Uses of the neutrophils-to-lymphocytes ratio

This 2021 review subject was recent history and uses of the neutrophils-to-lymphocytes ratio parameter to indicate systemic inflammation and stress:

“NLR is widely used across almost all medical disciplines as a reliable and easily available marker of immune response to various infectious and non-infectious stimuli. NLR reflects dynamic relationships between innate (neutrophils) and adaptive (lymphocytes) cellular immune response during illness and various pathological states.

A normal range of NLR is between 1–2, and values higher than 3.0 and below 0.7 in adults are pathological. NLR between 2.3–3.0 may serve as an early warning of cancer, atherosclerosis, infection, inflammation, psychiatric disorders, and stress.

nlr

Neutrophils play a pivotal role in innate immune response including phagocytosis, and release of a variety of cytokines and molecule mediators. Lymphocytopenia is a hallmark of stress while inflammation is due to demargination, redistribution, and accelerated apoptosis.

Opposite changes in neutrophil and lymphocyte counts are a multifactorial dynamic process depending on regulation of various immunologic, neuroendocrine, humoral, and biologic processes such as margination / demargination, mobilization / redistribution, accelerated / delayed apoptosis, influence of stress hormones, and sympathetic / parasympathetic nervous system imbalance. NLR is the best expression of a tight functional relation between two fundamental immunocompetent leukocyte populations.”

https://doi.org/10.4149/bll_2021_078 “Neutrophil-to-lymphocyte ratio, past, present and future perspectives”


The fourth study of Uses of the lymphocytes-to-monocytes ratio also commented on NLR.

Sunday morning stroll

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Is liver damage reversible?

This 2021 rodent study measured sequential liver changes caused by a high-fat diet:

“Using a longitudinal mouse study of diet-induced obesity in male mice, we investigated kinetics of hepatic DNA methylation and gene expression compared to those of obesity-induction to assess if they could be causal for development of insulin resistance. We aimed to find out if these changes were reversed by massive weight loss induced by vertical sleeve gastrectomy or metformin treatment.

We identified two CpG sites within exon 1 of Fgf21 that became gradually hypomethylated upon HFD feeding. DNA demethylation started between week two and four, to become significant at week five, and significantly correlated with hepatic Fgf21 gene expression.

These DNA methylation changes preceded development of insulin resistance, and were potentially causally involved in increased Fgf21 expression and plasma levels associated with insulin resistance. This points to a key regulatory function of gene body DNA methylation, which was eventually a compensatory response to counteract the developing insulin resistance.

HFD-induced decrease in Fgf21 DNA methylation could not be reversed by vertical sleeve gastrectomy or metformin treatment. As soon as weight loss slowed down or mice started to re-gain weight, differences in DNA methylation were no longer detected compared to sham-operated mice.

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As the altered DNA methylation pattern was acquired during adulthood in differentiated cells, our data emphasize that metabolic programming via DNA methylation is dynamic and not restricted to fetal development. This supports the concept that individuals can actively influence their DNA methylation patterns by lifestyle choices.

Our data indicate that DNA methylation alterations in key metabolic tissues can be acquired by an obesogenic diet, and not easily be reversed by interventions common in obese and diabetic subjects.”

https://www.sciencedirect.com/science/article/pii/S0955286321003272 “Dietary induction and reversal of obesity and insulin resistance is associated with changes in Fgf21 DNA methylation in liver of mice”


This study attempted two interventions that didn’t have desired effects. All about the betaine mentioned others that may reverse liver epigenetic changes.

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Eat broccoli sprouts for your liver

This 2021 rodent study investigated sulforaphane pretreatment’s role in reducing liver injuries:

“As a double blood supply organ of the portal vein and artery, the liver is highly sensitive to ischemia, and is one of the common organs to suffer from hepatic ischemia-reperfusion injury (HI/RI). HI/RI leads to overproduction of reactive oxygen species (ROS).

  • Overdoses of ROS promote reaction of lipid peroxidation and generation of the extremely aggressive oxidants nitric oxide (NO) and malondialdehyde (MDA).
  • HI/RI decreased antioxidant enzyme activity of glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD).
  • Sulforaphane (SFN) intervention significantly decreased levels of MDA and NO by increasing activity of GSH, CAT, and SOD.

