Week 144 of Changing to a youthful phenotype with sprouts

Two papers, starting with a 2023 study that investigated the same red radish cultivar as Sulforaphene, a natural analog of sulforaphane:

“Availability of microgreen products is constantly rising, i.e., they are offered for sale in local farmers markets, specialty stores, and in chain grocery stores. Due to the low demands required for their cultivation and easily available LED settings, microgreens are increasingly grown on a small scale in homes and after harvesting, they are stored in kitchen refrigerators at 4 °C.

The aim of this study was to simulate such cultivation and storage conditions to examine antioxidant capacity of home-grown radish microgreens. Seven-day-old radish microgreens, grown under purple and white LED light, were harvested and stored at 4 °C for two weeks.

Measurements of total antioxidant capacity and bioactive substances were conducted on the harvesting day and on the 3rd, 7th, and 14th day of storage. All three radish cultivars (Raphanus sativus L.) with different leaf colorations:

  • Purple radish (R. sativus cult. China Rose, cvP);
  • Red radish (R. sativus cult. Sango, cvR); and
  • Green radish (Raphanus sativus var. longipinnatus, Japanese white or daikon radish, cvG)

were purchased commercially from a local supplier.

The highest contents of total soluble phenolics, proteins, and sugars, dry matter, and monomeric anthocyanin content, as well as higher overall antioxidant capacity determined in the red radish cultivar (cvR), distinguished this cultivar as the most desirable for human consumption regardless of the cultivation light spectrum.”

https://www.mdpi.com/2311-7524/9/1/76 “Antioxidant Capacity and Shelf Life of Radish Microgreens Affected by Growth Light and Cultivars”


A 2021 review summarized what was known about radishes up to then. Here’s part of its Discussion section:

“It is worth considering radish’s organoleptic characteristics since its particular flavor can influence its acceptability among consumers. The main compound associated with its characteristic pungent flavor is raphasatin, which we have found to be the most reported isothiocyanate produced from the breakdown of glucoraphasatin.

Glucoraphasatin ranked as one of the most concentrated glucosinolates in radish, particularly in its sprouts, but also present in other parts like roots and seeds. Pungency differs among radish cultivars, environmental growth factors, agronomic, and cooking practices.”

1-s2.0-S0924224421003058-gr3_lrg

https://www.sciencedirect.com/science/article/pii/S0924224421003058 “Nutritional and phytochemical characterization of radish (Raphanus sativus): A systematic review”


Seeds I’ve sprouted this year so far, left to right – red radish (Sango), broccoli, red cabbage (Red Acre), yellow mustard, oat (Avena sativa):

PXL_20230106_121548495

Red radish had similar growth characteristics as broccoli. Started with 3.6 grams of seeds, which increased to 22.2 g after three days using the same soaking and rinsing protocol I use for other sprouts.

PXL_20230109_121815772

The taste of red radish was too sharp for me to eat by themselves, so I combined them with my broccoli / red cabbage / mustard sprout mix. Bumped up microwaving time to 48 seconds in a 1000 W microwave while staying short of the 60°C (140°F) myrosinase cliff.

The whole mix still had a strong radish taste, though. It was as if two whole red radishes were sliced into a small salad.

Can’t add anything more to dampen that taste and expect beneficial compounds to be unaffected. From Week 19:

A 2018 Netherlands study Bioavailability of Isothiocyanates From Broccoli Sprouts in Protein, Lipid, and Fiber Gels found:

Compared to the control broccoli sprout, incorporation of sprouts in gels led to lower bioavailability for preformed sulforaphane and iberin.”

IAW, eating protein, fats, and fiber along with microwaved broccoli sprouts wouldn’t help. A 2018 review with some of the same researchers Isothiocyanates from Brassica Vegetables-Effects of Processing, Cooking, Mastication, and Digestion offered one possible explanation for protein acting to lower broccoli sprout compounds’ bioavailability:

“In vitro studies show that ITCs can potentially react with amino acids, peptides, and proteins, and this reactivity may reduce the ITC bioavailability in protein‐rich foods. More in vivo studies should be performed to confirm the outcome obtained in vitro.”

Mixing in red radish sprouts also gave me an upset stomach five of the six mornings. So I won’t continue to sprout red radish.

That said, I’d definitely consider sprouting red radish again to accelerate isothiocyanate treatment of problems where symptoms are much worse than an upset stomach, such as:

  • Neurogenerative diseases with their cognitive decline;
  • Immune system disorders;
  • Bacterial and viral infections; and
  • Other damage caused by oxidative stress conditions in eyes, vascular system, kidney function, etc.

Piping in the New Year

PXL_20230101_193048797

Eat mushrooms every day?

Three 2022 papers on amino acid ergothioneine, starting with a human study:

“We examined temporal relationships between plasma ergothioneine (ET) status and cognition in a cohort of 470 elderly subjects attending memory clinics in Singapore. All participants underwent baseline plasma ET measurements as well as neuroimaging for cerebrovascular disease (CeVD) and brain atrophy. Neuropsychological tests of cognition and function were assessed at baseline and follow-up visits for up to five years.

Lower plasma ET levels were associated with poorer baseline cognitive performance and faster rates of decline in function as well as in multiple cognitive domains including memory, executive function, attention, visuomotor speed, and language. In subgroup analyses, longitudinal associations were found only in non-demented individuals.

Mediation analyses showed that effects of ET on cognition seemed to be largely explainable by severity of concomitant CeVD, specifically white matter hyperintensities, and brain atrophy. Our findings support further assessment of plasma ET as a prognostic biomarker for accelerated cognitive and functional decline in pre-dementia and suggest possible therapeutic and preventative measures.”

https://www.mdpi.com/2076-3921/11/9/1717 “Low Plasma Ergothioneine Predicts Cognitive and Functional Decline in an Elderly Cohort Attending Memory Clinics”


Earlier this year, two of the study’s coauthors put together a collection of 11 ergothioneine papers:

“One catalyst for this upsurge of interest was the discovery in 2005 of a transporter for ET (OCTN1, often now called the ergothioneine transporter, ETT), which accounts for the fact that animals (including humans) take up and avidly retain ET from the diet. The presence of a specific transporter together with the avid retention of ET in the body implies that this compound is important to us.

