Fight Aging! Newsletter, November 8th 2021

Fight Aging! Newsletter
November 8th 2021

Fight Aging! publishes news and commentary relevant to the goal of ending all age-related disease, to be achieved by bringing the mechanisms of aging under the control of modern medicine. This weekly newsletter is sent to thousands of interested subscribers. To subscribe or unsubscribe from the newsletter, please visit: https://www.fightaging.org/newsletter/

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Contents

How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Khavinson Peptides for Thymic Regrowth Atherosclerosis, the As Yet Undefeated Monster An Example of the Growth in Investment Funds Dedicated to the Longevity Industry Cardiovascular Disease is Perhaps Less Well Understood than is Widely Perceived to be the Case VitaDAO, a Novel Approach to Crowdfunding Life Science Research Exosome Treated Macrophages Improve Regeneration Following Spinal Cord Injury in Mice The NLRP3 Inflammasome in Osteoporosis Exercise as an Approach to Slow Alzheimer's Disease Glial Cells and the Propagation of Tau through the Brain in Tauopathies A Possible Feedback Loop Between Abnormal Brain Activity and Neuroinflammation in Alzheimer's Disease Glutathione Delivery via Iontophoresis Increases Gluthathione Levels in Blood Samples Finding Cautions in the Ease with which it is Possible to Create Epigenetic Clocks The Tumor Suppression Theory of Aging Telomerase Reverse Transcriptase Improves Mitochondrial Function Summarizing What is Known of the INDY Longevity Gene in Flies

How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Khavinson Peptides for Thymic Regrowth
https://www.fightaging.org/archives/2021/11/how-to-plan-and-carry-out-a-simple-self-experiment-a-single-person-trial-of-khavinson-peptides-for-thymic-regrowth/

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess whether a nine month course of injected peptides will significantly and beneficially affect thymus size and cellularity. The thymus atrophies steadily with age. This organ is where maturation of T cells takes place, a complex process of selection to enable T cells to recognize foreign molecule without attacking the body's own component parts. Regrowth of active thymus tissue should improve immune function for the long term by increasing the supply of new T cells, slowing or reversing some of the age-related decline of the immune system.

The peptides in question are three of the Khavinson peptides, the subject of a long-running line of research and development originating in the Russian biomedical community. The original Khavinson peptides were sourced from animal organs, and then over the years synthetic forms were manufactured instead. Some of these natural and synthetic peptides have undergone human trials and have been approved for use as therapies in Russia. The peptides epitalon, thymogen, and vilon have been used to generate animal data to suggest that they improve immune function, at least partially via some degree of regrowth of the aging thymus. But this latter point of thymic regrowth has not been assessed in humans.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it, though the existing human data for the Khavinson peptides suggests a minimal side-effect profile. If you come away thinking that you should just jump in, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.

Contents

Why Self-Experiment with Methods of Thymic Regrowth? Caveats in More Detail Summarizing the Use of Thymus-Relevant Khavinson Peptides Establishing Dosage An Introduction to Injections Considering Autoinjectors Obtaining a Needle-Free Injection System Obtaining Vials of the Correct Size Preparing Khavinson Peptides for Injection Obtaining Khavinson Peptides Storing Khavinson Peptides Validating the Purchased Peptides Establishing Tests and Measures Guesstimated Costs Practice Before Working with Peptides Schedule for the Self-Experiment Where to Publish?

Why Self-Experiment with Methods of Thymic Regrowth?

Thymic involution with age, the process of atrophy that replaces active thymic tissue with fat, is an important contributing cause of immune system aging. The thymus plays a vital role: thymocytes created in the bone marrow migrate to the thymus, where they mature through a complex process of selection in order to become T cells of the adaptive immune system. As active thymic tissue atrophies, the supply of new T cells diminishes. This lack of continued reinforcements leads to an aged adaptive immune system that is ever more populated by exhausted, senescent, and malfunctioning T cells.

Notably, the decline of the immune system is slow. The thymus loses 1% of its active tissue with each passing year of adult life, and is near all fat by the time most individuals are in their 50s. The adaptive immune system takes another decade after that to become notably problematic. Given a way to wholly replenish the T cell complement of the adaptive immune system, one could likely gain an additional decade of function. Equally, regrowing 10% of the original youthful thymic tissue would also likely gain about a decade of additional immune function. Given the great importance of the immune system to health, a restored thymus seems a goal worthy of pursuit.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this self-experiment involves injecting peptides that, while having a fair amount of use outside the US and in self-experimenter communities, come with comparatively little easily accessible primary sources of published human data. Much of that data is actually quite challenging to access, being both dated and from the Russian medical community, so the only English language references are in the form of reviews that describe only the outline of an earlier study. So while the materials that can be reviewed suggest a good safety profile, it is more of a leap into the unknown than is experimenting with compounds that have gone through clinical trials in the English-language world and where the primary sources are easily accessible.

Secondly, obtaining peptides in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is permitted to use peptides that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Summarizing the Use of Thymus-Relevant Khavinson Peptides

Most of the primary research materials relating to Khavinson peptides are largely in Russian, and largely not available online. The book Peptides in the Epigenetic Control of Aging provides a decent summary of the peptides and what has been done with them, but lacks many of the specifics that would be provided in the original clinical trial materials and study papers. There are 16 Khavinson peptides, derived from different organ tissues, and claimed to have numerous beneficial effects in aged individuals, but only three are of interest for animal data suggesting an effect on the thymus, or an increase in life span, or both. These are epitalon (derived from pineal gland tissue peptide extracts called epithalamin), thymogen (derived from thymus tissue peptide extracts called thylamin), and vilon (also derived from thylamin). While the details are not well understood, evidence tentatively suggests cross-talk between the pineal gland and thymus, and an involvement for pineal signaling in thymic involution.

Epitalon and vilon been claimed to extend life in rodents, while thymogen has been claimed to be geroprotective in rodents. Researchers have reported lower cancer incidence in treated mice. Further evidence suggests benefits to immune function, but the claimed benefits to health are very broad, including reduced incidence of many different diseases. Similar reports exist for the animal organ extracts epithalamin and thylamin from which the more modern Khavinson peptides are derived. Near all of this is from Russian scientific literature, or from old literature that is not fully available online, or from second-hand reports of the same, so should be taken as less robust than would otherwise be the case. A great deal is claimed, and these peptides have been used in human trials, but such claims are by no means as rigorously proven as would be the case if the English-language regulators had been involved, or the work carried out more recently.

Establishing Dosage

The Khavinson peptides have been used orally and via subcutaneous or intramuscular injection. The latter path should provide a greater bioavailability of the peptide. In the reported animal studies, doses vary widely. Where human trials are discussed, there are more useful examples. For example, "Peptides of pineal gland and thymus prolong human life", published in 2003 and hard to find online, reports a trial in which patients underwent intermittent 10 day courses of injections of 10mg per day of thymlamin or epithalamin. The intervals between courses were 6-12 months, with a trial duration of up to 3 years, with 5 years of following observation. Vilon, thymogen, and other later synthetic Khavinson peptides have similarly been reported to have undergone a variety of human trials.

After looking through the unfortunately sparse literature, the dose selected for this self-experiment is 10mg epitalon, 10mg thymogen, and 10mg vilon daily. That dose is to be split between two intramuscular or intravenous injections, carried out 12 hours apart (morning and evening). That dose schedule is repeated for 10 consecutive days, once every 3 months, for 9 months. This choice is aimed to (a) not go too far beyond what has been done in terms of a single injected dose at any one time to minimize the risk of side-effects, while (b) providing a sufficiently high dose and intensive schedule to argue, should no increase in thymus cellularity be observed, that this failure was not due to using too small a dose or too sparse a dose schedule.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1 ml, and intramuscular is limited to 2-3 ml depending on location. These are all very fuzzy numbers, but these upper limits don't really matter for the purposes of injecting 10 μg of a protein: it can be dissolved in a very small amount of liquid, 0.5 ml or less.