Inflammation is the most serious secondary injury experienced in HI/RI.

  • Monocyte chemotactic protein 1 (MCP-1) is involved in an inflammatory reaction with regulation of monocytes, T lymphocytes, and NK cells. MCP-1 can also increase infiltration of inflammatory cells by activating NF-κB.
  • Tumor necrosis factor-α (TNF-α) is a promoter of the inflammatory response. Interleukin-6 (IL-6) is an inflammatory mediator in the acute reaction period.
  • SFN treatment significantly decreased HI/RI-induced expression of TNF-a, IL-6, and MCP-1.

sulforaphane and liver inflammation

In conclusion, SFN has a protective effect on HI/RI. The mechanism is associated with activating Nrf2/HO-1 signaling to suppress oxidative stress and inflammation.”

https://www.sciencedirect.com/science/article/abs/pii/S0966327421000794 “Sulforaphane alleviates hepatic ischemia–reperfusion injury through promoting the activation of Nrf-2/HO-1 signaling” (not freely available)


A human equivalent of this study’s 5 mg / kg sulforaphane dose was (.161 x 5 mg) x 70 kg ≈ 56 mg. For comparison, my estimated daily sulforaphane intake from microwaved sprouts is 52 mg.

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Sprout bioaccessibility

Twin 2021 in vitro studies of cruciferous sprout bioaccessibility, with the first addressing hydroxycinnamic acids and flavonols:

“The present work studies effects of physicochemical and enzymatic characteristics of gastrointestinal digestion on two major groups of phenolic compounds – flavonols and cinnamoyl derivatives – on red radish, red cabbage, broccoli, and white mustard sprouts. Effects of gastrointestinal digestion on release and stability of phenolic compounds depends on different factors, such as physicochemical traits of the food matrix, pH, temperature, or enzymatic activity.

Although initial concentrations of phenolic acids in red radish were lower than in other sprouts, their bioaccessibility after digestion was higher, followed by red cabbage, white mustard, and broccoli. Most degradation of phenolic compounds corresponded to the flavonol fraction, which was almost erased during digestion (with the exception of digestion products of broccoli sprouts, which retained around 30% of the original flavonol concentration):

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Red radish sprouts exhibited the greatest bioaccessibility.

Gastric digestion prepares the food matrix for more efficient polyphenol extraction during intestinal digestion, in which the highest release and stability of these compounds takes place. Hydroxycinnamic acids reach higher concentrations than flavonols, making them tentatively more available to be absorbed at the intestinal level.”

https://www.mdpi.com/2072-6643/13/11/4140/htmIn Vitro Evidence on Bioaccessibility of Flavonols and Cinnamoyl Derivatives of Cruciferous Sprouts”


A cited predecessor used similar methods to study glucosinolate breakdown products like sulforaphane, iberin, and indole-3-carbinol:

“Significantly higher bioaccessibility of isothiocyanates (ITCs) and indoles from glucosinolates (GSLs) of red cabbage sprouts were observed. Bioaccessibility of GSLs from Brasicaceae sprouts is not exclusively associated with initial content of these compounds in plant material (almost negligible), but also with release of GSLs and ongoing breakdown reactions during gastric and intestinal phases of digestion, respectively:

ITCs

The intestinal phase was the most relevant for bioaccessibility of ITCs. Aliphatic GSLs provided higher bioaccessibility of their corresponding ITCs in comparison to indolic and aromatic GSLs.”

https://www.mdpi.com/1422-0067/22/20/11046/htm “Evidence on the Bioaccessibility of Glucosinolates and Breakdown Products of Cruciferous Sprouts by Simulated In Vitro Gastrointestinal Digestion”


Gastric and intestinal simulations were instructive. But rather than depending on digestion for ITCs, I “enzymatically convert to SF before oral intake” per A follow-on study to 3-day-old broccoli sprouts have the optimal yields.

Regarding phenolic compound digestion, my focus this year has been to give my gut microbiota what they want. I expect and get reciprocity from treating them well with whole oats, broccoli-red cabbage-mustard-oat sprouts, blackberries-blueberries-strawberries, quercetin from capers, etc. polyphenols. Not to mention inulin, artichoke hearts, and yeast cell wall β-glucan. Haven’t considered sprouting red radish seeds.