To quote an old phrase ‘correlation does not imply causation.’ Low ET levels may predispose to disease, but disease could also lead to low ET levels. Possible reasons could include:

  • Alterations in diet due to illness so that less ET is consumed;
  • Decreases in ETT activity in the gut (leading to less ET uptake) or kidney (impairing ET reabsorption) with age and disease.
  • Changes in gut microbiota might influence uptake and accumulation in the body.
  • ET is being consumed as it scavenges oxygen radicals and other reactive oxygen species, the production of which is known to increase in these diseases and during ageing in general.

Only the gold standard of placebo-controlled double-blinded clinical studies can definitively establish the value (if any) of ET in preventing or treating human disease. Several such trials are being planned or in progress; we await the results with interest, and a streak of optimism.”

https://febs.onlinelibrary.wiley.com/doi/10.1002/1873-3468.14350 “Ergothioneine, where are we now?”


One of the collection’s papers focused on what ETT research findings could or could not be replicated:

“ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable. We review substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes.

Comparison of transport efficiency (TE) for acknowledged substrates of the ETT. Bar length represents approximate TE of wild-type human ETT.

feb214269-fig-0001-m

We have not found in the literature any other ET transporters. However, it is highly probable that additional ET transporters work in the human body:

  • Uptake of ET from the small intestine into epithelial cells occurs through apically localized ETT. The very hydrophilic ET cannot then exit these cells toward the blood without help – a basolateral efflux transporter is required.
  • After oral administration of 3H-ET, a considerable amount of ET was still absorbed into the body in the ETT KO [knockout] mice. There must be another transporter for apical uptake at least in the small intestine of the mouse.
  • When ET was administered intravenously, ETT KO mice showed no change in ET concentration in the brain compared to wild type. The little ET that enters the brain must therefore pass through the BBB via a different transporter.”

https://febs.onlinelibrary.wiley.com/doi/10.1002/1873-3468.14269 “The ergothioneine transporter (ETT): substrates and locations, an inventory”


It’s persuasive that there’s an evolutionarily conserved transmitter specific to ergothioneine. It isn’t persuasive that this compound once consumed is almost always in stand-by mode to do: what?

Ergothioneine isn’t a substitute for the related glutathione, especially since its supply isn’t similarly available from an endogenous source. It isn’t an active participant in day-to-day human life.

Still, I hedge my bets. I eat ergothioneine every day via white button mushrooms in AGE-less chicken vegetable soup at a cost of about $1.30.

PXL_20221210_191511270

Eat broccoli sprouts to epigenetically regulate histones

Five papers on beneficial effects from sulforaphane inhibiting histone deacetylases (HDACs), starting with a 2022 rodent cell study:

“Sulforaphane (SFN) has tissue specificity for subtypes of HDACs that are downregulated. For example:

  • In breast cancer cells, HDAC1-3 are inhibited by SFN to induce cell apoptosis;
  • In skin cells, HDAC1-4 are regulated by SFN [anti-skin cancer]; and
  • In the cochlea, SFN inhibits HDAC2, 4, and 5 [attenuates hearing loss].

In the present study, SFN significantly inhibited HDAC2, 3, and 5 expression and HDACs activity in cardiomyocytes, thereby increasing H3 acetylation levels in the Nrf2 promoter and upregulating Nrf2 expression. Mechanism by which SFN prevents Ang II-induced cardiomyocyte apoptosis:

  • Ang II activates oxidative stress by increasing ROS leading to inflammation, oxidative stress and fibrosis in cardiomyocytes.
  • SFN prevents Ang II-induced cardiomyocyte apoptosis by inhibiting HDACs to activate Nrf2 and downstream antioxidant genes.

aging-14-204247-g007

SFN activates Nrf2 by inhibiting HDACs expression and activation.”

https://www.aging-us.com/article/204247/text “Sulforaphane inhibits angiotensin II-induced cardiomyocyte apoptosis by acetylation modification of Nrf2”


A 2021 rodent study found:

“SFN significantly attenuated diabetes-induced renal fibrosis in vivo. SFN inhibited diabetes-induced increase in HDAC2 activity.

Bone morphologic protein 7 (BMP-7) has been shown to reduce renal fibrosis induced by transforming growth factor-beta1. SFN protects against diabetes-induced renal fibrosis through epigenetic up-regulation of BMP-7.”

dmj-2020-0168f7

https://e-dmj.org/journal/view.php?doi=10.4093/dmj.2020.0168 “Sulforaphane Ameliorates Diabetes-Induced Renal Fibrosis through Epigenetic Up-Regulation of BMP-7”


A 2019 human osteosarcoma cell study found:

“SFN inhibits mTOR in a concentration- and time-dependent manner. This inhibition occurs in the presence or in the absence of NRF2.

SFN inhibits HDAC6 and decreases catalytic activity of AKT, which partially explains the mechanism by which SFN inhibits mTOR.”

https://www.sciencedirect.com/science/article/pii/S0944711319302284 “The isothiocyanate sulforaphane inhibits mTOR in an NRF2-independent manner”


A 2022 review cited a 2018 cell study:

“HDAC expression and activity are dysregulated in various diseases including asthma, chronic obstructive pulmonary disease, cancer, cardiac hypertrophy, and neurodegenerative and psychological disorders. HDAC inhibitors could be a potential therapeutic target for many diseases.

In hypertension, aortic stiffness is usually increased and vascular smooth muscle cells (VSMCs) contribute to vascular stiffness. We used VSMCs to test the degree of acetylation of histones in this study.

Sulforaphane weakly inhibited HDAC2 and effectively inhibited HDAC9.”

https://www.sciencedirect.com/science/article/pii/S0006295222002052 “Zinc-dependent histone deacetylases: Potential therapeutic targets for arterial hypertension”

https://www.sciencedirect.com/science/article/abs/pii/S0753332217364636 “Inhibition of class IIa histone deacetylase activity by gallic acid, sulforaphane, TMP269, and panobinostat” (not freely available)


PXL_20221125_211516042

What do we know about human aging from mouse models?

Here is a 2021 rodent study and relevant parts from 3 of its 26 citing papers:

“A long line of evidence has established the laboratory mouse as the prime model of human aging. However, relatively little is known about detailed behavioral and functional changes that occur across their lifespan, and how this maps onto the phenotype of human aging.