Different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

It seems sensible to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections. In the case of flagellin, it makes sense to stick with the human trial approach of intramuscular injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately largely not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. When initially looking at the market a few years ago, PharmaJet was the only available needle-free system capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized 0.5 ml syringes are built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing Khavinson Peptides for Injection

If using a needle-free injection system, you will likely be limited to injecting 0.5ml amounts. The objective here is to produce doses of the peptides dissolved in 0.5 ml of phosphate buffered saline in sealed vials, ready to be used with the injection system, with as little contamination as possible from the environment, and stored a freezer until it is ready to use. Depending on the size of the vial, it might be able to contain doses for multiple injections, but it is better to stick to one dose per vial. Peptides are sensitive to free-thaw cycles, so you want as few of those as possible.

When ordering epitalon, thymogen, and vilon, they will arrive as lyophilized (freeze-dried) crystals or powder. Thymogen must be shipped on ice, as it is not very stable at room temperature. (a) Divide the lyophized peptides into 100mg amounts, and place into a freezer. (b) Every month pull out 100mg of each peptide, and dissolve all three 100mg amounts into the same 10ml of phosphate buffered saline, with the addition of five drops of DMSO to aid in solubility. Thymogen is a very light, fine powder, and 10ml is right on the edge of what is required to dissolve 100mg. You may see a few flecks remaining. (c) Split the 10ml solution into 20 vials of 0.5ml each. This is a lot of precision pipetting, so practice first! (d) Seal the vials and place them into the freezer, for use in injections that month.

Keeping Things Sterile is Very Important

Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have unfortunate consequences.

Obtaining Khavinson Peptides

A number of peptide vendors worldwide sell epitalon, the most popular of the Khavinson peptides. A much smaller number keep a stock of thymogen and vilon. Most reputable peptide manufacturers could also run up a custom order, but that is much more expensive. An advantage of the companies that advertise epitalon, thymogen, and vilon in their catalog is that they will have established mass spectra and other data sheets for the compound that can be compared with one another, or used as a basis for evaluating the quality of the product. Prices are often outrageous, however. Chinese companies, on the other hand, have a comparatively low price for their offerings, significant lower than that of even the most aggressively competitive companies in the US and Europe. Among European and US synthesis companies out there, Pepscan and Genscript are options, with Genscript recommended by a number of sources.

Without focusing on any of the specific vendors mentioned above, the easiest way to obtain cost-effective synthesis of peptides is to find and connect with smaller-sized suppliers in China. There are a good many reputable peptide synthesis concerns in that part of the world. Sadly, finding such companies has become great deal more challenging, since Alibaba removed all such commerce from their platform in 2021.

As noted at the outset of this post, all of these efforts to obtain, ship, and use any random protein for self-experimentation are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary protein isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Finding Overseas Manufacturers

It used to be the case that Alibaba was the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers of peptides and other compounds. Unfortunately, the company banned all such manufacturers from their marketplace as of mid-2021, for reasons that have not yet become clear. Reputable manufacturers exist in China and other countries, but finding them is now more of an exercise without an initial set of connections to work from. Smaller companies are desired, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a protein, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Chinese manufacturers are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized proteins. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.

Storing Khavinson Peptides

Peptides are commonly shipped in a solid freeze-dried (lyophilised) form. While in this form they are easily stored in a refrigerator for the short-term or in a freezer for the long term. Some are fairly stable at room temperature in this state, and some are not - it varies widely from peptide to peptide. A peptide has a much shorter life span once it has been mixed with liquid for injection, and should be kept frozen, used within a matter of a few months at the most, and not subject to repeated freeze-thaw cycles.

Validating the Purchased Peptides

A peptide may have been ordered, but that doesn't mean that what turns up at the door is either the right one or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a protein is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

At one point, Science Exchange was a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Unfortunately, their service is now expensive and restricted to industry. Thus we must fall back on more laborious approaches to finding small laboratory service companies or university core services to work with. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Rates vary, but 200 per sample is a fair price for LC-MS to check the purity of a compound.

Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by other sources. Finding those sources through PubChem is not hard for more widely used compounds.

Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor or other sources.

Establishing Tests and Measures

There are a few options for testing before and after effects of an intervention that may provoke the regrowth of active thymus tissue, and thereby increase the production of T cells.

CT Scan

The most direct approach is to undergo a CT scan of the chest, with the aid of a cooperative physician, and obtain a copy of the imaging data to work with. There are a number of freely available viewers for scan data, such as Gingko CADx. The thymus is clearly identifiable in such scans, and one can perform manual or pixel-counting analysis to get some idea as to whether cellularity or size has changed. In humans, the imagery provided in the Intervene Immune trial results suggest that changes in cellularity and thus density are the more likely outcome than any growth in size. It will require some reading around the literature in order to understand the CT images one obtains from a provider. There are a number of useful papers containing sample images from individuals at various stages of thymic involution. See "Normal CT characteristics of the thymus in adults" for example.

Monocyte to Leukocyte Ratio

The monocyte to leukocyte ratio should be altered towards an increased number of leukocytes if T cell production by the thymus is upregulated. This ratio can be obtained from the counts provided in normal bloodwork. There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Though note that some US states require the involvement of a physician!

Naive T Cell and Recent Thymic Emigrant T Cell Counts

Given a cooperative physician who is knowledgable regarding immunology, it should be possible to order tests that count (a) naive T cells and (b) recent thymic emigrants, T cells with characteristics that fade within a few weeks of leaving the thymus. Both populations should be increased by a more active thymus. These are less common, more expensive assays, but are more compelling than a monocyte to leukocyte ratio measure.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

Baseline bloodwork for monocyte and leukocyte counts from WellnessFX: 220 / test Naive T cell assay via a medical provider: 1500+ / test Recent thymic emigrant assay via a medical provider: 1500+ / test CT scan: 1000 / scan Business mailbox, such as from UPS: 250 / year Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: 60 Phosphate buffered saline and DMSO: 100 Small pack of 13mm sterile serum vials: 35 PharmaJet Needle-free Injection Kit: 1020 Comfort-in Needle-free Injection Kit: 470 Bulk 13mm serum vial parts and capping tools: 750 100mg of each of epitalon, thymogen, and vilon from Chinese peptide manufacturers: 600 Shipping and LC-MS analysis of samples: 600

Practice Before Working with Peptides

Do you think you can reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with saline solution rather than finding out that your manual dexterity and methods are lacking while handling an expensive protein. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of peptides to take a couple of months. During that process, also obtain the baseline assessments such as CT scan. Once all of the decisions are made and the materials are in hand, pick a start date. The self-experiment will last for 9 months, with 10 days of twice-daily injections every 3 months. On each injection day, space the injections 12 hours apart. After completion of the self-experiment, rerun the selected assays.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results! Publish whatever the outcome.

Atherosclerosis, the As Yet Undefeated Monster
https://www.fightaging.org/archives/2021/11/atherosclerosis-the-as-yet-undefeated-monster/

As you may know, I co-founded Repair Biotechnologies, a company presently focused on developing an approach to rapidly reverse the cholesterol content of atherosclerotic lesions, a goal that is impossible to achieve using the existing panoply of treatments for atherosclerosis. We use gene therapy techniques to provide cells with the ability to safely break down excess cholesterol, enabling the removal of pathological levels of intracellular cholesterol and localized deposits of extracellular cholesterol that characterize conditions such as atherosclerosis (in blood vessel walls) and NASH (in the liver). Atherosclerosis is an important consequence of aging: the structural weakness and narrowing in blood vessels caused by the growth of cholesterol-laden lesions is the cause of death for a quarter of humanity. Closer to half of humanity were cancer somehow removed from the human condition.