Per Red cabbage effects on gut microbiota, a related research group had an in vitro system that included gut microbiota. Maybe these researchers will get together in a future study?

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Saving bees by regulating epigenetics

This 2021 study investigated an epigenetic treatment for bees forgetting about their hives:

“Over the last few decades, numbers of both wild and managed bee pollinators have been declining. Although reasons for this decline are under debate, it is highly likely that a combination of multiple stressors is to blame, in particular, deformed wing virus (DWV).

Histone deacetylase inhibitors (HDACi) are a class of compounds which prevent deacetylation of histones and therefore increase gene expression. The present study found that HDACi sodium butyrate (NaB) significantly increased survival and reversed the learning / memory impairment of DWV-infected bees. We demonstrated the mechanism of how epigenetic regulation can resume honeybees’ memory function.

bee survival rates

  • When bees were infected with DWV, 50% of bees died by the end of day 2 and only 10% survived to the end of day 5.
  • When NaB was added to the diet prior to DWV infection, survival rate of DWV-infected bees (N/D group) remained >90% after 5 days.
  • Under laboratory rearing conditions, around 30% of control bees died over a period of 5 days.
  • When NaB was included in uninfected bees’ diet, less than 15% of bees died.

These results indicate that feeding bees with NaB could significantly increase survival with or without DWV infection.”

https://www.cell.com/iscience/fulltext/S2589-0042(21)01024-5 “Real-time monitoring of deformed wing virus-infected bee foraging behavior following histone deacetylase inhibitor treatment”


Interesting that these researchers didn’t attempt to eliminate either the virus cause of bee behavior or parasitical mites that carried the virus. They mainly depended on bees’ endogenous systems providing beneficial responses when stimulated.

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Save the bees with Brassicaceae seeds

Two 2021 papers, with the first studying bee infections:

“Fungus Nosema ceranae represents one of the primary bee infection threats worldwide, and antibiotic fumagillin is the only registered product for nosemosis disease control. Natural bioactive compounds deriving from glucosinolate–myrosinase (GSL–MYR) in Brassicaceae plants, mainly isothiocyanates (ITCs), are known for antimicrobial activity against numerous pathogens and health-protective effects in humans.

This work explored Brassica nigra [black mustard] and Eruca sativa [arugula] defatted seed meal (DSM) GSL-containing diets against natural Nosema infection in honeybees. Feeding was administered in May to mildly N. ceranae-infected colonies for four weeks at 250 g/week.

  • N. ceranae abundance showed a slight but significant decrease.
  • No significant effects on colony development and bee mortality were observed compared to controls.
  • MYR activity was detected both in bees fed DSMs and controls.
  • ITCs were found in gut tissues from bees treated with DSMs, corroborating presence of a MYR-like enzyme capable of hydrolyzing ingested GSLs.

Use of DSMs containing GSLs represents a promising alternative to fumagillin as it would overcome the problem of toxic bee product residues encountered with antibiotic treatment.”

https://www.mdpi.com/2218-273X/11/11/1657/htm “Glucosinolate Bioactivation by Apis mellifera Workers and Its Impact on Nosema ceranae Infection at the Colony Level”


A review was cited for “ITCs are GSL hydrolysis products known for their broad-spectrum biological activities against pests and soil/food-borne fungi, bacteria, and human microorganisms.”

“ITCs are efficient agents against a wide range of fungal strains. Many plant and human pathogens, as well as other fungi, were shown to be inhibited in vitro by these agents.

ITC-containing, chemically-characterized plant matrices used to test antifungal activity are summarized. The same activities were tested in pure ITCs; in fact, several well-designed studies did both approaches.

Although there is no one-size-fits-all rule to predict antifungal activity, in general, less polar compounds are usually more potent in solution, but lag behind more volatile compounds with small molecule size in vapor-phase applications. The main biochemical targets seem to be directly related to chemical reactivity, with antioxidant machinery a well-identified target.

Excellent studies on transcriptomics show general stress responses. Inhibiting production of fungal toxins is shown in many real-life applications as well.

Additional applications include:

  • Inhibition of fungal growth, pathogenesis, and/or toxin production in a variety of stored plants and grains;
  • Inhibition of disease on post-harvest fruits; as well as
  • Increasing shelf-life of different food products.

What is more, decrease in decay is frequently accompanied by the lack of measurable changes in various quality characteristics.

