PNAS politics in the name of science

This 2019 Germany/Canada human fetal cell study was a Proceedings of the National Academy of Sciences of the United States of America direct submission:

“In a human hippocampal progenitor cell line, we assessed the short- and long-term effects of GC [glucocorticoid] exposure during neurogenesis on messenger RNA expression and DNA methylation profiles. Our data suggest that early exposure to GCs can change the set point of future transcriptional responses to stress by inducing lasting DNAm changes.”


The study’s basic finding was that cells had initial responses to stressors that primed them for subsequent stressors. Since this finding wasn’t new, the researchers tried to make it exciting by applying it to novel contexts that were yet circumscribed by official paradigms.

Hypothesis-seeking associations of human fetal hippocampal cell behaviors with human behaviors were flimsy stretches, as were correlations to placental measurements. These appeared to have been efforts to find headline-making effects.

There wasn’t even a hint of the principle described in Epigenetic variations in metabolism:

“Because of the extreme interconnectivity of cell regulatory networks, even at the cellular level, predicting the impact of a sequence variant is difficult as the resultant variation acts:

  • In the context of all other variants and
  • Their potential additive, synergistic and antagonistic interactions.

This phenomenon is known as epistasis.”

It would have condemned pet models of reality to admit that a cell exists in multiple contexts of other cells with potential additive, synergistic, and antagonistic interactions.

A research proposal to trace a specific cell type’s behaviors – while isolated from their extremely interconnected networks – to trillion-celled human behaviors would be rejected in less-politicized organizations.

Sanctioned speculations manifested in this paper with phrases such as “although not significant..” and “although not directly tested..” The study’s title was probably a disappointment in that it conformed to the study’s evidence.

Involvements of psychiatry departments at the pictured Kings College, Harvard, etc., as part of PNAS entrenched politics, retard advancements of science past approved paradigms.

This is my final curation of PNAS papers.

https://www.pnas.org/content/pnas/early/2019/08/08/1820842116.full.pdf “Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation”

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Developmental disorders and the epigenetic clock

This 2019 UK/Canada/Germany human study investigated thirteen developmental disorders to identify genes that changed aspects of the epigenetic clock:

“Sotos syndrome accelerates epigenetic aging [+7.64 years]. Sotos syndrome is caused by loss-of-function mutations in the NSD1 gene, which encodes a histone H3 lysine 36 (H3K36) methyltransferase.

This leads to a phenotype which can include:

  • Prenatal and postnatal overgrowth,
  • Facial gestalt,
  • Advanced bone age,
  • Developmental delay,
  • Higher cancer predisposition, and, in some cases,
  • Heart defects.

Many of these characteristics could be interpreted as aging-like, identifying Sotos syndrome as a potential human model of accelerated physiological aging.

This research will shed some light on the different processes that erode the human epigenetic landscape during aging and provide a new hypothesis about the mechanisms behind the epigenetic aging clock.”

“Proposed model that highlights the role of H3K36 methylation maintenance on epigenetic aging:

  • The H3K36me2/3 mark allows recruiting de novo DNA methyltransferases DNMT3A (in green) and DNMT3B (not shown).
  • DNA methylation valleys (DMVs) are conserved genomic regions that are normally found hypomethylated.
  • During aging, the H3K36 methylation machinery could become less efficient at maintaining the H3K36me2/3 landscape.
  • This would lead to a relocation of de novo DNA methyltransferases from their original genomic reservoirs (which would become hypomethylated) to other non-specific regions such as DMVs (which would become hypermethylated and potentially lose their normal boundaries),
  • With functional consequences for the tissues.”

The researchers improved methodologies of several techniques:

  1. “Previous attempts to account for technical variation have used the first 5 principal components estimated directly from the DNA methylation data. However, this approach potentially removes meaningful biological variation. For the first time, we have shown that it is possible to use the control probes from the 450K array to readily correct for batch effects in the context of the epigenetic clock, which reduces the error associated with the predictions and decreases the likelihood of reporting a false positive.
  2. We have confirmed the suspicion that Horvath’s model underestimates epigenetic age for older ages and assessed the impact of this bias in the screen for epigenetic age acceleration.
  3. Because of the way that the Horvath epigenetic clock was trained, it is likely that its constituent 353 CpG sites are a low-dimensional representation of the different genome-wide processes that are eroding the epigenome with age. Our analysis has shown that these 353 CpG sites are characterized by a higher Shannon entropy when compared with the rest of the genome, which is dramatically decreased in the case of Sotos patients.”