To better understand age-related changes across the lifespan, we characterized functional aging in male C57BL/6J mice of five different ages (3, 6, 12, 18, and 22 months of age) using a multi-domain behavioral test battery. Assessment of functional aging in humans and mice: age-related patterns were determined based on representative data (Table 2), and then superimposed onto survival rate. (A) Body weight, (B) locomotor activity, (C) gait velocity, (D) grip strength, (E) trait anxiety, (F) memory requiring low attention level, and (G) memory requiring high attention level.

fnagi-13-697621-g012

These functional alterations across ages are non-linear, and patterns are unique for each behavioral trait. Physical function progressively declines, starting as early as 6 months of age in mice, while cognitive function begins to decline later, with considerable impairment present at 22 months of age.

Functional aging of male C57BL/6J mice starts at younger relative ages compared to when it starts in humans. Our study suggests that human-equivalent ages of mice might be better determined on the basis of its functional capabilities.”

https://www.frontiersin.org/articles/10.3389/fnagi.2021.697621/full “Functional Aging in Male C57BL/6J Mice Across the Life-Span: A Systematic Behavioral Analysis of Motor, Emotional, and Memory Function to Define an Aging Phenotype”


“Studies in mice show that physical function (i.e., locomotor activity, gait velocity, grip strength) begins to deteriorate around post-natal day (PND) 180, but cognitive functions (i.e., memory) do not exhibit impairment until roughly PND 660. Our results should be considered within the context of behavior changing throughout vole adulthood. Caution should be taken to avoid categorizing the oldest age group in our study as ‘elderly’ or ‘geriatric.'”

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0276897 “Behavioral trajectories of aging prairie voles (Microtus ochrogaster): Adapting behavior to social context wanes with advanced age”


“We used adult mice ranging in age from 5-6 months, not enough to modify experimental autoimmune encephalomyelitis progression. Mice are considered adult after 8 weeks; however, rapid growth for most biological processes is observed until 3 months of age, while past 6 months, mice might be affected by senescence.”

https://www.frontiersin.org/articles/10.3389/fimmu.2022.1036680/full “Age related immune modulation of experimental autoimmune encephalomyelitis in PINK1 knockout mice”


“Locomotor activity and gait velocity of 12 months old male C57BL/6 correlates with an elderly human being aged 60 or older, supporting that the ~15 months old mice we used in our study were aged mice at the time of tissue collection.”

https://www.mdpi.com/1422-0067/23/20/12461 “Genomic Basis for Individual Differences in Susceptibility to the Neurotoxic Effects of Diesel Exhaust”


PXL_20221122_200643133

Broccoli sprouts activate the AMPK pathway, Part 4

Today someone viewed the 2020 Part 3 of Broccoli sprouts activate the AMPK pathway which lacked citations at the time. Checking again, here are three citing 2022 papers, starting with a review:

“Nrf2 is an important transcription factor that regulates expression of a large number of genes in healthy and disease states. Nrf2 regulates expression of several key components of oxidative stress, mitochondrial biogenesis, mitophagy, autophagy, and mitochondrial function in all organs of the human body, and in the peripheral and central nervous systems.

Overall, therapeutic drugs including sulforaphane that target Nrf2 expression and Nrf2/ARE pathway are promising. This article proposes additional research in Nrf2’s role within Parkinson’s disease, Huntington’s disease, and ischemic stroke in preclinical mouse models and humans with age-related neurodegenerative diseases.”

https://www.sciencedirect.com/science/article/pii/S1568163722001982 “Role of Nrf2 in aging, Alzheimer’s and other neurodegenerative diseases” (not freely available) Thanks to Dr. P. Hemachandra Reddy for providing a copy.


One of the Part 3 study’s coauthors contributed to this very detailed review:

“Due to observed overlapping cellular responses upon AMPK or NRF2 activation and common stressors impinging on both AMPK and NRF2 signaling, it is plausible to assume that AMPK and NRF2 signaling may interdepend and cooperate to readjust cellular homeostasis.

1-s2.0-S089158492200497X-gr3_lrg

The outcome and underlying signaling events of AMPK-NRF2 crosstalk may diverge between:

  1. in vitro and in vivo studies (one cell type in isolation vs inter-organ crosstalk in living organisms);
  2. Different cell types/organs/organisms of different cultivation conditions, genetic background, age or sex;
  3. Different stress-regimens (chronic vs acute, nature of stress (lipotoxicity, redox stress, xenobiotic, starvation, etc));
  4. Different modes of Nrf2 or AMPK activation and inhibition (genetic vs pharmacological, constitutive vs transient/intermittent, systemic vs organ-specific, electrophilic vs PPI, allosteric vs covalent, or pan vs subtype-specific);
  5. Different target genes with distinct promoter and enhancer structure; or
  6. Different timing of activation.

The latter should deserve increased attention as Nrf2 is one of the most cycling genes under control of the circadian clock. Feeding behavior, metabolism and hence AMPK activity follow and substantiate the biological clock, indicating an entangled circadian regulation of metabolic and redox homeostasis.”

https://www.sciencedirect.com/science/article/pii/S089158492200497X “AMPK and NRF2: Interactive players in the same team for cellular homeostasis?”


A third citing paper was a study of lens cells that provided an example of similar metformin effects noted in Part 2 of Broccoli sprouts activate the AMPK pathway:

“Loss of Nrf2 and Nrf2 antioxidant genes expression and activity in aging cells leads to an array of oxidative-induced deleterious responses, impaired function, and aging pathologies. This deterioration is proposed to be the primary risk factor for age-related diseases such as cataracts.

AMPK regulates energy at physiological levels during metabolic imbalance and stress. AMPK is a redox sensing molecule, and can be activated under cellular accumulation of reactive oxygen species, which are endogenously produced due to loss of antioxidant enzymes.

The therapeutic potential of AMPK activation has context-dependent beneficial effects, from cell survival to cell death. AMPK activation was a requisite for Bmal1/Nrf2-antioxidants-mediated defense, as pharmacologically inactivating AMPK impeded metformin’s effect.