ITCs’ natural origin and biodegradability make them good candidates for a wide range of possible applications. Long-term studies show effects are delivered usually without apparent side-effects to plants.”

https://www.mdpi.com/2309-608X/7/7/539/htm “Effects of Glucosinolate-Derived Isothiocyanates on Fungi: A Comprehensive Review on Direct Effects, Mechanisms, Structure-Activity Relationship Data and Possible Agricultural Applications”


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Reworking evolutionary theory

Dr. Michael Skinner coauthored a 2021 review arguing for inclusion of epigenetic transgenerational inheritance into evolutionary theory:

“Over the past 50 years, molecular technology has been used to investigate evolutionary biology. Many examples of finding no correlated genetic mutations or a low frequency of DNA sequence mutations suggest that additional mechanisms are also involved.

  • Identical twins have essentially the same genetics, but generally develop discordant disease as they age.
  • Only a low frequency (generally 1% or less) of individuals that have a specific disease have a correlated genetic mutation.
  • Dramatic increases in disease frequency in the population cannot be explained with genetics alone.

DNA methylation, histone modifications, changes to chromatin structure, expression of non-coding RNA, and RNA methylation can directly regulate gene expression independent of DNA sequence. These different epigenetic factors do not only act independently, but integrate with each other to provide a level of epigenetic complexity to accommodate the needs of cellular development and differentiation.

dvab012f1

Environmental epigenetics is the primary molecular mechanism in any organism that is used to promote physiological and phenotypic alterations. Actions of environmental factors early in development can permanently program the cellular molecular function, which then impacts later life disease or phenotypes.

dvab012f2

Integration of epigenetics and genetics contribute to a Unified Theory of Evolution that explains environmental impacts, phenotypic variation, genetic variation, and adaptation that natural selection acts on. The current review expands this proposed concept and provides a significant amount of supporting literature and experimental models to support the role of environmentally induced epigenetic transgenerational inheritance in evolution.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8557805/ “Role of environmentally induced epigenetic transgenerational inheritance in evolutionary biology: Unified Evolution Theory”


Organisms cited in this review’s references are similar to humans in ancestral influences and developmental influences during the first 1000 days of our lives. Humans are different in that even after all these influences, we can choose to influence our own change and individually evolve. We can also change our internal environments per Switch on your Nrf2 signaling pathway and An environmental signaling paradigm of aging.

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Remembering encounters provides future benefits

Two 2021 papers on trained immunity, with the first a review:

“Effective memory immune responses rely on interaction between innate and adaptive immune cells. While activation of innate immunity provides the first line of defense against infections, it also primes the adaptive immune response.

Adaptive immunity can enhance antimicrobial machinery of innate cells, making them more effective at clearing pathogenic microorganisms. An additional layer of complexity adds to this network of interactions, with innate cells adopting a memory phenotype, which used to apply to only adaptive immunity. Furthermore, non-immune cells can develop some features of this memory-like phenotype.

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Cell subsets in which trained immunity has been described. Different stimuli including Bacillus Calmette Guerin (BCG), β-glucan, cytokines, cytomegalovirus (CMV), and bacterial components can induce a trained immunity phenotype. A common hallmark of trained immunity in these cases is H3K4me3 in promoters of genes encoding for different cytokines.

  • Mechanisms Underlying Establishment of Trained Immunity
  • Trained Immunity in Neutrophils
  • Trained Immunity in Monocytes and Macrophages: General Features
  • Metabolic Pathways Involved in Training of Monocytes and Macrophages
  • Hormonal Control of Trained Immunity Responses in Monocytes and Macrophages
  • Trained Immunity on Alveolar Macrophages and Involvement of Resident Cells
  • Trained Immunity in NK Cells
  • Trained Immunity in Innate Lymphoid Cells
  • Trained Immunity on Hematopoietic Stem Cells
  • Trained Immunity in Bronchial Epithelial Cells
  • Trained Immunity in Skin Stem Cells
  • Trained Immunity in the Gastrointestinal Tract
  • Immunity Training Against Protozoan-Mediated Pathologies
  • Trained Immunity in Non-Infectious Pathologies

Many gaps of knowledge remain in this field. For example, how long changes associated to trained immunity last, and if, in addition to epigenetic modulation, there are other post-translational modifications on proteins relevant for induction of trained immunity.”

