (10M) treatment using Zeiss LSM700 with 63x/1.4 oil objective. For experiments in Fig.2df, cells were treated with 10M CCCP or equivalent DMSO for 30min. Next, cells were washed with iNS media, and then incubated with 250l of JC1 media (1:100 in iNS) for 30min in the incubator. The JC1 media was then removed, cells were washed again, and 2ml of new iNS was added to the dish. The dish was then transferred to the live cell chamber and imaged. Analysis was carried out using ImageJ with sum projections of red and green channels. The red fluorescence from the tdTomato expressed by the hRGCs was much less intense than the JC1 red staining of mitochondria, thus the red fluorescence measured from the cytoplasm was considered background and subtracted out of the measurements. For the green fluorescence, background was measured from outside the cell. Red-to-Green ratios were calculated by dividing the red intensity by green intensity. These values were then normalized to the control condition, hRGCWT-before (Fig.2c) or average DMSO ratio for each cell line (Fig.2f).

The 96-well seahorse plate was coated with MG and hRGCs were seeded at 250,000 cells per well and maintained for 2 days. 24h before measurements, media was exchanged with 100l iNS with 1g/ml BX795 or equivalent vehicle control DMSO. A day prior to the assay, the sensor cartridge was fully submerged with 200l of sterile water to hydrate it overnight in a non-CO2, 37C incubator. The next day, sterile water was replaced with pre-warmed XF calibrant buffer(Agilent) and the sensors were again submerged and incubated in the non-CO2, 37C incubator for 4560min. Seahorse media was made by adding stock solutions to XF DMEM to have final concentrations of 21.25mM glucose, 0.36mM sodium pyruvate, and 1.25 mM L-glutamine (Agilent), with pH adjusted to 7.387.42. Depending upon the assay, 20M Oligomycin (Oligo), 20M FCCP, 2.5g/ml Rotenone plus 5M Antimycin A (Rot/AA), and/or 175mM 2-deoxy-d-glucose (2-DG) solutions were then prepared in Seahorse media. In the Seahorse plate with hRGCs, iNS media was carefully exchanged to Seahorse media by removing, 60l iNS from all wells and adding 140l of Seahorse media. Next, 140l of the mixed media was removed by pipette and then an additional 140l seahorse media was added to each well to have a final volume of 180l. 180l of Seahorse media was then added to any empty wells. The plate was placed in Incucyte S3 (Sartorius) and one image of each well was taken for cell area normalization using brightfield and red fluorescence (tdTomato) channels. The hRGC seahorse plate was then placed into a non-CO2, 37C incubator for at least 45min. The reagents were added into their respective ports in the cartridge with the final concentrations in the wells as follows. For ATP rate assay, 2.0M Oligo and 0.25g/ml Rotenone plus 0.5M Antimycin A; for the glycolytic rate assay, 0.25g/ml Rotenone plus 0.5M Antimycin A and 17.5mM 2-DG; and for the Mito stress test, 2.0M Oligo, 2.0M FCCP (optimal concentration from FCCP titration), and 0.25g/ml Rotenone plus 0.5M Antimycin A. After loading all ports, the cartridge was placed into a non-CO2, 37C incubator while the experiment was setup in WAVE software (Agilent). The cartridge was then placed into the XFe96 analyzer (Agilent) and run for calibration. After the machine calibrations were successful, the hRGC seahorse plate was placed into the machine and the assay was run (ATP rate assay, glycolytic rate assay, or Mito stress test). Cell area from Incucyte images were measured using Image J and extrapolated for the total cell area in each well for normalization. The assay results were then exported into the appropriate excel macro using Seahorse Wave Desktop software (Agilent) for analysis.

hRGCs were seeded at 25,000 cells per well of a 96-well clear bottom black-walled plate and maintained for 3 days. The cells were then treated with indicated molecules for the designated time points. The caspase activity was measured using the ApoTox-Glo Triplex assay kit (Promega). 100l of Caspase-Glo 3/7 reagent was added to all wells and incubated for 30minutes at room temperature before measuring luminescence (Caspase). Measurements were normalized to DMSO control.

hRGCs were seeded at 250,000 cells per well on MG-coated 6-well plates and maintained for 3 days. The cells were then treated with 1 g/ml BX795 or equivalent DMSO for 24h. Media was aspirated, 500 l of fixative solution (3% Glutaraldehyde, 0.1M Cacodylate) was added, and the cells were incubated for 15min. Next, hRGCs were scraped and pelleted by centrifugation at 10,000xg for 20minutes. The pellets were fixed overnight at 4C, then rinsed the next day in 0.1M cacodylate buffer, followed by post fixation with 1% osmium tetroxide, 0.1M cacodylate buffer for 1h. After rinsing again with 0.1M cacodylate buffer, the cell pellets were dehydrated through a series of graded ethyl alcohols from 70 to 100%, and 2 changes of 100% acetone. The cell pellets were then infiltrated with a 50:50 mixture of acetone and embedding resin (Embed 812, Electron Microscopy Sciences, Hatfield, PA) for 72h. Specimen vial lids were then removed, and acetone allowed to evaporate off for 3h. Then the pellets were embedded in a fresh change of 100% embedding resin. Following polymerization overnight at 60C the blocks were then ready for sectioning. All procedures were done in centrifuge tubes including the final embedding. Sections with cut at 85nm, placed on copper grids, stained with saturated uranyl acetate, viewed, and imaged on a Tecnai Spirit (ThermoFisher, Hillsboro, OR) equipped with an AMT CCD camera (Advanced Microscopy Techniques, Danvers, MA). 49000X images were analyzed using Image J to measure mitochondrial parameters as explained in Fig.5.