Using lens epithelial cell lines (LECs) of human or mouse aging primary LECs along with lenses as model systems, we demonstrated that metformin could correct deteriorated Bmal1/Nrf2/ARE pathway by reviving AMPK-activation and transcriptional activities of Bmal1/Nrf2, resulting in increased antioxidants enzymatic activity and expression of Phase II enzymes. Results uncovered crosstalk between AMPK and Bmal1/Nrf2/antioxidants mediated by metformin for blunting oxidative/aging-linked pathobiology.”

https://www.mdpi.com/2073-4409/11/19/3021/htm “Obligatory Role of AMPK Activation and Antioxidant Defense Pathway in the Regulatory Effects of Metformin on Cellular Protection and Prevention of Lens Opacity”


PXL_20221027_185754842

All about walnuts’ effects

Five 2022 papers focusing on walnuts, starting with a comparison of eight tree nuts:

“The aim of the present study was to examine 8 different popular nuts – pecan, pine, hazelnuts, pistachio, almonds, cashew, walnuts, and macadamia. Total content of phenolic compounds in nuts ranged from 5.9 (pistachio) to 432.9 (walnuts) mg/100 g.

Walnuts had the highest content of polymeric procyanidins, which are of great interest as important compounds in nutrition and biological activity, as they exhibit antioxidant, anti-inflammatory, antimicrobial, cardio- and neuroprotective action. Walnuts are good sources of fatty acids, especially omega-3 and omega-6.”

https://www.sciencedirect.com/science/article/pii/S2590157522002164 “Nuts as functional foods: Variation of nutritional and phytochemical profiles and their in vitro bioactive properties”


A second study compared the same eight tree nuts plus Brazil nuts and peanuts:

“The highest total content of all analyzed flavonoids was determined in walnuts (114.861 µg/g) with epicatechin the most abundant, while the lowest was in almonds (1.717 µg/g). Epicatechin has antioxidant, anti-inflammatory, antitumor, and anti-diabetic properties. Epicatechin has beneficial effects on the nervous system, enhances muscle performance, and improves cardiac function.”

https://www.mdpi.com/1420-3049/27/14/4326/htm “The Content of Phenolic Compounds and Mineral Elements in Edible Nuts”


Next, two systematic reviews and meta-analyses of human studies:

“We carried out a systematic review of cohort studies and randomized controlled trials (RCTs) investigating walnut consumption, compared with no or lower walnut consumption, including those with subjects from within the general population and those with existing health conditions, published from 2017 to 5 May 2021.

  • Evidence published since 2017 is consistent with previous research suggesting that walnut consumption improves lipid profiles and is associated with reduced CVD risk.
  • Evidence pointing to effects on blood pressure, inflammation, hemostatic markers, and glucose metabolism remains conflicting.
  • Evidence from human studies showing that walnut consumption may benefit cognitive health, which is needed to corroborate findings from animal studies, is now beginning to accumulate.”

https://academic.oup.com/nutritionreviews/advance-article/doi/10.1093/nutrit/nuac040/6651942 “Walnut consumption and health outcomes with public health relevance – a systematic review of cohort studies and randomized controlled trials published from 2017 to present”


“We aimed to perform a systematic review and meta-analysis of RCTs to thoroughly assess data concerning effects of walnut intake on selected markers of inflammation and metabolic syndrome in mature adults. Our findings showed that:

  • Walnut-enriched diets significantly decreased TG, TC, and LDL-C concentrations, while HDL-C levels were not significantly affected.
  • No significant changes were noticed on anthropometric, cardiometabolic, and glycemic indices after higher walnut consumption.
  • Inflammatory biomarkers did not record statistically significant results.”

https://www.mdpi.com/2076-3921/11/7/1412/htm “Walnut Intake Interventions Targeting Biomarkers of Metabolic Syndrome and Inflammation in Middle-Aged and Older Adults: A Systematic Review and Meta-Analysis of Randomized Controlled Trials”


Finishing with a rodent study that gave subjects diabetes with a high-fat diet, then mixed two concentrations of walnut extract in with the treatment groups’ chow:

“This study was conducted to evaluate the protective effect of Gimcheon 1ho cultivar walnut (GC) on cerebral disorder by insulin resistance, oxidative stress, and inflammation in HFD-induced diabetic disorder mice. After HFD feed was supplied for 12 weeks, samples were orally ingested for 4 weeks to GC20 and GC50 groups (20 and 50 mg/kg of body weight, respectively).

  • Administration of GC improved mitochondrial membrane potential function, and suppressed oxidative stress in the brain.
  • GC inhibited hepatic and cerebral lipid peroxidation and the formation of serum AGEs, and increased serum antioxidant activity to improve HFD-induced oxidative stress.
  • The HFD group showed significant memory impairment in behavioral tests. On the other hand, administration of GC showed improvement in spatial learning and memory function.

walnut brain effects

Based on these physiological activities, GC showed protective effects against HFD-induced diabetic dysfunctions through complex and diverse pathways.”

https://www.mdpi.com/1420-3049/27/16/5316/htm “Walnut Prevents Cognitive Impairment by Regulating the Synaptic and Mitochondrial Dysfunction via JNK Signaling and Apoptosis Pathway in High-Fat Diet-Induced C57BL/6 Mice”


How do you like my sand art?PXL_20221016_154923750

Measuring epigenetic DNA causes

This 2022 human cell study investigated DNA methylation and aging:

“Models based on DNA methylation can be used to predict the age of biological samples, but their interpretability is limited due to the lack of causal inferences. Neither existing epigenetic clocks nor DNA methylation changes are enriched in causal CpG sites. Causal CpGs include similar numbers of sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits.

One general approach for developing anti-aging interventions is to identify molecular changes during aging and use these changes as targets to modulate the aging process. A similar idea has also been applied to evaluate potential longevity interventions. However, this logic is intrinsically flawed, as correlation does not imply causation, and age-related changes are not necessarily causal to age-associated declines.

We developed a framework for integrating causal knowledge into epigenetic clock models and constructed DamAge and AdaptAge that measure age-related damaging and adaptive changes, respectively. DamAge acceleration is associated with various adverse conditions (e.g., mortality risk), whereas AdaptAge acceleration is related to beneficial adaptations.

causality clocks

We found that transcription factor (TF)-binding sites of BRD4 and CREB1 are enriched with CpG sites whose methylation levels promote healthy longevity, and TF-binding sites for HDAC1 are enriched with CpG sites whose methylation levels decrease healthy longevity.