https://www.frontiersin.org/articles/10.3389/fimmu.2021.745332/full “Molecular and Cellular Mechanisms Modulating Trained Immunity by Various Cell Types in Response to Pathogen Encounter”


This second paper was a human study cited for its glutathione findings as follows:

  • “Plasma concentration of IL-1β from BCG-vaccinated individuals are positively associated with serum glutathione concentrations.
  • Trained immunity up-regulates expression of genes involved in glutathione metabolism, suggesting an increase in glutathione synthesis and a higher glutathione recycling rate.
  • Single nucleotide polymorphisms in these genes are associated with changes in pro-inflammatory cytokine production after in vitro training by β-glucan and BCG.

Enzymes whose activities are dependent on glutathione could be used as novel targets to modulate trained immunity.”

IL-1β production

“We found a positive association between plasma glutathione concentration and ex vivo IL-1β production 90 days after BCG vaccination upon in vitro exposure to heterologous stimulus Staphylococcus aureus. Up-regulation of IL-1β production by BCG vaccination was also positively associated with circulating concentrations of other metabolites involved in glutathione metabolism, such as methionine, cysteine, glutamate, and glycine.

GSH metabolism was associated with trained immunity traits in 278 healthy individuals. Trained immunity mechanisms that are shaped by GSH metabolism remain to be further explored.”

https://www.mdpi.com/2073-4409/10/5/971/htm “Glutathione Metabolism Contributes to the Induction of Trained Immunity”


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Intergenerational epigenetic inheritance of trained immunity

I’ll curate this 2021 rodent study Transmission of trained immunity and heterologous resistance to infections across generations (not freely available) through two instances of its news coverage:

“Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. Progeny of trained mice exhibited cellular, developmental, transcriptional, and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment.

Progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous [elicits a reaction in a nonspecific antibody] Escherichia coli and Listeria monocytogenes infections. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.”


Its most frequent coverages were repetitions of a press release from an institution that funded this research:

“How does this transmission of immunization to subsequent generations work? In contrast to the classical theory of evolution, which assumes slow adaptation through changes in genetic code, this involves very rapid changes via epigenetic regulation of gene activities, irrespective of genetic code.”

https://medicalxpress.com/news/2021-10-epigenetics-immunization-offspring.html “Epigenetics: Immunization is passed on to offspring”

Not much objectivity in a sponsor’s press release. May as well ask a coworker if they had a good vacation.


And the second:

“When either parent was subjected to real or simulated infection, offspring showed a stronger immune response to potential pathogens, including E. coli bacteria, than controls whose parents hadn’t been subjected to an immune system challenge. They had lower numbers of the bacteria in their lungs and liver, as well as higher concentrations of immune cells and pro-inflammatory cytokines. The effect persisted further: offspring of these second-generation mice also showed a lower bacterial burden after infection.

One weakness of the study is that results do not clearly show how enhanced immunity is being transferred from parent to offspring. The study found that fungal infection induced changes in sperm DNA methylation. But female mice who recovered from infection also produced offspring with fortified immune systems.”

https://www.the-scientist.com/news-opinion/mice-that-survive-infection-pass-on-stronger-immunity-69324 “Mice that Survive Infection Pass on Stronger Immunity”

This reporter gathered good comments from unassociated researchers, but whiffed overall by misinterpreting intergenerational epigenetic inheritance as transgenerational epigenetic inheritance. Per definitions in A review of epigenetic transgenerational inheritance of reproductive disease and Transgenerational effects of early environmental insults on aging and disease, for the term “transgenerational transmission” to apply, researchers need to provide evidence in at least the next 2 male or non-gestating female generations and/or 3 gestating female generations of:

“Altered epigenetic information between generations in the absence of continued environmental exposure.”

I’ll ask about their plans regarding continuing to a F3 generation for further epigenetic inheritance evidence. The interviewed coauthor didn’t indicate that was their current direction, though.

While we wait, train your body’s antioxidant response elements and immune system every day. Exercise your endogenous responses with weak pro-inflammatory isothiocyanates in broccoli sprouts, and your gut with weak antigens in yeast cell wall β-glucan. Daily drills will keep your body’s systems tuned up and ready for both these specific challenges as well as others per this study’s referenced heterologous findings.