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1753-9 “Screening for genes that accelerate the epigenetic aging clock in humans reveals a role for the H3K36 methyltransferase NSD1”

Too cheap for clinical trials

Let’s compare and contrast a 2019 meta-analysis and a 2017 review of using acetyl-L-carnitine to treat diabetic neuropathy.

A 2019 Brazilian meta-analysis Acetyl‐L‐carnitine for the treatment of diabetic peripheral neuropathy of four previous trials stated:

  • “The risk of bias was high in both trials of different ALC doses and low in the other two trials.
  • No included trial measured the proportion of participants with at least moderate (30%) or substantial (50%) pain relief.
  • At doses greater than 1500 mg/day, ALC reduced pain more than placebo. This subgroup analysis should be viewed with caution as the evidence was even less certain than the overall analysis, which was already of very low certainty.
  • The placebo-controlled studies did not measure functional impairment and disability scores.
  • No study used validated symptom scales.
  • Two studies were funded by the manufacturer of ALC and the other two studies had at least one co-author who was a consultant for an ALC manufacturer.

Authors’ conclusions:

  • We are very uncertain whether ALC causes a reduction in pain after 6 to 12 months treatment in people with DPN, when compared with placebo, as the evidence is sparse and of low certainty.
  • Data on functional and sensory impairment and symptoms are lacking, or of very low certainty.
  • The evidence on adverse events is too uncertain to make any judgements on safety.”

A 2017 Italian review Effects of acetyl-L-carnitine in diabetic neuropathy and other geriatric disorders stated:

“A long history of diabetes mellitus and increasing age are associated with the onset of diabetic neuropathy, a painful and highly disabling complication with a prevalence peaking at 50% among elderly diabetic patients. The management of diabetic neuropathy is extremely difficult: in addition to the standard analgesics used for pain control, common treatments include opioids, anticonvulsants, antidepressants, and local anesthetics, alone or in combination. Such therapies still show a variable, often limited efficacy, however.

Many patients do not spontaneously report their symptoms to physicians, but, if asked, they often describe having experienced a persistent and non-abating pain for many years. The prevalence of painful symptoms is just as high in patients with mild neuropathy as in those with more advanced DPN.

Through the donation of acetyl groups, ALC exerts a positive action on mitochondrial energy metabolism. ALC has cytoprotective, antioxidant, and antiapoptotic effects in the nervous system.

ALC has also been proposed for the treatment of other neurological and psychiatric diseases, such as mood disorders and depression, dementia, Alzheimer’s disease, and Parkinson’s disease, given that synaptic energy states and mitochondrial dysfunctions are core factors in their pathogenesis. Compared to other treatments, ALC is safe and extremely well tolerated.”

“In nerve injury, the mGlu2 receptor overexpressed by ALC binds the glutamate, reducing its concentration in the synapses with an analgesic effect. ALC may improve nerve regeneration and damage repair after primary nerve trauma.”


Where will the money come from to realize what the 2017 review promised, as well as provide what the 2019 meta-analysis required?

Do we prefer the current “limited efficacy” treatments of “opioids, anticonvulsants, antidepressants, and local anesthetics?”

Who will initiate clinical trials of a multiple of the normal dietary supplement dose (500 mg at $.25 a day, retail)? How profitable is a product whose hypothetical effective dosage for diabetic neuropathy (3000 mg) sells for only $1.50 a day?

Effects of advanced glycation end products on quality of life and lifespan

This 2018 Chinese review concerned advanced glycation end products (AGE) mobility interventions:

“Only a limited number of studies have focused on measuring the effects of low AGEs levels or AGEs inhibitors on mobility, although many observational human studies and in vitro studies have reported the correlation of AGEs with and the contribution of AGEs to mobility, particular in diseases such as:

  • osteoporosis,
  • cartilage degradation,
  • osteoarthritis and
  • sarcopenia.