  • BRD4 contributes to cell senescence and promotes inflammation, and higher DNA methylation at BRD4 binding sites may inhibit the downstream effects of BRD4 and promote healthy longevity.
  • CREB1 is related to type II diabetes and neurodegeneration, and mediates the effect of calorie restriction. Our data suggest that higher methylation at CREB1-binding sites may support its longevity effects.
  • HDAC1 is a histone deacetylase, and its activity increases with aging and may promote age-related phenotypes. Increased DNA methylation at HDAC1 binding sites may causally inhibit healthy longevity.

Our causality-informed clock models provide novel insights into the aging mechanisms and testing interventions that delay aging and reverse biological age.”

https://www.biorxiv.org/content/10.1101/2022.10.07.511382v1 “Causal Epigenetic Age Uncouples Damage and Adaptation”


PXL_20221001_224441637_exported_1932

Minds of their own

It’s the weekend, so it’s time for: Running errands? Watching sports? Other conditioned behavior?

Or maybe broadening our cognitive ability with Dr. Michael Levin’s follow-ups to his 2021 Basal cognition paper and 2020 Electroceuticals presentation with a 2022 paper and presentation starting around the 13:30 mark:

Michael Levin - Cell Intelligence in Physiological and Morphological Spaces

“A homeostatic feedback is usually thought of as a single variable such as temperature or pH. The set point has been found to be a large-scale geometry, a descriptor of a complex data structure.”


His 2022 paper Technological Approach to Mind Everywhere: An Experimentally-Grounded Framework for Understanding Diverse Bodies and Minds:

“It is proposed that the traditional problem-solving behavior we see in standard animals in 3D space is just a variant of evolutionarily more ancient capacity to solve problems in metabolic, physiological, transcriptional, and morphogenetic spaces (as one possible sequential timeline along which evolution pivoted some of the same strategies to solve problems in new spaces).

Developmental bioelectricity works alongside other modalities such as gene-regulatory networks, biomechanics, and biochemical systems. Developmental bioelectricity provides a bridge between the early problem-solving of body anatomy and the more recent complexity of behavioral sophistication via brains.

This unification of two disciplines suggests a number of hypotheses about the evolutionary path that pivoted morphogenetic control mechanisms into cognitive capacities of behavior, and sheds light on how Selves arise and expand.

While being very careful with powerful advances, it must also be kept in mind that existing balance was not achieved by optimizing happiness or any other quality commensurate with modern values. It is the result of dynamical systems properties shaped by meanderings of the evolutionary process and the harsh process of selection for survival capacity.”


PXL_20220904_102050409

An inflammation clock

Here are six 2022 papers that either cited the second study of Variable aging measurements, or provided further evidence for its findings. Let’s start with a citing study:

“This study aimed to investigate expression patterns and prognostic values of the inflammatory aging clock (iAge) in glioblastoma (GBM), and its relations with stem cells. Similar to epigenetic clocks and transcriptomic clocks, iAge could track multifaceted aging phenotypes and have clinical significance in translation medicine.

iAge was positively correlated with chronological age, and highly associated with immune cells and inflammatory activities. iAge could serve as a prognostic biomarker for overall survival, and could precisely predict GBM stem cells stemness.

We identified the physiological importance and function of iAge in GBM, and provided novel insights into how iAge is a critical event for development of GBM.”

https://www.frontiersin.org/articles/10.3389/fgene.2022.925469/full “Inflammatory aging clock: A cancer clock to characterize the patients’ subtypes and predict the overall survival in glioblastoma”


Beginning with a human osteoporosis study, five papers investigated cytokine CXCL9, which the iAge study found to be “clearly actionable as shown by our experiments in CXCL9 where we can reverse aging phenotypes.”

“We assessed whether levels of CXCL9 and CXCL10 were elevated in human serum samples of older adults who had incident hip fractures. Our findings revealed higher serum levels of CXCL9 in pre-fracture blood samples of men with subsequent hip fractures, compared with their non-fracture controls. There was no such difference in CXCL9 serum levels between cases and controls in women.

Serum CXCL9 improved the prediction of osteoporotic hip fracture in men. The association between CXCL10 and hip fracture risk was not statistically significant in either sex.

While our epidemiologic findings are supported by experimental data providing the mechanistic pathway for CXCL9 in regulating osteoclast recruitment, further studies are needed to confirm validity of our findings and determine their generalizability to other study populations. Underlying biological mechanisms that limit our findings to men but not women require further investigation.”

https://asbmr.onlinelibrary.wiley.com/doi/10.1002/jbmr.4646 “CXCL9 Predicts the Risk of Osteoporotic Hip Fracture in a Prospective Cohort of Chinese Men—A Matched Case–Control Study”


Two immune-mediated skin diseases, with a vitiligo review:

“Current findings emphasize the critical role of immune cells and their mediators in the immunopathogenesis of vitiligo. IFN-γ [interferon gamma] is the primary cytokine mediator that activates the JAK/STAT pathway, causing keratinocytes to produce the key chemokines CXCL9 and CXCL10.

Interactions between immune and non-immune cells finally result in apoptosis of melanocytes. Additional investigations of these pathways may provide an opportunity for finding possible therapeutic targets, as there are currently no targeted biological drugs available for treatment of vitiligo.”

https://www.mdpi.com/2227-9059/10/7/1639/htm “Current Concepts of Vitiligo Immunopathogenesis”

and a study of psoriasis:

“CXCL9 is an important chemokine involved in T cell recruitment, and is up-regulated in plasma of patients with psoriasis. Increased CXCL9 expression can aggravate the progression of psoriasis.

cxcl9 expression

IL-1β and CXCL9 were up-regulated and CLDN8 was down-regulated in psoriasis with statistically significant differences. Identification of potential key molecular markers and signaling pathways provides potential research directions for further understanding molecular mechanisms of psoriasis.”

https://www.wjgnet.com/2307-8960/full/v10/i18/5965.htm “Identification of potential key molecules and signaling pathways for psoriasis based on weighted gene co-expression network analysis”


Two lung-related studies, first, an editorial for a human lung transplant study that isn’t freely available:

“CXCL9 and CXCL10 are chemokines that bind to the shared receptor CXCR3, potentiating T cells, mononuclear cells, and natural killer (NK) cells. Previous studies demonstrated that presence of these chemokines in bronchoalveolar lavage samples preceded development of chronic lung allograft dysfunction (CLAD).