Seconds to sunrise

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Do genes determine monogamy / polygamy?

This 2021 rodent study developed epigenetic clocks for deer mice:

“We have undertaken a genome-wide analysis of DNA methylation in Peromyscus, spanning different species, stocks, sexes, tissues, and age cohorts. We present CpGs and enriched pathways that relate to different conditions such as chronological age, high altitude, and monogamous behavior.

  • Analysis involved tails, whole brain, and liver samples that are not major target tissues for sex hormones. This implies that sex-specific patterns of methylation are inflicted early during development, and persist at adulthood.
  • Altitude-specific age-related changes are adjacent to genes that play a role in brain development, immune system functioning, and T-cell development.
  • Comparison of brain specimens between older P. leucopus and P. maniculatus indicated that in the latter, coordination of the unfolded protein response is compromised, and evidence of neurodegenerative pathology was obtained.
  • Our study involved three monogamous (P. californicus, P. polionotus, and P. eremicus) and two polygamous (P. maniculatus and P. leucopus) species. The most significant EWAS hits for monogamy included decreased methylation in Zeb2 intron, a key regulator of midbrain dopaminergic neuron development. These results derived from tail tissues, suggesting that inherent differences in bonding behavior instruct specific epigenetic changes in peripheral tissues that may be translated into distinct physiological outcomes. Whether this is due to differential regulation of specific neurohormonal circuits in response to hormones and neurotransmitters related to bonding, and what the exact physiological outputs are, remains to be determined.

Our study provided the first epigenetic clock for Peromyscus, and illustrated the hierarchical association between various biological variables in determining methylation profiles across different scales of biological organization.”

https://link.springer.com/article/10.1007/s11357-021-00472-5 “Methylation studies in Peromyscus: aging, altitude adaptation, and monogamy”


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All about the betaine, Part 2

Continuing Part 1 by curating a partial outline of a 2021 review:

“This review focuses on the biological and beneficial effects of dietary betaine (trimethylglycine), a naturally occurring and crucial methyl donor.

Betaine has a neuroprotective role, preserves myocardial function, and prevents pancreatic steatosis. Betaine also attenuates oxidant stress, endoplasmic reticulum stress, inflammation, and cancer development.

  • Betaine Protects against Development of Alcohol-Induced Hepatic Steatosis
  • Betaine Protects against Detrimental Effects of HCV and Ethanol on Innate Immunity
  • Betaine Maintains Intestinal Epithelial Barrier Integrity
  • Betaine Maintains Adipose Function

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Human intervention studies showed no adverse effects with 4 g/day supplemental administration of betaine in healthy subjects. However, overweight subjects with metabolic syndrome showed a significant increase in total and LDL-cholesterol concentrations. These effects were not observed with 3 g/day of betaine administration.

We suggest betaine as a promising therapeutic for clinical use to treat these aforementioned diseases as well as other liver-/non-liver-related diseases and conditions.”

https://www.mdpi.com/2079-7737/10/6/456/htm “Beneficial Effects of Betaine: A Comprehensive Review”



This review cited a 2020 study Transgenerational Inheritance of Betaine-Induced Epigenetic Alterations in Estrogen-Responsive IGF-2/IGFBP2 Genes in Rat Hippocampus (not freely available):

“Hippocampal expression of aromatase, estrogen receptor α, and estrogen-related receptor β is downregulated in F1, together with estrogen-responsive insulin-like growth factor 2/insulin-like growth factor binding protein 2 (IGF-2/IGFBP2) genes. However, all these genes are upregulated in F2, which follows the same pattern of F0.

Imprinting control region of IGF-2 gene is hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the promoter DNA methylation status of all affected genes is hypermethylated in F1 but hypomethylated in F2 and F0.”

Intergenerational flip-flops of F0 phenotypes to opposite F1 phenotypes back to F0 phenotypes in the F2 generation can’t conclusively demonstrate transgenerational epigenetic inheritance of alterations due to betaine consumption during pregnancy. Those researchers had to continue on to a F3 female generation for transgenerational results, because F2 generation cells were present in F1 fetuses, and were potentially affected during pregnant F0 treatments.

I came across this paper through a citation chain initiated by Dr. Paul Clayton’s blog post Foie Gras:

“Thanks to our modern diet and lifestyle, nonalcoholic fatty liver disease (NAFLD) is now reckoned to affect an astonishing quarter of the world’s population.”