The Foresight Institute runs salons these days, and publishes a range of interesting presentations from people in the biotechnology, molecular nanotechnology, and artificial general intelligence fields. Earlier this year, the Foresight Institute staff were kind enough to invite me to present on the work taking place at Repair Biotechnologies, and the understanding of atherosclerosis that informs that work.

I titled the presentation "Atherosclerosis, the As Yet Undefeated Monster" in part as a reaction to a certain type of conversation that recurs when talking to venture capitalists. Many biotech investors, and others too, seem to think that atherosclerosis is a solved problem, and therefore a poor choice of field in which to see a return by supporting the development of new approaches. Many of the best-selling small molecule drugs are statins that have been deployed for decades to treat atherosclerosis by lowering LDL cholesterol in the bloodstream. Physicians reflexively prescribe statins to older individuals. Everyday people obsess about their blood cholesterol levels. New LDL cholesterol lowering drugs that employ modern technologies such as monoclonal antibodies and siRNA are being approved for use on an ongoing basis.

Yet in this environment of decades of ever more attention given to the reduction of LDL cholesterol levels in the bloodstream, atherosclerosis still kills a quarter of humanity. Atherosclerosis is very far removed from being a solved problem! There is an enormous unmet need and ongoing mortality, on a par with the global burden of cancer.

LDL cholesterol reduction as a basis for therapy can lower late life mortality by 20% at most, with many large, robust clinical trials failing to obtain even that degree of benefit. It just isn't the right mechanism if the goal to produce a cure. It doesn't matter how efficiently a therapy reduces LDL cholesterol in the bloodstream, that 20% mortality reduction appears to be a ceiling. PCSK9 inhibitors do no better than statins when it comes to lowered mortality following control of LDL cholesterol, despite being a much more modern and capable technology. I feel that perhaps the research and development community has been encouraged in its near monomaniacal fixation on lowering LDL cholesterol by the discovery of human gene variants (in PCSK9, ANGPTL3, and so forth) that result in lower LDL cholesterol and up to 50% lower cardiovascular mortality. But that result is obtained due to a full lifetime of lowered LDL cholesterol. Therapies built on those discoveries cannot match that outcome.

Atherosclerotic lesions grow over time at a pace that is influenced by LDL cholesterol levels, by the pace at which cholesterol arrives from the bloodstream to a lesion. Reducing that pace via LDL cholesterol lowering therapy cannot reverse established lesions, or even stop them from further growth and eventual rupture, however. Once a lesion is established, the more important mechanism is the dysfunction of the macrophage cells responsible for clearing cholesterol from blood vessel walls. Those cells are overwhelmed, become inflammatory, attract more macrophages, and die, adding their mass to the lesion. The lesion becomes a macrophage graveyard.

To actually reverse atherosclerotic lesions, to actually produce a treatment that could legitimately be called a cure for atherosclerosis, one needs to protect the function of macrophages in the hostile, inflammatory, cholesterol-laden atherosclerotic plaques. If macrophages can be made invulnerable to excess cholesterol, and other harms such as oxidized LDL particles, then given enough time they will do their jobs and repair the blood vessel wall. That is our goal at Repair Biotechnologies, to make a real and meaningful inroad towards that goal.

Atherosclerosis: The As Yet Undefeated Monster

So why are macrophages not able to do their job later in life? We need to understand cholesterol transport first. Cholesterol isn't created or destroyed in near all cells, it is rather ingested and excreted. Cells don't break down or get rid of the cholesterol they don't want locally, they hand it off to other cells and parts of the system when they no longer need it. Cholesterol is created in the liver, gets stuck onto LDL particles and goes into the bloodstream, gets stuck in a blood vessel wall, macrophages eat it and then throw it back into the bloodstream to attach to the HDL particles that flow back into the liver. LDL and HDL particles do pretty much the same work when you're young and old, it's the macrophages that stop doing their job. So why exactly do they stop doing their job? Due to a variety of issues - namely systemic inflammation, systemic oxidative stress, and too much cholesterol, although the last is probably not the worst of those three. What this leads to is a feedback loop. Your plaque is a macrophage graveyard, and the signaling of that draws in even more macrophages trying to fix the problem. That is the underlying cause of atherosclerosis. As you are aware, there's an entire research community and pharmaceutical industry focused only on lowering LDL cholesterol - taking that part of cholesterol transport from the liver to the rest of your body and turning it down. This probably helps a little, since you're reducing oxidized LDL and altered cholesterols, you end up with less altered cholesterol in the plaques, so you're giving macrophages a little bit more breathing room. But it doesn't work to a great enough degree, even if you reduce LDL cholesterol to 10-20% of what is normal in humans, you won't get rid of the plaques, you won't reverse it. What are the alternatives? Let's start with those that don't work. I mentioned that systemic inflammation is one of the problems leading to atherosclerosis. But if you reduce inflammation systemically, studies suggest you get about the same benefit to mortality as you would get from lowering LDL cholesterol. Which doesn't mean that somebody cannot come up with a way that could do this in a better and more targeted fashion, but the tools available for control of systemic inflammation are really blunt right now. The second alternative sounds much better, if limited to mice. Reverse cholesterol transport is the pathway wherein a macrophage sucks up cholesterol and hands it off to HDL particles in the bloodstream. There are a number of genes involved in this - macrophages use ABCA1 to hand off cholesterol to the HDL particle initially and then ABCG1 helps add more cholesterol to the particle. Then the particle heads to the liver and is excreted and ejected from the body. Anything you do in mice to make one or more parts of this system work better, it all works great - up to 50% reversal of plaque lipid content in some cases. But every time it was tried in humans, it failed - there's a whole list of clinical trials over the last 20 years that tried and failed. That tells us that we don't understand something very important about the way in which cholesterol transport is rate-limited in its different steps in humans vs in mice. So our approach is to make macrophages resilient to the environment in old tissues. There have been a number of people trying this, some of it hasn't made it very far, some of it is interesting, and sometimes there is overlap between those two. There is a recent paper with a hypothesis of effect they showed is that if you target lysosomes in macrophages with antioxidants, it prevents the oxidized LDL particles from messing things up, and therefore more macrophages are doing their job - reversing the plaque by 50% in a mouse model. It's entirely possible their hypothesis is wrong and delivering antioxidants is improving something else in the picture, but it's certainly a result that self-experimenters should pay attention to because these antioxidants are easily available. Secondly there is the Underdog Pharmaceuticals approach - sequestration of 7-ketocholesterol, which is a highly toxic altered cholesterol, thought to play a big role in atherosclerosis. Lastly there is our approach - engineering macrophages to give the ability to degrade excess cholesterol, whether or not it is altered. The company is named Repair, since I believe that if you're going to address aging and you can't point to something you are actually repairing - a form of damage or dysfunction, where you can clearly say that you are fixing this - then you might not be doing the right thing. In summary, what we're doing is allowing macrophages to degrade cholesterol and then stepwise approaching the various atherosclerotic conditions in order of number of patients. So starting with an orphan condition - homozygous familial hypercholesterolemia, then you go into larger patient groups as you gain experience doing this. Unlike most therapies, we can actually apply ourselves to any form of atherosclerosis, whether or not it has a genetic cause, we don't care how you got plaques, we just break them down. We've demonstrated AAV delivery of our cholesterol degrading protein - has a very large effect of 48% reversal of plaque lipids in a month, which is large in the scheme of things as compared to other approaches. Our goal is to produce a universal macrophage cell therapy. As I said, atherosclerosis is basically the encounter of an aged macrophage with cholesterol, at which point you get a lot of cell death and cholesterol-based plaque. If you overwhelm the existing systems of normal macrophages with excess cholesterol, they can't do anything with it and bbecome pathological foam cells - they don't have an way to deal with that level of cholesterol. So with that picture in mind, the whole spectrum of LDL lowering cholesterol drugs really only lowers that input to the problem. And they can't lower it more than a little, because the macrophage is in the plaque, not in the bloodstream, and the plaque is packed full of cholesterol and toxic horrible nastiness, so you're not really getting a lot of boost from lowering the input from bloodstream. The problem is the plaque that's sitting there. You can't reverse it by undertaking this LDL lowering approach, you still have macrophages exposed to excess cholesterol and becoming pathological foam cells as a result. And the pathological foam cells leading to your plaque brings us to this point, one that has to be made to a lot of people, unfortunately. Your risk of death is not due to LDL cholesterol, it's due how much plaque you have. It's exactly how much plaque you have and how much high risk plaque - the soft plaque laden with cholesterol. That determines your mortality. LDL cholesterol, while widely accepted as a surrogate marker, is not the cause of your death. That's why different people can have different levels of cholesterol in their bloodstream and have quite divergent mortality rates. The point of the exercise is to figure out what we should do differently - and that is making macrophages invulnerable to the plaque-based environment as best as we can. Our idea of "as best we can" is to give a macrophage the capability to break down cholesterol safely in situ. I should say that this is not a trivial thing to do, because a cell is basically an enormous lump of cholesterol - our body uses cholesterols everywhere in the cell membrane. The reason why we never evolved to break down cholesterol when it's harming us is probably because our cells have cholesterol everywhere. So you couldn't evolve something that just chews cholesterol whenever it sees it. And that's why delivering things like the known cyclodextrins that bind to cholesterol is not quite simple either, because the first thing that will happen if you dump a bunch of cyclodextrins into somebody is that their blood turns to mush, because it will consume all your blood cells by hooking all the cholesterol out of cell walls. So the objective is a safe way of breaking down cholesterol, but only the excess cholesterol, which is what we achieve by putting in these specific mechanisms into the cells we're working with. We can demonstrate that by putting these mechanisms into any old cell, and the output is exactly the same - we get a catabolite that is safe and more soluble, and quickly leaves the cell, departing into the bloodstream where it is gotten rid of. What this means is that we can take macrophages and give them the ability to express our cholesterol degrading proteins, and then if you dump cholesterol on those cells they remain competent and able to ingest cholesterol and dispose of it - that's what you want in your plaque. So going forward, we take induced pluripotent stem cells (iPSCs) from mice or humans, the lines are then disrupted in certain ways to make them universal (you get rid of the surface markers that make them recognizable - a very important technology that leads to off the shelf lines of universal cells, you can look at recent reports from Sana Biotechnology, of the delivery of universal iPSCs to primates, for example). We then differentiate iPSCs into macrophages that express cholesterol degrading proteins, and this is the way we produce a cost effective cell therapy. We've injected mice with the first of these cells over the last month or so and we should have initial data by the end of the year. And then what we do with this is a stepwise approach through the orphan indication of homozygous familial hypercholesterolemia with very few patients and a much easier FDA process, then to the heterozygous familial hypercholesterolemia indication with more patients, and then to the large high risk subpopulations of atherosclerosis, possibly tens of millions of patients at the end of the day. These are all people who will have medical imaging carried out to show the presence of high-risk, cholesterol-laden plaques. Ultimately we think you can take the lion's share of death - of that 27% by atherosclerotic diseases - and use technology such as ours to remove that cause of death from the human condition. How long is it going to take? Who knows, but the most high-risk population is where we start.