Acute rejection and acute lung injury are known risk factors to the development of CLAD, yet this study found that increased risk was dependent on the presence of CXCL9/CXCL10 plasma elevation. Early identification of patients at risk, possibly during the active inflammatory phase, rather than once abnormal wound healing pathways dominate resulting in irreversible injury, provides an attractive opportunity for intervention.”

https://onlinelibrary.wiley.com/doi/10.1111/ajt.17135 “CXCL9 and CXCL10 plasma levels: Potential keys to unlocking CLAD risk”

and a study of smoking effects:

“We collected blood samples from 78 healthy male volunteers aged 18–60, including non-smokers (n = 30), current smokers (n = 30), and ex-smokers (n = 18). Expression levels of CXCL9/MIG [monokine induced by IFN-γ] and sIL-6R significantly increased after smoking, and continued to increase after quitting smoking.

cxcl9 smoking

Changes in related cytokines after smoking cessation are mainly restorative, while some cytokines further strengthen the trend of smoking-related changes.”

https://www.mdpi.com/1420-3049/27/12/3715/htm “Effects of Smoking on Inflammatory-Related Cytokine Levels in Human Serum”


PXL_20220904_100859449.NIGHT

Epigenetic effects of plasma concentrate

“We use data from a safety study (n = 18, mean age 74) to investigate whether human umbilical cord plasma concentrate (hereinafter Plasma Concentrate) injected weekly (1 ml intramuscular) into elderly human subjects over a 10-week period affects different biomarkers, including epigenetic age measures, standard clinical biomarkers of organ dysfunction, mitochondrial DNA copy number (mtDNA-CN), and leukocyte telomere length.

More than 20 clinical biomarkers were significantly and beneficially altered. Telomere length and mtDNA-CN were not significantly affected by treatment.

An increase in entropy means that the methylome becomes noisier. We found that entropy was significantly decreased after treatment. Decreased entropy may implicate rejuvenation of the epigenetic landscape after plasma concentrate treatments.

changes in methylation entropy

Treatment reduced DNA methylation-based GrimAge by an average of 0.82 years, suggesting a reduction in morbidity and mortality risk. By contrast, no significant results could be observed for epigenetic clocks that estimate chronological age.

Our study lends credence to the notion that there are youth-promoting factors in the secretome of umbilical cord plasma. This conclusion has also been reached by other researchers that have provided treatment with stem cells, which do not work by plasma dilution but primarily by providing humoral factors and changing the microenvironment of cells and tissues. While there may be youth-promoting microvesicles or humoral factors that are at work, we do not want to rule out the possibility that it is ‘young and undamaged’ albumin that leads to the improvements noted, especially in light of recent evidence for such a mechanism.

This first human epigenetic clock study of plasma concentrate treatments revealed age-reversal effects according to a well-established DNA methylation-based estimator of morbidity and mortality risk. Future placebo-controlled replication studies are warranted with a larger number of participants over a longer study period, which our laboratory has undertaken to pursue.”

https://onlinelibrary.wiley.com/doi/10.1111/acel.13696 “Umbilical cord plasma concentrate has beneficial effects on DNA methylation GrimAge and human clinical biomarkers”


PXL_20220903_101625819

Non-patentable boron benefits

To follow up Is boron important to health? I’ll highlight a 2022 review of boron intake:

“Boron is essential for activity of several metabolic enzymes, hormones, and micronutrients. It is important for growth and maintenance of bone, reduction in inflammatory biomarkers, and increasing levels of antioxidant enzymes.

The average person’s daily diet contains 1.5 to 3 milligrams of boron. Boron intakes of 1–3 mg/day have been shown to improve bone and brain health in adults when compared to intakes of 0.25–0.50 mg/day.

One week of 10 mg/d boron supplementation resulted in a 20% reduction in inflammatory biomarkers TNF-α, as well as significant reductions (nearly 50%) in plasma concentrations of hs-CRP and IL-6. Calcium fructoborate, a naturally occurring, plant-based boron-carbohydrate complex, had beneficial effects on osteoarthritis (OA) symptoms. A double-blind study in middle-aged patients with primary OA found that all groups except the placebo group saw a reduction in inflammatory biomarkers after 15 days of food supplementation with calcium fructoborate.

Dietary boron intake significantly improves brain function and cognitive functioning in humans. Electroencephalograms showed that boron pharmacological intervention after boron deficiency improved functioning in older men and women, such as less drowsiness and mental alertness, better psychomotor skills (for example, motor speed and dexterity), and better cognitive processing (e.g., attention and short-term memory). Boron compounds can help with both impaired recognition and spatial memory problems.

We discussed the role of boron-based diet in memory, boron and microbiome relation, boron as anti-inflammatory agents, and boron in neurodegenerative diseases. Boron reagents will play a significant role to improve dysbiosis.”

https://www.mdpi.com/1420-3049/27/11/3402/htm “The Role of Microbiome in Brain Development and Neurodegenerative Diseases”


PXL_20220814_101418757

If you lose mobility, you lose cognitive function

This 2022 human study used four epigenetic clocks to assess aging:

“This cohort study was a secondary analysis of 3 Women’s Health Initiative (WHI) ancillary studies among 1813 women eligible to survive to age 90 years by end of study period. The study found that increased epigenetic age acceleration (EAA) as measured by 4 epigenetic clocks was associated with lower odds of survival to age 90 years with intact mobility; results were similar when including intact cognitive functioning.

This study benefited from a large, racially and ethnically diverse sample of women who were followed up to at least age 90 years with detailed longitudinal data on a host of lifestyle and health history factors. This study is generalizable to WHI women owing to use of IPW weights, and may be generalizable to a large range of women in the United States.

zoi220662t1_1658260078.05222

Among 1813 women, there were:

  • 464 women who survived to age 90 years with intact mobility and cognitive functioning;
  • 420 women who survived to age 90 years without intact mobility and cognitive functioning; and
  • 929 women who did not survive to age 90 years.