Samples were treated with CCCP or BX795 at different time points as independent biological samples. Statistical tests between two independent datasets were done by Students t-test, each data point within a dataset is from an independent culture wellor cell (Figs.1cf, h, 1m; j, l, 2c, f, 3ad, fi, 4bd, fi, 5b, 6b, e, hl, Supplementary Figs.3b, 4bc). We used t-test rather than ANOVA because we did not want to assume that each group has the same variance. For non-normal data distribution, we performed MannWhitney U test to compare between two independent data sets (Fig.5ce, Supplementary Fig.5b). Graphs were made using GraphPad Prism 9.0 softwareand figures were made using Adobe Illustrator.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

Read the original:
Enhanced mitochondrial biogenesis promotes neuroprotection in ... - Nature.com

Will future computers run on human brain cells? Breaking ground on … – Science Daily

A "biocomputer" powered by human brain cells could be developed within our lifetime, according to Johns Hopkins University researchers who expect such technology to exponentially expand the capabilities of modern computing and create novel fields of study.

The team outlines their plan for "organoid intelligence" today in the journal Frontiers in Science.

"Computing and artificial intelligence have been driving the technology revolution but they are reaching a ceiling," said Thomas Hartung, a professor of environmental health sciences at the Johns Hopkins Bloomberg School of Public Health and Whiting School of Engineering who is spearheading the work. "Biocomputing is an enormous effort of compacting computational power and increasing its efficiency to push past our current technological limits."

For nearly two decades scientists have used tiny organoids, lab-grown tissue resembling fully grown organs, to experiment on kidneys, lungs, and other organs without resorting to human or animal testing. More recently Hartung and colleagues at Johns Hopkins have been working with brain organoids, orbs the size of a pen dot with neurons and other features that promise to sustain basic functions like learning and remembering.

"This opens up research on how the human brain works," Hartung said. "Because you can start manipulating the system, doing things you cannot ethically do with human brains."

Hartung began to grow and assemble brain cells into functional organoids in 2012 using cells from human skin samples reprogrammed into an embryonic stem cell-like state. Each organoid contains about 50,000 cells, about the size of a fruit fly's nervous system. He now envisions building a futuristic computer with such brain organoids.

Computers that run on this "biological hardware" could in the next decade begin to alleviate energy-consumption demands of supercomputing that are becoming increasingly unsustainable, Hartung said. Even though computers process calculations involving numbers and data faster than humans, brains are much smarter in making complex logical decisions, like telling a dog from a cat.

"The brain is still unmatched by modern computers," Hartung said. "Frontier, the latest supercomputer in Kentucky, is a $600 million, 6,800-square-feet installation. Only in June of last year, it exceeded for the first time the computational capacity of a single human brain -- but using a million times more energy."

It might take decades before organoid intelligence can power a system as smart as a mouse, Hartung said. But by scaling up production of brain organoids and training them with artificial intelligence, he foresees a future where biocomputers support superior computing speed, processing power, data efficiency, and storage capabilities.

"It will take decades before we achieve the goal of something comparable to any type of computer," Hartung said. "But if we don't start creating funding programs for this, it will be much more difficult."

Organoid intelligence could also revolutionize drug testing research for neurodevelopmental disorders and neurodegeneration, said Lena Smirnova, a Johns Hopkins assistant professor of environmental health and engineering who co-leads the investigations.

"We want to compare brain organoids from typically developed donors versus brain organoids from donors with autism," Smirnova said. "The tools we are developing towards biological computing are the same tools that will allow us to understand changes in neuronal networks specific for autism, without having to use animals or to access patients, so we can understand the underlying mechanisms of why patients have these cognition issues and impairments."

To assess the ethical implications of working with organoid intelligence, a diverse consortium of scientists, bioethicists, and members of the public have been embedded within the team.

Johns Hopkins authors included: Brian S. Caffo, David H. Gracias, Qi Huang, Itzy E. Morales Pantoja, Bohao Tang, Donald J. Zack, Cynthia A. Berlinicke, J. Lomax Boyd, Timothy DHarris, Erik C. Johnson, Jeffrey Kahn, Barton L. Paulhamus, Jesse Plotkin, Alexander S. Szalay, Joshua T. Vogelstein, and Paul F. Worley.

Other authors included: Brett J. Kagan, of Cortical Labs; Alysson R. Muotri, of the University of California San Diego; and Jens C. Schwamborn of University of Luxembourg.

See the rest here:
Will future computers run on human brain cells? Breaking ground on ... - Science Daily