An Example of the Growth in Investment Funds Dedicated to the Longevity Industry
https://www.fightaging.org/archives/2021/11/an-example-of-the-growth-in-investment-funds-dedicated-to-the-longevity-industry/

Cambrian Biopharma started as a venture fund, but is a business development company now. The most important difference between those two business models is that a fund waits for startup companies to form and be ready for investment, while a development company sets out to create startups. The longevity industry is still comparatively small, and the arrival of new investment opportunities is thought by many, including the Cambrian Biopharma principals, to be too sparse to sustain larger funds. The solution, in an environment rich with promising scientific projects, is to create those opportunities: bring together scientists and entrepreneurs, license the relevant technologies from the universities, and fund the resulting startup company.

As noted in today's publicity materials, Cambrian has assembled a sizable fund, joining other large funds and business development companies such as the Longevity Vision Fund, Life Biosciences, Juvenescence, and Kizoo Technology Ventures. As Juvenescence and the Longevity Vision Fund illustrate, much of the investment in the early stages of growing the longevity industry has gone to less promising projects, or biotech and medical opportunities unrelated to the treatment of aging. Running a fund has a timeline built in, and investments must be made on a schedule. This, coupled with the small size of the longevity industry, is why we see a sizable fraction of the larger investment ventures in this space formed as business development companies rather than pure venture funds. In general, one should expect a business development company run by knowledgeable people to be more likely than a standard venture fund to invest in projects that are relevant to treating aging as a medical condition.

Cambrian Biopharma's 100 million Series C will advance healthspan-boosting therapeutics with financing co-led by Anthos Capital and SALT Fund

Longevity biotech Cambrian Biopharma has announced the close of an oversubscribed Series C financing, which raised 100 million. The financing was co-led by Anthos Capital and SALT Fund, with participation from existing investors Apeiron Investment Group, Future Ventures, Moore Capital and others, to develop therapeutics to combat the biological drivers of aging, treat and prevent age-related diseases and lengthen healthspan. With this financing, Cambrian has raised approximately 160 million since the company was founded in 2019. Age-related diseases account for more than two-thirds of all deaths worldwide, taking 41 million lives every year, or nearly one death every second. Existing approaches to these diseases are almost exclusively reactive - waiting for people to get sick and only then using the rapidly expanding knowledge of biology to try to treat very sick patients. Cambrian believes the future of medicine lies in approaching these diseases proactively, removing damage at a cellular level before a person becomes a patient. Each therapeutic in Cambrian's pipeline targets a different type of damage that builds up with age and will be tested for clinical safety and efficacy in an acute indication before using running multi-disease prevention trials. Cambrian operates as a Distributed Development Company designed to bridge the gap that exists today between academic discovery and drug development. This unique drug discovery model combines the advantages of a venture capital firm and a big pharmaceutical company, with the nimbleness of a biotech startup. Cambrian's hypothesis-driven approach and industry-leading pipeline of drug candidates provides for reduced risk and multiple "shots on goal". To date, Cambrian has 14 novel therapeutics in development across its majority-held pipeline companies. Proceeds from the financing will support the advancement and expansion of a diversified pipeline of novel therapies, each with the potential to both treat and prevent age-related diseases, with the goal of extending human healthspan. Cambrian expects to initiate clinical trials for three programmes in the next 18 months.

Cardiovascular Disease is Perhaps Less Well Understood than is Widely Perceived to be the Case
https://www.fightaging.org/archives/2021/11/cardiovascular-disease-is-perhaps-less-well-understood-than-is-widely-perceived-to-be-the-case/

There is, in general, a growing appreciation of the relevance of previously dismissed mechanisms of damage in the pathology of common age-related disease, such as cardiovascular conditions. The biggest challenge in aging and age-related disease is perhaps less the identification of relevant mechanisms, but rather understanding the relative importance of those mechanisms. Take transthyretin amyloidosis for example, the accumulation of misfolded transthyretin aggregates that occurs over the course of aging. It is only comparatively recently that the research community has established that this universal process of aging contributes meaningfully to a sizable fraction of heart failure.

Work on treatments for the inherited, accelerated version of transthyretin amyloidosis caused by mutation has produced a drug, tafamidis, that can at least slow the condition, and can also be applied to the vast majority of individuals lacking that mutation. Joining the dots will lead to this treatment being used in heart failure patients with notable amyloidosis. But again, this is all very recent. The lesson to take away from all of this is that improvements in the understanding of even well studied, common age-related conditions are ongoing. Slowly, the mechanisms of aging are being linked to conditions, and that leads to progress towards the development of therapies.