There is insufficient information from previous animal and human studies for use as a reference to determine the intervention period. Although serum AGEs levels can be easily affected by a lower AGEs diet or AGEs inhibitors, it may take longer to see the changes in certain organs or tissues, as a result of a reduction in AGEs accumulation.”

 

“Effect of AGEs on apoptosis signalling. AP-1, activator protein 1; ERK, extracellular signal-regulated protein kinases; IGF-I, insulin-like growth factor I; IL-6, interleukin-6; JAK, Janus kinase; JNK, c-Jun N-terminal kinases; MEK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; p38 MAPK, p38 mitogen-activated protein kinase; RAGE, receptor for AGEs; STAT3, signal transducers and activators of transcription 3; TGF-β, transforming growth factor-β”


Citations aren’t validations of the reference’s quality and strength of evidence. This review would have benefited from not citing reviews that contained misrepresentations, such as one mentioned in Wikipedia is a poor source of information on advanced glycation end products (AGEs).

I came across this review as a result of it citing the excellent 2008 rodent study Oral Glycotoxins Determine the Effects of Calorie Restriction on Oxidant Stress, Age-Related Diseases, and Lifespan which found:

“Higher levels of oxidant AGEs in offspring of Reg-F0 dams may be attributable to placental transmission from mothers with high AGE levels. These high intrauterine AGE levels may predispose the offspring to the development of chronic inflammation and diseases in adulthood, such as insulin resistance and diabetes.

Increasing the intake of AGEs in the diet erases the benefits of CR [calorie restriction]. OS [oxidant stress] can be reduced, and healthspan increased, in mice fed a diet that is restricted in the content of AGEs.

The beneficial effects of a CR diet may be partly related to reduced oxidant intake rather than decreased energy intake.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180645/ “Role of advanced glycation end products in mobility and considerations in possible dietary and nutritional intervention strategies”

A drug that countered effects of a traumatizing mother

This 2019 US rodent study concerned transmitting poor maternal care to the next generation:

“The quality of parental care received during development profoundly influences an individual’s phenotype, including that of maternal behavior. Infant experiences with a caregiver have lifelong behavioral consequences.

Maternal behavior is a complex behavior requiring the recruitment of multiple brain regions including the nucleus accumbens, bed nucleus of the stria terminalis, ventral tegmental area, prefrontal cortex, amygdala, and medial preoptic area. Dysregulation within this circuitry can lead to altered or impaired maternal responsiveness.

We administered zebularine, a drug known to alter DNA methylation, to dams exposed during infancy to the scarcity-adversity model of low nesting resources, and then characterized the quality of their care towards their offspring.

  1. We replicate that dams with a history of maltreatment mistreat their own offspring.
  2. We show that maltreated-dams treated with zebularine exhibit lower levels of adverse care toward their offspring.
  3. We show that administration of zebularine in control dams (history of nurturing care) enhances levels of adverse care.
  4. We show altered methylation and gene expression in maltreated dams normalized by zebularine.

These findings lend support to the hypothesis that epigenetic alterations resulting from maltreatment causally relate to behavioral outcomes.”


“Maternal behavior is an intergenerational behavior. It is important to establish the neurobiological underpinnings of aberrant maternal behavior and explore treatments that can improve maternal behavior to prevent the perpetuation of poor maternal care across generations.”

The study authors demonstrated intergenerational epigenetic effects, and missed an opportunity to also investigate transgenerational epigenetically inherited effects. They cited reference 60 for the first part of the above quotation, but that reviewer misused the transgenerational term by applying it to grand-offspring instead of the great-grand-offspring.

There were resources available to replicate the study authors’ previous findings, which didn’t show anything new. Why not use such resources to uncover evidence even more applicable to humans by extending experiments to great-grand-offspring that have no potential germline exposure to the initial damaging cause?

Could a study design similar to A limited study of parental transmission of anxiety/stress-reactive traits have been integrated? That study’s thorough removal of parental behavior would be an outstanding methodology to confirm by falsifiability whether parental behavior is both an intergenerational and a transgenerational epigenetic inheritance mechanism.

Rodent great-grand-offspring can be studied in < 9 months. It takes > 50 years for human studies to reach the transgenerational generation. Why not attempt to “prevent the perpetuation of poor maternal care across generations?”