Only 29 women were reclassified from the healthy longevity group to surviving to age 90 years without intact mobility and cognitive functioning. Although it was of great interest to investigate the association between EAA and survival to age 90 years with intact cognitive function independently, this study population did not have sufficient numbers of women who experienced loss of cognitive function (without loss of mobility) to do so.”

https://jamanetwork.com/journals/jamanetworkopen/fullarticle/2794706 “Analysis of Epigenetic Age Acceleration and Healthy Longevity Among Older US Women”


Early humans who lost mobility in our African savanna ancestral environment during the Pleistocene Epoch (approximately 2.6M to 12K years ago) were prey. I highly doubt that immobile individuals successfully became our ancestors.

I downgraded this study because these researchers misguidedly soiled worthwhile findings with BMI and education level non-causal associations. They intentionally did this, as several of them were coauthors of the execrable Epigenome-wide meta-analysis of BMI in nine cohorts: examining the utility of epigenetic BMI in predicting metabolic health.

See Findings, or fun with numbers? and Does a societal mandate cause DNA methylation? for opposing research.


PXL_20220813_102515183

Broccoli sprouts and your brain

A 2022 review of Nrf2 signaling hilariously avoided mentioning sulforaphane, although of ~4,000 sulforaphane published articles, two were cited. I’ll curate it anyway to highlight referenced brain effects.

“A good stability of NRF2 activity is crucial to maintain redox balance and therefore brain homeostasis. In this review, we have gathered recent data about the contribution of the NRF2 pathway in the healthy brain as well as during metabolic diseases, ageing, and ageing-related neurodegenerative diseases.

A functional NRF2 system is important to regulate both neuroinflammation, i.e., activation of microglia and astrocytes, and oxidative stress in the brain. NRF2 and NF-κB transcription factors regulate cellular responses to inflammation and oxidative stress in order to maintain brain homeostasis. Both pathways have been described to inhibit each other.

Nrf2 brain aging

Future challenges will be to establish novel therapies to:

  • Increase NRF2 activation in specific cell types and/or brain regions; and
  • Modulate NRF2 pathway in senescent cells.

Modulation of NRF2 signalling pathway by using specific food products [like unmentioned broccoli sprouts] and phytochemicals [like unmentioned sulforaphane], dietary supplements [like unmentioned Vitamin D3], drugs, and epigenetic modifiers, alone or in combination, will help to limit inflammatory diseases, ageing process, and subsequently ageing-related diseases.”

https://www.mdpi.com/2076-3921/11/8/1426/htm “Normal and Pathological NRF2 Signalling in the Central Nervous System”


PXL_20220808_095334058

The goddess of rainbows

Two 2022 papers, starting with a review of irisin:

“This article is an overview of irisin generation, secretion, and tissue distribution. Its targeting of tissues or organs for prevention and treatment of chronic diseases is systematically summarized, with discussion of underlying molecular mechanisms.

Irisin is an exercise-induced myokine expressed as a bioactive peptide in multiple tissues and organs. Exercise and cold exposure are major inducers for its secretion.

Mechanistic studies confirm that irisin is closely correlated with lipid metabolism, insulin resistance, inflammation, ROS, endocrine, neurotrophic factors, cell regeneration and repairing, and central nervous system regulation. Irisin decreases with age, and is closely associated with a wide range of aging-related diseases.

A number of studies in elderly humans and animal models have shown that exercise can promote the body’s circulation and increase irisin levels in some tissues and organs. Resistance, aerobic, or combined exercise seem to play a positive role. However, exercise could not change serum irisin in some reported studies.

irisin human studies

There are large individual differences in exercise training in the elderly population. Since the half-life of irisin in the body is less than 1 h, it is necessary to pay attention to the time of blood sampling after a single exercise intervention. Some factors that impede detection of irisin levels in vivo include the half-life of irisin protein, sampling time, different tissues, and different health statuses before and after intervention.

It is worth noting that high-intensity exercise shows higher irisin levels even with the same energy expenditure during exercise. Precision studies of irisin in elderly subjects following exercise intervention need to be further clarified.”

https://www.sciencedirect.com/science/article/pii/S1568163722001222 “Irisin, An Exercise-induced Bioactive Peptide Beneficial for Health Promotion During Aging Process” (not freely available) Thanks to Dr. Ning Chen for providing a copy.


A second paper was a human study too recent to be cited by the first paper. I’ll highlight its irisin findings:

“We investigated the complex relationship among DNAm based biomarkers of aging, including DNAmFitAge, a variety of physiological functioning variables, blood serum measures including cholesterol, irisin level, and redox balance, and the microbiome on 303 healthy individuals aged between 33 and 88 years with a diverse level of physical fitness. Regular exercise was associated with younger biological age, better memory, and more protective blood serum levels.

Our research intends to show that regular physical exercise is related to microbiota and methylation differences which are both beneficial to aging and measurable. Our research provides the first investigation between microbiome derived metabolic pathways and DNAm based aging biomarkers.

Irisin levels decrease with age (0.23 average decrease for every 1 year older). We found age-related decreases in irisin levels were attenuated by exercise training. The link between irisin to GrimAge Acceleration and FitAge Acceleration is a novel observation.

HDL is positively associated with irisin. HDL and irisin have complex roles in physiology, and the positive relationship we observe between physical exercise and HDL and irisin align with protective effects seen between HDL and irisin with glucose homeostasis.

This work further supports the biological importance of irisin to the aging process. It is possible our research motivates interventions to boost irisin, like through physical exercise, as possible anti-aging therapies.”

https://www.medrxiv.org/content/10.1101/2022.07.22.22277842v1 “DNA methylation clock DNAmFitAge shows regular exercise is associated with slower aging and systemic adaptation


PXL_20220725_095201761

Variable aging measurements

Two papers on aging measurements, starting with a 2022 human study:

“We collected longitudinally across the adult age range a comprehensive list of phenotypes within four domains (body composition, energetics, homeostatic mechanisms and neurodegeneration / neuroplasticity) and functional outcomes. We integrated individual deviations from population trajectories into a global longitudinal phenotypic metric of aging.

blsa participant ages

We demonstrate that accelerated longitudinal phenotypic aging is associated with faster physical and cognitive decline, faster accumulation of multimorbidity, and shorter survival.”

https://www.nature.com/articles/s43587-022-00243-7 “Longitudinal phenotypic aging metrics in the Baltimore Longitudinal Study of Aging”


I disagree with this study’s methodology.