Cardiovascular Diseases That Have Emerged From the Darkness

It is important for both the patient and physician communities to have timely access to information recognizing rapid progress in the diagnosis and treatment of familiar but relatively uncommon cardiovascular diseases. Patients with three cardiovascular diseases, i.e. hypertrophic cardiomyopathy, pulmonary arterial hypertension, and transthyretin (TTR) cardiac amyloidosis (ATTR), once considered rare without effective management options and associated with malignant prognosis, have now benefited substantially from the development of a variety of innovative therapeutic strategies. In addition, in each case, enhanced diagnostic testing has expanded the patient population and allowed for more widespread administration of contemporary treatments. In hypertrophic cardiomyopathy, introduction of implantable defibrillators to prevent sudden death as well as high-benefit:low-risk septal reduction therapies to reverse heart failure have substantially reduced morbidity and disease-related mortality (to 0.5% per year). For pulmonary arterial hypertension, a disease once characterized by a particularly grim prognosis, prospective randomized drug trials with aggressive single (or combined) pharmacotherapy have measurably improved survival and quality of life for many patients. In cardiac amyloidosis, development of disease-specific drugs can for the first time reduce morbidity and mortality, prominently with breakthrough ATTR-protein-stabilizing tafamidis. In conclusion, in less common and visible cardiovascular diseases, it is crucial to recognize substantial progress and achievement, given that penetration of such information into clinical practice and the patient community can be inconsistent. Diseases such as hypertrophic cardiomyopathy, pulmonary arterial hypertension, and ATTR cardiac amyloidosis, once linked to a uniformly adverse prognosis, are now associated with the opportunity for patients to experience satisfactory quality of life and extended longevity.

VitaDAO, a Novel Approach to Crowdfunding Life Science Research
https://www.fightaging.org/archives/2021/11/vitadao-a-novel-approach-to-crowdfunding-life-science-research/

How to crowdfund the development of products, with variants such as crowdfunding to purchase equity in a company rather than a product, is a solved problem. Enabled by the internet, crowdfunding clearly works well when those who provide the funds will obtain something of concrete value in the near term as a result, be it a product or equity. Unfortunately, the established approaches to crowdfunding, exemplified by platforms such as Kickstarter, fail to work at scale when the goal is to fund scientific research. People have tried, numerous times, to make a Kickstarter for scientific research, with Experiment being perhaps the best of the resulting failures. The fundamental issue is that without the quid pro quo of a product or equity, some concrete value obtained, the incentives and feedback loops that make Kickstarter and its ilk work are absent.

The only type of crowdfunding that works to fund scientific research is small in scale, a matter of enabling passionate special interest communities to fund favored projects in ways that are not appreciably different from pre-Internet non-profit pledge drives. I'd like to think that our own, still comparatively small longevity-focused community has proven to be fairly adept at this sort of thing, with groups such as Lifespan.io enabling a number of crowdfunded projects to proceed over the years.

Still, much more is needed. Enter VitaDAO, which is structured in a way made possible by blockchain technology, and which probably could not have arisen absent the present fervor for blockchain implementations such as cryptocurrencies. The core ideas on how to make crowdfunding of research actually work, implemented by VitaDAO, could just as well be put into practice by an ordinary non-profit organization with centralized administrative software, absent a blockchain implementation, however. One could argue that the primary use for blockchains here was to enable VitaDAO to start out with a very large warchest of funds via a token launch, without the need to convince the traditional, highly skeptical sources of non-profit funding to back such a project. That is important!

The core ideas appear good, and VitaDAO does indeed have deep pockets with which to fund research. Stripped down, the concept is to build a self-sustaining long term research fund in which: (a) people buy into the organization, putting in funds to obtain voting rights on which research projects to fund; (b) deals are struck with universities and startups to provide funding for specific projects in exchange for a cut of later patents, royalties, equity, or similar flows of funds; (c) successfully commercialized research will lead to funds flowing back into VitaDAO for investment into research. There are numerous ways in which VitaDAO could perhaps help to make such commercialization easier in the future, such as by building a marketplace akin to, say, BioNeex or similar.

The additions to this core idea made possible by the use of blockchain technologies revolve around decentralization of governance, keeping track of ownership, and perhaps later allowing trading on interests in research programs. But the current administrators have structured the organization and its work, quite carefully, in order to avoid any appearance of conducting a stock offering or producing a trading platform. Regulatory compliance is very important in this part of the field. The core of it is the virtuous feedback loop of funds, governance, and income from successful commercialization - a slow loop, given the timelines for moving from research to clinical, but there nonetheless.

VitaDAO is focused on the longevity industry and aging research aimed at lengthening the healthy human life span, an endeavor with strong support in the broader cryptocurrency community, as illustrated by large donations to organizations such as the SENS Research Foundation in recent years. But there is no reason as to why this model couldn't work for any arbitrary field of research with a strongly interested community of laypeople cheering it on. Environmental science, for example, or many others.

VitaDAO is in its early stages, but they have passed the first few most difficult hurdles. Firstly, they have obtained significant funds to deploy to research programs via a token offering. Secondly they have structured the first deals with a university, laying the groundwork for future similar agreements. After that, the rest is largely a matter of attracting attention and keeping the wheels turning. Any researcher or entrepreneur who is looking for a 250,000 grant in exchange for a modest interest in later patents or royalties can step up today and pitch the VitaDAO community and leadership on their project. We live in interesting times!

VitaDAO

VitaDAO is a new cooperative vehicle for community-governed and decentralized drug development. Our core mission is the acceleration of R&D in the longevity space and the extension of human life and healthspan. To achieve this, VitaDAO utilizes a combination of novel governance (distributed autonomous organizations - DAOs), digital assets (non-fungible tokens - NFTs), and financial market frameworks (automated market makers - AMMs). Value creation in biopharma centers heavily around intellectual property assets and patents as core drivers for funding and innovation. Yet intellectual property ownership as a business model has barely evolved in the past century. Current biopharma business models carry severe limitations and R&D inefficiencies that cost those who should be the core stakeholders: patients and researchers. We believe the future of biopharma is transparent, collaborative, and open source. VitaDAO is an open cooperative that anyone can join, with the goal to acquire, support and finance new therapeutics and research data in the longevity space. The VitaDAO collective will directly hold legal IP rights to these projects and may develop a growing portfolio of assets represented as NFTs. Members of the public can join VitaDAO and become owners of its IP by purchasing VITA tokens through contributing funds, work, or valuable research data or IP assets. Ownership and governance of VitaDAO requires VITA tokens. VITA tokens enable their holder to engage in decision-making and governance of VitaDAO's research, signal support for specific initiatives, and govern its data repositories and IP portfolio. VitaDAO will acquire and commission research, as well as own, develop and monetize the resulting intellectual property assets. VitaDAOs portfolio consists of: 1) NFTs representing intellectual property, patents and licenses to therapeutic research projects; 2) Data assets generated by funding R&D around its research projects and NFTs. Vetted longevity research projects will request funds from VitaDAO, and members will vote to grant or raise those funds in exchange for ownership in the resulting IP. To fund more research and to provide long term funding for the DAO operations, there are several options to monetize owned data and IP. VitaDAO can enter co-development deals with private companies or other DAOs. in single or multi-license agreements, VitaDAO could license data and IP to 3rd parties, or could sell to the highest suitable bidder.

Exosome Treated Macrophages Improve Regeneration Following Spinal Cord Injury in Mice
https://www.fightaging.org/archives/2021/11/exosome-treated-macrophages-improve-regeneration-following-spinal-cord-injury-in-mice/

Exosomes are membrane-wrapped parcels of molecules used by cells to pass signals between one another. Researchers here note that exosomes harvested from stem cells provide a way to induce macrophages to adopt the M2 phenotype that suppresses inflammation and is involved in tissue regeneration. These macrophages can then be injected into mice in order to spur a greater degree of regeneration following spinal cord injury than would otherwise take place. It is far from a complete recovery, but it is certainly better than the alternative.