Isn’t it a plausible hypothesis that humans “with a history of maltreatment mistreat their own offspring?” Isn’t it worth the extra effort to extend animal research to investigate this unfortunate chain?

https://www.nature.com/articles/s41598-019-46539-4 “Pharmacological manipulation of DNA methylation normalizes maternal behavior, DNA methylation, and gene expression in dams with a history of maltreatment”

A better method of measuring neurogenesis

One of the references cited in Linking adult neurogenesis to Alzheimer’s disease was https://www.nature.com/articles/s41591-019-0375-9 “Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease” (not freely available).

This 2019 Spanish human study used improved techniques to find:

“Adult hippocampal neurogenesis (AHN), confers an unparalleled degree of plasticity to the entire hippocampal circuitry. Direct evidence of AHN in humans has remained elusive. Determining whether new neurons are continuously incorporated into the human dentate gyrus (DG) during physiological and pathological aging is a crucial question with outstanding therapeutic potential.

By combining human brain samples obtained under tightly controlled conditions and state-of-the-art tissue processing methods, we identified thousands of immature neurons in the DG of neurologically healthy human subjects up to the ninth decade of life. These neurons exhibited variable degrees of maturation along differentiation stages of AHN. In sharp contrast, the number and maturation of these neurons progressively declined as AD advanced.

These results demonstrate the persistence of AHN during both physiological and pathological aging in humans and provide evidence for impaired neurogenesis as a potentially relevant mechanism underlying memory deficits in AD that might be amenable to novel therapeutic strategies.”


The control group was 13 neurologically healthy deceased people aged 43 to 87. The AD group was 45 deceased people, distributed among the six Braak stages of the pathology, aged 52 to 97.

Linking adult neurogenesis to Alzheimer’s disease

This 2019 Spanish human study compared DNA methylation, chromatin and histone modifications in the hippocampus of deceased Alzheimer’s disease patients with controls:

“A significant percentage of the differentially methylated genes were related to neural development and neurogenesis. It was astounding that other biological, cellular, and molecular processes generally associated with neurodegeneration such as apoptosis, autophagy, inflammation, oxidative stress, and mitochondrial or lysosomal dysfunction were not overrepresented.

The results of the present study point to neurogenesis-related genes as targets of epigenetic changes in the hippocampus affected by AD. These methylation changes might be built throughout life due to external and internal cues and would represent an example of epigenetic interaction between environmental and genetic factors in developing AD.

As an alternative explanation, these epigenetic marks might also represent the trace of DNA methylation alterations induced during early developmental stages of the hippocampus, which would remain as a fingerprint in the larger proportion of hippocampal neurons that are not exchanged. This second hypothesis would link AD to early life stages, in concordance with recent studies that revealed abnormal p-tau deposits (pre-tangles) in brains of young individuals under 30, suggesting AD pathology would start earlier in life than it was previously thought. The influence of the genetic risk for AD has also been postulated to begin in early life, and other AD risk factors may be influenced by in utero environment.”


The study cited references to adult neurogenesis:

“Though strongly related to brain development, neurogenesis is also maintained in the adult human brain, mainly in two distinct areas, i.e., the subventricular zone and the subgranular zone of the dentate gyrus in the hippocampus. There is substantial neurogenesis throughout life in the human hippocampus as it is estimated that up to one third of human hippocampal neurons are subject to constant turnover.

Adult neurogenesis is linked to hippocampal-dependent learning and memory tasks and is reduced during aging. Recent evidence suggests that adult neurogenesis is altered in the neurodegenerative process of AD, but it is still controversial with some authors reporting increased neurogenesis, whereas others show reduced neurogenesis. In the human hippocampus, a sharp drop in adult neurogenesis has been observed in subjects with AD.”

One of the study’s limitations was its control group:

“There was a significant difference in age between controls [12, ages 50.7 ± 21.5] and AD patients [26, ages 81.2 ± 12.1], being the latter group older than the former group. Although we adjusted for age in the statistical differential methylation analysis, the accuracy of this correction may be limited as there is little overlap in the age ranges of both groups.”

https://clinicalepigeneticsjournal.biomedcentral.com/track/pdf/10.1186/s13148-019-0672-7 “DNA methylation signature of human hippocampus in Alzheimer’s disease is linked to neurogenesis”