1. Although it acknowledged individual variability, nothing was done to positively adjust to those facts. What could have been done per A review of biological variability was:

“Obtain a measurement of variability that is independent of the mean to ensure to not confound changes in variability with shifts in mean.”

2. A usual research practice is to take at least three measurements, and use their average as representative. That wasn’t done here, maybe because of time and expense considerations?

3. An important measurement for physical function was the time to finish a 400 meter walk. I walk more than ten times that almost every day. I use the first 400 meters as a warmup period while getting to the beach to walk eastward and enjoy the predawn light and water animal activity. I concentrate on gait speed during the last third while walking westward on a straightaway bike path.

This study would measure my gait speed as a sometimes old person during the first 400 meters, rather than a gait speed that usually approaches a young person’s during the last 400 meters. Even if I tried to walk my fastest right out of the gate, I wouldn’t be surprised to find a decade or two difference by this study’s measurements between a morning walk’s first and last 400 meter gait speeds.

4. An important cognitive function measurement was the Digital Symbol Substitution Test, apparently taken during subjects’ fasted state? Sometimes after exercising, I’m okay cognitively when starting work in a fasted state at 6:30 a.m., and other times I’m tired.

Two days ago during the last hour of work 1:30-2:30 p.m., I did outstanding work, four hours after eating whole oats for breakfast, and after drinking two coffees and three teas. I took time to put together pieces of puzzles into proper contexts for management’s attention. My bosses weren’t too pleased with the story it told, but it is what it is.

5. Are measurements of how you start what matters? Or is it how you finish, as is common in competitive sports?

This study would measure my cognitive function as a sometimes old person, rather than performance that approaches a young person’s later in the workday. For both physical and cognitive function, my abilities to ramp up and come close to young people’s capabilities are features that I work on, not random, inconvenient measurement variability.

6. Blood measurements were downgraded as having “limited coverage of the four phenotypic domains.” These were taken to fit into specific paradigms and epigenetic clocks. They predictably failed to show causality, as acknowledged with:

“Our analysis showed strong associations between global longitudinal phenotypic score and changes in physical and cognitive function. We did not have sufficient observations to fully separate these two dimensions over time, which would have strengthened the assumption of causality.”

Nowhere in this study was it hinted that all measurements were downstream effects of unmeasured causes. A follow-on study could reanalyze these subjects’ blood samples, MRI, and other measurements for originating upstream factors of signaling pathways and cascades per Signaling pathways and aging and An environmental signaling paradigm of aging.


Reference 35 of this first study was a 2021 human and rodent study that was tossed in as a limitation with:

“We might not have all of the relevant phenotypic measures (for example, more detailed immune profiles) for all participants.”

Its findings included:

“From the blood immunome of 1,001 individuals aged 8–96 years, we developed a deep-learning method based on patterns of systemic age-related inflammation. The resulting inflammatory clock of aging (iAge) tracked with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians.

Canonical markers of acute infection such as IL-6 and tumor necrosis factor-α were not major contributors to iAge, indicating that, except for IL-1β, infection-driven inflammatory markers of the acute inflammatory response do not contribute to age-related chronic inflammation.

We conducted a follow-up study in an independent cohort of 97 extremely healthy adults (aged 25–90 years) matched for cardiovascular risk factors (including conserved levels of high-sensitivity C-reactive protein), selected from a total of 151 recruited participants using strict selection criteria. In this healthy cohort, inflammation markers were measured using a 48-plex cytokine panel. Only 6 circulating immune proteins were significantly correlated with age, with CXCL9 again the largest contributor to age-related inflammation.

CXCL9 is a T-cell chemoattractant induced by IFN-γ and is mostly produced by neutrophils, macrophages and endothelial cells (ECs). We find that CXCL9 is mainly produced by aged endothelium and predicts subclinical levels of cardiovascular aging in nominally healthy individuals.

We did not find any significant correlation between known disease risk factors reported in the study (BMI, smoking, dyslipidemia) and levels of CXCL9 gene or protein expression. We hypothesize that one root cause of CXCL9 overproduction is cellular aging per se, which can trigger metabolic dysfunction.

As ECs but not cardiomyocytes expressed the CXCL9 receptor, CXCR3, we hypothesize that this chemokine acts both in a paracrine fashion (when it is produced by macrophages to attract T cells to the site of injury) and in an autocrine fashion (when it is produced by the endothelium) creating a positive feedback loop. In this model, increasing doses of CXCL9 and expression of its receptor in these cells leads to cumulative deterioration of endothelial function in aging.

IFN-γ did not increase in expression in our cellular aging RNA-seq experiment, suggesting that there are triggers of CXCL9 (other than IFN-γ) that play a role in cellular senescence in the endothelium that are currently unknown. However, in our 1KIP study, IFN-γ was in fact the second-most important negative contributor to iAge, which could be explained by the cell-priming effect of cytokines, where the effect of a first cytokine alters the response to a different one.

iAge derived from immunological cytokines gives us an insight into the salient cytokines that are related to aging and disease. A notable difference compared to other clocks is that iAge is clearly actionable as shown by our experiments in CXCL9 where we can reverse aging phenotypes. More practical approaches range from altering a person’s exposomes (lifestyle) and/or the use of interventions to target CXCL9 and other biomarkers described here.

Our immune metric for human health can identify within healthy older adults with no clinical or laboratory evidence of cardiovascular disease, those at risk for early cardiovascular aging. We demonstrate that CXCL9 is a master regulator of vascular function and cellular senescence, which indicates that therapies targeting CXCL9 could be used to prevent age-related deterioration of the vascular system and other physiological systems as well.”

https://www.nature.com/articles/s43587-021-00082-y “An inflammatory aging clock (iAge) based on deep learning tracks multimorbidity, immunosenescence, frailty and cardiovascular aging”


PXL_20220721_093128925.NIGHT