The spinal cord injury is a site of severe central nervous system (CNS) trauma and disease without an effective treatment strategy. Neurovascular injuries occur spontaneously following spinal cord injury (SCI), leading to irreversible loss of motor and sensory function. Bone marrow mesenchymal stem cell (BMSC)-derived exosome-educated macrophages (EEM) have great characteristics as therapeutic candidates for SCI treatment. It remains unknown whether EEM could promote functional healing after SCI. The effect of EEM on neurovascular regeneration after SCI needs to be further explored. We generated M2-like macrophages using exosomes isolated from BMSCs, which were known as EEM, and directly used these EEM for SCI treatment. We aimed to investigate the effects of EEM using a spinal cord contusive injury mouse model in vivo combined with an in vitro cell functional assay and compared the results to those of a normal spinal cord without any biological intervention, or PBS treatment or macrophage alone (MQ). Neurological function measurements and histochemical tests were performed to evaluate the effect of EEM on angiogenesis and axon regrowth. In the current study, we found that treatment with EEM effectively promoted the angiogenic activity of HUVECs and axonal growth in cortical neurons. Furthermore, exogenous administration of EEM directly into the injured spinal cord could promote neurological functional healing by modulating angiogenesis and axon growth. EEM treatment could provide a novel strategy to promote healing after SCI and various other neurovascular injury disorders.

The NLRP3 Inflammasome in Osteoporosis
https://www.fightaging.org/archives/2021/11/the-nlrp3-inflammasome-in-osteoporosis/

The NLRP3 inflammasome is a part of the complex regulatory system that controls inflammatory responses, the rousing of the immune system to action. NLRP3 has become an topic of interest to researchers as it seems, potentially, a point of intervention to suppress inflammatory signaling and maladative cell responses to that signaling. While regulatory adjustment is a poor substitute for removal of the causes of chronic inflammation in aging, interfering in NLRP3 activity may diminish the downstream consequences of chronic inflammation, slowing or reversing the progression of inflammatory conditions in later life. Whether the benefit is large enough to merit the effort can only be discovered by making the attempt.

Osteoporosis is a systemic bone metabolism disease that often causes complications, such as fractures, and increases the risk of death. The NLRP3 inflammasome is an intracellular multiprotein complex that regulates the maturation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18, mediates inflammation, and induces pyroptosis. The chronic inflammatory microenvironment induced by aging or estrogen deficiency activates the NLRP3 inflammasome, promotes inflammatory factor production, and enhances the inflammatory response. In this review, we summarize the related research and demonstrate that the NLRP3 inflammasome plays a vital role in the pathogenesis of osteoporosis by affecting the differentiation of osteoblasts and osteoclasts. IL-1β and IL-18 can accelerate osteoclast differentiation by expanding inflammatory response, and can also inhibit the expression of osteogenic related proteins or transcription factors. In vivo and in vitro experiments showed that the overexpression of NLRP3 protein was closely related to aggravated bone resorption and osteogenesis deficiency. In addition, abnormal activation of NLRP3 inflammasome can not only produce inflammation, but also lead to pyroptosis and dysfunction of osteoblasts by upregulating the expression of Caspase-1 and gasdermin D (GSDMD). In conclusion, NLRP3 inflammasome overall not only accelerates bone resorption, but also inhibits bone formation, thus increasing the risk of osteoporosis. Thus, this review highlights the recent studies on the function of NLRP3 inflammasome in osteoporosis, provides information on new strategies for managing osteoporosis, and investigates the ideal therapeutic target to treat osteoporosis.

Exercise as an Approach to Slow Alzheimer's Disease
https://www.fightaging.org/archives/2021/11/exercise-as-an-approach-to-slow-alzheimers-disease/

Exercise is beneficial at every age, but most people do not undertake enough physical activity. In a sedentary world, structured exercise programs look like a decent therapy, because that exercise corrects a harmful deficiency in the operation of metabolism. Thus the studies showing a reduction in mortality resulting from exercise as an intervention in older individuals. Exercise improves mitochondrial function, amongst other changes, and these changes should be expected to modestly slow the progression of many age-related diseases.

Neurons are highly specialized post-mitotic cells that are inherently dependent on mitochondria due to their higher bioenergetic demand. Mitochondrial dysfunction is closely associated with a variety of aging-related neurological disorders, such as Alzheimer's disease (AD), and the accumulation of dysfunctional and superfluous mitochondria has been reported as an early stage that significantly facilitates the progression of AD. Mitochondrial damage causes bioenergetic deficiency, intracellular calcium imbalance, and oxidative stress, thereby aggravating β-amyloid (Aβ) accumulation and Tau hyperphosphorylation, and further leading to cognitive decline and memory loss. Although there is an intricate parallel relationship between mitochondrial dysfunction and AD, their triggering factors, such as Aβ aggregation and hyperphosphorylated Tau protein, are still unclear. Moreover, many studies have confirmed abnormal mitochondrial biosynthesis, dynamics, and functions will present once the mitochondrial quality control is impaired, thus leading to aggravated AD pathological changes. Accumulating evidence shows beneficial effects of appropriate exercise on improved mitophagy and mitochondrial function to promote mitochondrial plasticity, reduce oxidative stress, enhance cognitive capacity and reduce the risks of cognitive impairment and dementia in later life. Therefore, stimulating mitophagy and optimizing mitochondrial function through exercise may forestall the neurodegenerative process of AD.

Glial Cells and the Propagation of Tau through the Brain in Tauopathies
https://www.fightaging.org/archives/2021/11/glial-cells-and-the-propagation-of-tau-through-the-brain-in-tauopathies/

Tauopathies like Alzheimer's disease are characterized by the spread of tau aggregates through the brain. Tau is one of the few molecules in the body that can become altered in a way that encourages other copies of the same molecule to also alter, causing aggregates to form. These aggregates and their surrounding biochemistry are disruptive to cell function and toxic to cells. A number of neurodegnerative conditions are associated with protein aggregates of amyloid-β, α-synuclein, and tau. The mechanisms by which this spread occurs are debated, but researchers strongly suspect a role for glial cells in this process.

Dementia is one of the leading causes of death worldwide, with tauopathies, a class of diseases defined by pathology associated with the microtubule-enriched protein, tau, as the major contributor. Although tauopathies, such as Alzheimer's disease and frontotemporal dementia, are common amongst the ageing population, current effective treatment options are scarce, primarily due to the incomplete understanding of disease pathogenesis. The mechanisms via which aggregated forms of tau are able to propagate from one anatomical area to another to cause disease spread and progression is yet unknown. The prion-like hypothesis of tau propagation proposes that tau can propagate along neighbouring anatomical areas in a similar manner to prion proteins in prion diseases, such as Creutzfeldt-Jacob disease. This hypothesis has been supported by a plethora of studies that note the ability of tau to be actively secreted by neurons, propagated and internalised by neighbouring neuronal cells, causing disease spread. Surfacing research suggests a role of reactive astrocytes and microglia in early pre-clinical stages of tauopathy through their inflammatory actions. Furthermore, both glial types are able to internalise and secrete tau from the extracellular space, suggesting a potential role in tau propagation; although understanding the physiological mechanisms by which this can occur remains poorly understood.

A Possible Feedback Loop Between Abnormal Brain Activity and Neuroinflammation in Alzheimer's Disease
https://www.fightaging.org/archives/2021/11/a-possible-feedback-loop-between-abnormal-brain-activity-and-neuroinflammation-in-alzheimers-disease/

Researchers here put forward an interesting view of the progression of Alzheimer's disease, in which abnormal modes of brain activity are part of a feedback loop that between inflammation and pathological protein aggregation. Evidence strongly suggests that chronic inflammation in brain tissue is an important component of neurodegenerative conditions, and the aggregation of altered proteins such as tau is both caused by inflammation and contributes to it. It is interesting to see that view expanded out to encompass the neural activity of the brain as a part of the downward spiral of interacting dysfunctions.