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Eat oats and inulin to reverse effects of circadian disruption

This 2021 rodent study induced metabolic syndrome with a high-fat diet and switching light-dark cycles every week for 14 weeks. While continuing to disrupt circadian rhythms for ten more weeks, most metabolic effects were reversed by adding either 5% β-glucan, 5% inulin, or .05% melatonin to subjects’ high-fat diet:

“Both prebiotics (oat β-glucan and chicory inulin) and melatonin significantly reversed circadian disruption-induced metabolic syndrome (CDIMS) and alteration of gut microbiota composition. Both prebiotics also reversed increase in body weight and liver weight-to-body weight ratio, and decrease in fasting plasma insulin. Only oat β-glucan reduced plasma leptin and alleviated glucose intolerance.

body weight

All dietary interventions enhanced species richness. In altering gut microbiota, oat β-glucan reversed populations of 7 bacterial genera and increased butyrate producers including Ruminococcaceae and Lachnospiraceae which enhance gut barrier protection and regulate glucose homeostasis.

Though melatonin cannot be fermented in the gut as prebiotics, oral administration of exogenous melatonin absorbed via melatonin receptors concentrated in the intestine has been demonstrated for its effects on shaping gut microbiota. There is currently no concrete mechanism explaining how melatonin affects gut microbial ecology. We postulate that the ability of melatonin to alleviate CDIMS is not governed by changes of SCFAs, but possibly a direct host effect which subsequently affects other metabolites such as bile acids.

In contrast with melatonin, oligomeric chicory inulin as a fermentable fiber mainly affects gut microbiota which affects the host indirectly. For polymeric oat β-glucan, our results suggested that it is probably a combination of both direct and indirect effects to the host, and this is a special property not yet evidenced in other polysaccharides.

Approximately 35% of human gut microbiota undergo temporal rhythmicity. We speculate that prebiotics may affect diurnal oscillations of gut microbiota, its capacity for energy harvest and production of metabolites, which subsequently affect host central circadian clocks through gut-microbiome-brain axis, in which gut microbes interact with central nervous system via nervous, endocrine, and immune signaling pathways.”

https://www.sciencedirect.com/science/article/abs/pii/S0144861721006032 “Circadian disruption-induced metabolic syndrome in mice is ameliorated by oat β-glucan mediated by gut microbiota” (not freely available)


Humans could avoid a high-fat diet, of course. My main experiences with circadian disruptions were 18-hour days of submarine life. That didn’t cause metabolic syndrome, just disorientation to the real world after surfacing.

The end of fig season

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Part 3 of Switch on your Nrf2 signaling pathway

To complement Parts 1 and 2, an informative and detailed video:

sulforaphane defense actions


My daily intake includes most of what’s mentioned in the video. For example, sulforaphane and other beneficial broccoli sprouts compounds, twice a day through 65.5 g of three-day-old broccoli / red cabbage / mustard sprouts microwaved to 60°C. And capers – a high source of quercetin (but see comparisons starting at 31:00, 80% vs. 4% bioavailability) – after soaking and rinsing them several times to reduce sodium content.

I arrived at this video via Dr. Houghton’s paper https://www.researchgate.net/publication/355201137_The_COVID_’Vaccine’_Dilemma_-_a_mechanistic_hypothesis “The COVID ‘Vaccine’ Dilemma – a mechanistic hypothesis” (registration required) posted yesterday.

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Broccoli sprouts and microRNAs

This 2021 in vitro study investigated microRNAs as potential causative factors for broccoli’s beneficial effects:

“A computational analysis was performed to explore processes and pathways associated with genes targeted either by:

  1. Host-expressed miRNAs (endogenous) modulated by bioactive compounds in broccoli; or
  2. miRNAs derived from broccoli (exogenous).

miRNAs are noncoding RNAs containing between 19 and 24 nucleotides, which act as regulators of gene expression both in plants and animals via degradation or inhibition of target mRNAs. miRNAs participate in several biological processes, such as apoptosis, cell growth, differentiation, proliferation, immune response, intercellular communication, RNA stability and processing, stress response, and others.

miRNAs reported in the literature as being upregulated or downregulated in response to broccoli bioactive compounds, with a significant change in expression of at least 2-fold, were selected and used to predict possible mechanisms exerted through miRNA-related actions.

pubmed vs scopus

Sixty-one genes were targeted by both exogenous and endogenous miRNAs, while 6143 and 87 target genes were unique to exogenous and endogenous miRNAs, respectively. Biological processes and molecular functions of genes targeted by both exogenous and endogenous miRNAs were also associated with chromatin, DNA, and RNA regulation.