Scientists have known for a while that Alzheimer's disease is associated with chronic inflammation in the brain. A driver of this inflammation appears to be the accumulation of amyloid proteins in the form of "plaques," a neuropathological hallmark of the illness. In a new study, researchers identified non-convulsive epileptic activity as another critical driver of chronic brain inflammation in an Alzheimer's-related mouse model. This subtle type of epileptic activity also occurs in a substantial proportion of people with Alzheimer's disease and can be a predictor of faster cognitive decline in the patients. "One way this subclinical epileptic activity may accelerate cognitive decline is by promoting brain inflammation." Researchers discovered that, when they reduced epileptic activity in the mouse brain, one of the inflammatory factors most affected was TREM2, which is produced by microglia, the brain's resident immune cells. People with genetic variants of TREM2 are two to four times more likely to develop Alzheimer's disease than people with normal TREM2, but scientists are still trying to decipher the precise roles this molecule plays in health and disease. The scientists first showed that TREM2 was increased in brains of mice with amyloid plaques, but reduced after suppression of their epileptic activity. To find out why, they examined whether TREM2 affects the susceptibility of mice to low doses of a drug that can cause epileptic activity. Mice with reduced levels of TREM2 showed more epileptic activity in response to this drug than mice with normal TREM2 levels, suggesting that TREM2 helps microglia to suppress abnormal neuronal activities. "TREM2 has been primarily studied in relation to pathological hallmarks of Alzheimer's disease such as plaques and tangles. Here, we found that this molecule also has a role in regulating neural network functions. The genetic variants of TREM2 that increase the risk for Alzheimer's disease appear to impair its function. If TREM2 doesn't work properly, it could be harder for immune cells to suppress neuronal hyperexcitability, which in turn might contribute to the development of Alzheimer's disease and accelerate cognitive decline."

Glutathione Delivery via Iontophoresis Increases Gluthathione Levels in Blood Samples
https://www.fightaging.org/archives/2021/11/glutathione-delivery-via-iontophoresis-increases-gluthathione-levels-in-blood-samples/

Glutathione is a mitochondrial antioxidant, and additional antioxidant capacity in mitochondria appears to be beneficial to long-term health, improving mitochondrial function and overall health. Mitochondria conduct the energetic process of producing ATP, used to power the cell, with a flux of oxidative molecules as a side-effect. With age, mitochondria tend to become less efficient and produce more oxidizing molecules, harmful to the cell. Glutathione levels decline with age, which may contribute to this age-related mitochondrial dysfunction.

Oral supplementation with gluthatione doesn't have any effect, unfortunately, but researchers recently published a small study of supplementation with large amounts of glutathione precursors. This caused increased gluthatione manufacture, increased glutathione in blood samples, and measurable benefits to health in old individuals. Here, researchers provide evidence for an iontophoresis approach for the delivery of glutathione through the skin via an electrical field, an intriguing option to be compared against intravenous administration when considering relative costs and benefits.

Glutathione (GSH) is the most abundant antioxidant in human cells. Reactive oxygen species (ROS) produced in the body can promote oxidative damage to cells and may cause genomic instability and mitochondrial dysfunction, two hallmarks of aging. The concentration of GSH has been shown to decrease with aging, resulting in reduced antioxidant activity in cells. Consequently, lower GSH levels have been associated with an increased risk of aging-associated diseases. Relatively higher blood levels of GSH, on the other hand, are associated with improved physical and mental health in older individuals. Supplementation of GSH may, therefore, protect against age-related morbidity and mortality. In recent years, intravenous supplementation has become a popular method to restore GSH levels. It is an effective method but has its limitations as it is only accessible in a specialty clinic setting and is expensive and inconvenient for patients. Two aging patients with low serum GSH levels were supplemented with GSH in our clinic using a non-invasive drug delivery device, the IontoPatch, to deliver GSH through the skin. The IontoPatch technology uses bipolar electric fields, iontophoresis, to deliver molecules across the skin into the underlying tissue. Iontophoresis is widely used in physical therapy for localized treatment of pain and inflammation. A 1 mL dose of a 200 mg/mL saline solution of GSH was added to the patch's negative electrode for each treatment. The patch was applied on the upper arm's skin and was worn for six consecutive days for at least four hours each day. Serum levels of GSH were assessed at baseline and days 7 and 23 after treatment was initiated. In both cases, serum GSH levels increased after seven days of treatment (64.4% and 21.8%). Serum GSH levels then decreased between days 7 and 23 to 44.5% and 17.2% above baseline. There were no adverse events reported in either case. More extensive studies should be conducted to determine the pharmacokinetics, safety of long-term supplementation, and supplementation health benefits.

Finding Cautions in the Ease with which it is Possible to Create Epigenetic Clocks
https://www.fightaging.org/archives/2021/11/finding-cautions-in-the-ease-with-which-it-is-possible-to-create-epigenetic-clocks/

In recent years, researchers have established that machine learning approaches can be used to produce any number of clocks from biological data that shifts with age, finding patterns that match chronological or biological age to a great enough accuracy to suggest that they can be useful assays for the assessment of potential age-slowing and rejuvenating therapies. It remains an open question as to whether and how exactly the assessed patterns correlate to the specific forms of molecular damage that cause aging, or to any of the specific downstream consequences of that damage. Scientists here raise the possibility that much of the epigenetic change of aging may not in fact be as useful as a basis for measurement as thought, and suggest that more fundamental research is required in order to robustly connect clocks with specific processes of aging.

Our meta-analysis of the largest available age-annotated methylation dataset to date found: 1) as much as one fifth of the measured cytosines contains age-predictive methylation patterns; 2) tissues show largely similar aging patterns despite having methylated regions that define their identity; 3) epigenetic clock sites are enriched in intergenic regions, gene enhancers, and sites near expression quantitative trait loci (eQTLs) and 4) are depleted in the regions generally thought to have the largest direct impact upon gene expression (e.g., CpG Islands and gene promoters); 5) patients with age-correlated diseases did not appear significantly age-accelerated according to the chronological epigenetic clock. The fact that many different sites can be used to create an epigenetic clock with minimal impact on predictive performance argues against the idea that methylation changes are either programmed or individually important. Yet, because the clock is robustly predictive and age-related methylation changes are mostly similar between tissues, this argues against entropy as a driving force. This could be reconciled by hypothesizing some genomic regions and/or features receive less methylation maintenance than others. Perhaps the changes occur in regions of the genome where they have no consequence, and instead, vary with absolute time such as in determining speciation time using pseudogene mutation rates. This "pseudomethylation" would be problematic for modeling aging biology, as they would likely not respond to aging intervention. Methylation maintenance mechanisms (e.g., DNMT1) serve as a counterbalance against entropy. However, if some genomic regions are less maintained than others, then we would expect the probability of a methylation state change with age to be correlated with the degree to which it is subject to methylation surveillance and maintenance. Because maintenance costs energy, it is reasonable to hypothesize the degree of maintenance correlates with the adverse impact an unregulated change in methylation would cause. If so, the probability a site's methylation will vary with age would inversely correlate with its impact on an organism's survival. Given that methylation changes with age are robust across tissues, yet small in magnitude, leads the field to question whether the "ticking" that drives them is due to changes in cell population composition, such as a reduction of pluripotent stem cells or an increase in senescent cells within every tissue, or possibly high magnitude effects in rare cell populations (e.g., immune cells in the central nervous system compared to astrocytes or neurons). In either case, it is not clear whether the phenomenon driving ticking clock sites is due to healthy compensatory changes or deleterious drift toward age-related fragility. In summary, the predictive power of the epigenetic clock is robust, but such a large fraction of the genome can be used to predict, the magnitude of the changes is small, and these regions tend to be depleted near genes. This leads us to hypothesize that the pan-tissue predictive loci are more likely to be molecularly "silent" methylation changes that accrue outside of strong regulatory regions due to entropy in methylation maintenance, which must be explored in the future studies. Furthermore, if current models inconsistently annotate patients with age-related diseases as "age-accelerated" and the confidence by which one can declare a sample age-accelerated is small, this argues against the idea that epigenetic clocks can disentangle biological age from chronological age.

The Tumor Suppression Theory of Aging
https://www.fightaging.org/archives/2021/11/the-tumor-suppression-theory-of-aging/

While they cannot explain aging as a whole, single cause theories of aging can be useful tools to frame discussion and investigation aimed at better understanding aging and its evolution. The theory presented here is aging viewed through the lens of cancer, two entwined processes. Aging is viewed as largely a consequence of tumor suppression mechanisms that evolved to keep cancer at a low enough incidence for successful selection and continuation of the species. Cancer and evolution are themselves in a dynamic, competing equilibrium. Evolution requires a certain minimal rate of spontaneous mutation, while cancer thrives on those mutations; the higher the rate, the higher the risk of cancer. An ever more complex arms race results, eventually resulting in the varied pace of mutation and aging, types of tumor suppression mechanisms, and incidence of cancer found across diverse species today.

Single cause theories of aging remain important in aging research. One obvious reason for this is the need for simplification. Another reason is the necessity to at least break up the aging process into potentially treatable parts, even if no single treatment can be expected to do much. Somatic mutations have long been proposed as a cause of aging and genomic instability is one of the four primary hallmarks of aging. The tumor suppression theory of aging outlined here differs from previous theories in that clonal expansion and malignancy is proposed as the relevant consequence of somatic mutation and that impairment, loss of cellular function, or cell death as a consequence of somatic mutation is largely irrelevant. To counter the tumorigenic potential of clonally expanding cells, we have evolved tumor suppression mechanisms that remove or limit proliferation of stem cells. Accumulating senescent cells and loss of capacity for self-renewal and repair eventually cause the phenotypes we experience in very old age. Obesity and caloric restriction accelerate and decelerate aging due to their effect on cell proliferation, during which most mutations arise. Most phenotypes of aging are merely tumor-suppressive mechanisms that evolved to limit malignant growth, the dominant age-related cause of death in early and middle life. Cancer limits life span for most long-lived mammals, a phenomenon known as Peto's paradox. Its conservation across species demonstrates that mutation is a fundamental but hard limit on mammalian longevity. Cell senescence and apoptosis and differentiation induced by oncogenes, telomere shortening, or DNA damage evolved as a second line of defense to limit the tumorigenic potential of clonally expanding cells, but accumulating senescent cells, senescence-associated secretory phenotypes and stem cell exhaustion eventually cause tissue dysfunction and the majority, if not most, phenotypes of aging. If the tumor suppression theory of aging would be correct, the only way to retard human aging would be a reduction of somatic mutation. Preventing aging would be the same as preventing cancer. Unfortunately, reduction or prevention of somatic mutation is something that remains thoroughly out of reach of current medical technology. The problem of aging will probably defy the assault of human ingenuity for some time to come. In the meantime, removal of senescent cells seems to offer a reasonable chance of alleviating many phenotypes of very old age. Cell senescence is antagonistically pleiotropic, and accumulation of senescent cells probably an evolutionary accident brought about by the unforeseen increase in average human life expectancy. Together with a second antagonistically pleiotropic phenomenon, the age-related emergence of systemic and excessive chronic sterile inflammation, these two phenomena might be mutually reinforcing examples of evolutionary accidents, responsible for many of the pathogenic processes promoting the irreversible functional decline of very old age. Even though they do not prevent aging per se, in terms of looking at realistic strategies for increasing human health span, these two processes are, compared to the primary prevention of mutation, probably lower hanging fruit and offer plentiful possibilities in postponing the dreaded symptoms of old age.

Telomerase Reverse Transcriptase Improves Mitochondrial Function
https://www.fightaging.org/archives/2021/11/telomerase-reverse-transcriptase-improves-mitochondrial-function/

Telomerase gene therapies usual deliver telomerase reverse transcriptase (TERT), which might be thought of as the most important part of the full telomerase complex. Most research has focused on the ability of telomerase to lengthen telomeres, and where overexpression of telomerase is seen to extend life span in animal models, lengthening of telomeres is the mechanism most explored by the research community. However, TERT also acts on mitochondria. Here, researchers advance the understanding of how mitochondrially localized TERT can improve mitochondrial function. Given the importance of mitochondria in aging, it is an interesting question as to the degree to which telomere lengthening versus improved mitochondrial function produce the improved health, lower cancer incidence, and extension of life span observed in mice as a result of telomerase gene therapies.

The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) has protective functions in the cardiovascular system. TERT is not only present in the nucleus, but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection and appropriate tools are missing to dissect this. We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart as well as cellular functions of cardiomyocytes, fibroblasts, and endothelial cells were determined. All mice were phenotypically normal. While respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wildtype mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after one, two and four weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. In conclusion, mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. We conclude that mitochondrial TERT is responsible for cardioprotection and its increase could serve as a therapeutic strategy.

Summarizing What is Known of the INDY Longevity Gene in Flies
https://www.fightaging.org/archives/2021/11/summarizing-what-is-known-of-the-indy-longevity-gene-in-flies/

That the INDY gene can influence life span was one of the earlier discoveries made once researchers begin to manipulate the life span of short-lived species, spurred by the study of slowed aging via calorie restriction, and searching in earnest for the mechanisms by which metabolism determines the pace of aging. Progress is very slow in this part of the scientific community. Reviews of what is known of INDY are not that different today then they were a decade ago, and it remains an open question as to how relevant this is to humans. That INDY has effects related to preserved intestinal function in flies may just be a reflection of the great importance of the intestine in fly aging, and not an indication of what to expect in mammals.

Reduced gene expression of fly Indy and its worm homologues extends their life span by altering metabolism in a manner similar to calorie restriction (CR). Fly INDY and homologues in worms and mammals share a preference for transporting citrate. By regulating cytoplasmic citrate levels, INDY acts as a metabolic regulator in modulating glucose and lipid levels, and energy production in mitochondria. Metabolic changes associated with Indy reduction in the fly midgut results in dramatic changes in midgut physiology that lead to preserved intestinal stem cell (ISC) homeostasis. This is vital for replacement of damaged cells and the maintenance of midgut function illustrated by preserved intestinal integrity. Indy reduction extends lifespan in male and female flies, but the effects of Indy reduction on ISC homeostasis have only been studied in female flies. Male and female flies have different gut pathologies and respond differently to stress and CR, with males having a delay in age-related gut pathology and lower ISC proliferation, while females respond better to stress and CR. Considering these differences, it would be of interest to determine the effects of Indy reduction on the midguts of male flies. ISC homeostasis is regulated by multiple signaling pathways including IIS, Notch, EGF, Wnt/wingless, BMP/Dpp, JNK, and JAK/STAT, among others. It would be important to assess the status of different signaling pathways in flies with reduced Indy expression, as metabolic changes might delay age-associated activation of these pathways and could contribute to preservation of ISC homeostasis and longevity. The data reviewed here support the role of INDY as a metabolic regulator: Indy expression changes in response to nutrient availability and requirements of the organism, which, by regulating citrate levels, controls energetic status of the organism to maintain tissue-specific metabolic requirements leading to preserved organismal health and homeostasis. Reduced INDY levels in the midgut could then prevent age-related ISC hyperproliferation by reducing the available energy for proliferation.

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