Cooking, frying, microwaving [2 minutes in a 800W microwave on maximum power], steaming, and blanching were tested along with raw broccoli heads and sprouts and juice. Raw broccoli sprouts showed higher miRNA levels [in half of those tested]. Nearly all treatments did not significantly reduce miRNA levels compared to raw broccoli.

Samples of raw or boiled broccoli, juice, and broccoli sprouts were subjected to in vitro digestion, simulating GI conditions. miRNA survival levels dropped to percentages ranging approximately between 0.1 and 10% at the end of in vitro digestion, although complete elimination was not observed in any case.

Overall, bioinformatic results show that anticarcinogenic and cancer-preventive properties attributed to cruciferous vegetables might be mediated, at least in part, through miRNA-related mechanisms. Moreover, results show that broccoli-derived miRNAs can survive common food-processing conditions and GI digestion.”

https://pubs.acs.org/doi/10.1021/acs.jafc.1c04087 “Connection between miRNA Mediation and the Bioactive Effects of Broccoli (Brassica oleracea var. italica): Exogenous miRNA Resistance to Food Processing and GI Digestion” (not freely available)


I included part of this study’s methods to demonstrate the futility of a PubMed search on any topic. For example, a “caffeic acid and miRNA” search on Scopus returned 499 potential papers. In comparison, that search on PubMed returned 13 papers, or 2.6% of potentially relevant research.

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Eat isoflavones for your nerves

This 2021 rodent study investigated effects of dietary isoflavones and gut microbiota:

“Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the central nervous system (CNS) that results in sensory, motor, and/or cognitive dysfunction. This is due to complex interactions of genetic and environmental factors that trigger activation of autoreactive T cells, leading to subsequent immune cell infiltration into the CNS, neurodegeneration, and axonal damage.

Genetic influences on MS have been well characterized, such as the strong association of certain human leukocyte antigen haplotypes with disease. In contrast, environmental factors – which account for around 70% of disease risk – remain understudied.

In humans, certain gut bacteria digest phytoestrogens, which are plant-based compounds that resemble estrogen. Isoflavones are a major class of phytoestrogens that are highly abundant in legumes such as soy. Humans do not have the necessary enzymes to break down isoflavones, and rely on gut microbiota to harvest these biologically active metabolites.

In the present study, we demonstrate that experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is suppressed in mice fed a diet supplemented with isoflavones.

isoflavones eae

Adlercreutzia equolifaciens and Parabacteroides distasonis, which metabolize isoflavones, were more abundant in mice on an isoflavone diet. Both genera were enriched in healthy individuals but depleted in patients with MS. Conversely, Akkermansia muciniphila was found in greater abundance in mice on an isoflavone-free diet, and this genus is commonly enriched in patients with MS compared to healthy individuals.

isoflavones gut microbiota

We demonstrate that bacterial therapy with P. distasonis and A. equolifaciens results in markedly different clinical disease scores depending on diet of the host. In the absence of isoflavones, isoflavone-metabolizing bacteria may begin to metabolize host products, such as mucins, resulting in a proinflammatory state.

Considering the interplay between diet and gut bacteria is critical when developing dietary and gut microbiome-based therapies for MS and other diseases.”

https://www.science.org/doi/10.1126/sciadv.abd4595 “Isoflavone diet ameliorates experimental autoimmune encephalomyelitis through modulation of gut bacteria depleted in patients with multiple sclerosis”


Parabacteroides distasonis is my second most abundant gut microbiota species at 11.076%. Its main function is to metabolize carbohydrates, which are the bulk of my diet. Haven’t focused on isoflavones.

If you want to increase isoflavones with a soy product like tofu, try to eat it raw, steamed, or simmered in soup. Broiling, grilling, or sautéing tofu causes a dramatic rise in AGEs.

I came across this study by its citation in Dr. Paul Clayton’s rambling blog post Stranger together.