Rejuvant®, a potential life-extending compound formulation with alpha-ketoglutarate and vitamins, conferred an average 8 year reduction in biological aging, after an average of 7 months of use, in the TruAge DNA methylation test.

The search continues for possible interventions that delay and/or reverse biological aging, resulting in extended healthspan and lifespan. Interventions delaying aging in animal models are well established; however, most lack validation in humans. The length of human lifespan makes it impractical to perform survival analysis. Instead, aging biomarkers, such as DNA methylation (DNAm) clocks, have been developed to monitor biological age. Herein we report a retrospective analysis of DNA methylation age in 42 individuals taking Rejuvant®, an alpha-ketoglutarate based formulation, for an average period of 7 months. DNAm testing was performed at baseline and by the end of treatment with Rejuvant® supplementation. Remarkably, individuals showed an average decrease in biological aging of 8 years (p-value=6.538x10-12). Furthermore, the supplementation with Rejuvant® is robust to individual differences, as indicated by the fact that a large majority of participants decreased their biological age. Moreover, we found that Rejuvant® is of additional benefit to chronologically and biologically older individuals. While continued testing, particularly in a placebo-controlled design, is required, the nearly 8-year reversal in the biological age of individuals taking Rejuvant® for 4 to 10 months is noteworthy, making the natural product cocktail an intriguing candidate to affect human aging.

Correction: 16S rRNA gene sequencing and healthy reference ranges for 28 clinically relevant microbial taxa from the human gut microbiome.

[This corrects the article DOI: 10.1371/journal.pone.0176555.].

16S rRNA gene sequencing and healthy reference ranges for 28 clinically relevant microbial taxa from the human gut microbiome.

Changes in the relative abundances of many intestinal microorganisms, both those that naturally occur in the human gut microbiome and those that are considered pathogens, have been associated with a range of diseases. To more accurately diagnose health conditions, medical practitioners could benefit from a molecular, culture-independent assay for the quantification of these microorganisms in the context of a healthy reference range. Here we present the targeted sequencing of the microbial 16S rRNA gene of clinically relevant gut microorganisms as a method to provide a gut screening test that could assist in the clinical diagnosis of certain health conditions. We evaluated the possibility of detecting 46 clinical prokaryotic targets in the human gut, 28 of which could be identified with high precision and sensitivity by a bioinformatics pipeline that includes sequence analysis and taxonomic annotation. These targets included 20 commensal, 3 beneficial (probiotic), and 5 pathogenic intestinal microbial taxa. Using stool microbiome samples from a cohort of 897 healthy individuals, we established a reference range defining clinically relevant relative levels for each of the 28 targets. Our assay quantifies 28 targets in the context of a healthy reference range and correctly reflected 38/38 verification samples of real and synthetic stool material containing known gut pathogens. Thus, we have established a method to determine microbiome composition with a focus on clinically relevant taxa, which has the potential to contribute to patient diagnosis, treatment, and monitoring. More broadly, our method can facilitate epidemiological studies of the microbiome as it relates to overall human health and disease.

Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis.

In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded RNA (dsRNA) to knockdown translation of targeted mRNAs. However, it has been shown that E. coli is mildly pathogenic to C. elegans and this pathogenicity might influence aging and the accuracy of the RNAi-screening during aging may as well be affected. Here, we describe a novel system that utilizes the non-pathogenic bacterium Bacillus subtilis, to express dsRNA and therefore eliminates the effects of bacterial pathogenicity from the genetic analysis of aging.

A dual role of the Wnt signaling pathway during aging in Caenorhabditis elegans.

Wnt signaling is a major and highly conserved developmental pathway that guides many important events during embryonic and larval development. In adulthood, misregulation of Wnt signaling has been implicated in tumorigenesis and various age-related diseases. These effects occur through highly complicated cell-to-cell interactions mediated by multiple Wnt-secreted proteins. While they share a high degree of sequence similarity, their function is highly diversified. Although the role of Wnt ligands during development is well studied, very little is known about the possible actions of Wnt signaling in natural aging. In this study, Caenorhabditis elegans serves, for the first time, as a model system to determine the role of Wnt ligands in aging. Caenorhabditis elegans has five Wnt proteins, mom-2, egl-20, lin-44, cwn-1, and cwn-2. We show that all five Wnt ligands are expressed and active past the development stages. The ligand mom-2/Wnt plays a major detrimental role in longevity, whereas the function of lin-44/Wnt is beneficial for long life. Interestingly, no evidence was found for Wnt signaling being involved in cellular or oxidative stress responses during aging. Our results suggest that Wnt signaling regulates aging-intrinsic genetic pathways, opening a new research direction on the role of Wnt signaling in aging and age-related diseases.

Developmental drift as a mechanism for aging: lessons from nematodes.

Aging is a universal biological process that afflicts every creature on this planet. To date, we have a very poor understanding of what actually causes this degeneration. A commonly held view is that aging is the result of damage accumulation over a lifetime. However, research has shown that aging is not only the result of wear and tear in the organism, but also of genetic programs involved in organismal development that go awry as selective pressure is released. This review focuses on Wnt signalling pathways and discusses how these genetic programs orchestrate changes in the organism that could cause aging.

Premature and accelerated aging: HIV or HAART?

Highly active antiretroviral therapy (HAART) has significantly increased life expectancy of the human immunodeficiency virus (HIV)-positive population. Nevertheless, the average lifespan of HIV-patients remains shorter compared to uninfected individuals. Immunosenescence, a current explanation for this difference invokes heavily on viral stimulus despite HAART efficiency in viral suppression. We propose here that the premature and accelerated aging of HIV-patients can also be caused by adverse effects of antiretroviral drugs, specifically those that affect the mitochondria. The nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral drug class for instance, is known to cause depletion of mitochondrial DNA via inhibition of the mitochondrial specific DNA polymerase-γ. Besides NRTIs, other antiretroviral drug classes such as protease inhibitors also cause severe mitochondrial damage by increasing oxidative stress and diminishing mitochondrial function. We also discuss important areas for future research and argue in favor of the use of Caenorhabditis elegans as a novel model system for studying these effects.

An elt-3/elt-5/elt-6 GATA transcription circuit guides aging in C. elegans.

To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and sod-3. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-3, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage.

An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase.

Protein kinases are important mediators of much of the signal transduction that occurs in eukaryotic cells. Unfortunately, the identification of protein kinase substrates has proven to be a difficult task, and we generally know few, if any, of the physiologically relevant targets of any particular kinase. Here, we describe a sequence-based approach that simplified this substrate identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. In this method, the evolutionary conservation of all PKA consensus sites in the S. cerevisiae proteome was systematically assessed within a group of related yeasts. The basic premise was that a higher degree of conservation would identify those sites that are functional in vivo. This method identified 44 candidate PKA substrates, 5 of which had been described. A phosphorylation analysis showed that all of the identified candidates were phosphorylated by PKA and that the likelihood of phosphorylation was strongly correlated with the degree of target site conservation. Finally, as proof of principle, the activity of one particular target, Atg1, a key regulator of autophagy, was shown to be controlled by PKA phosphorylation in vivo. These data therefore suggest that this evolutionary proteomics approach identified a number of PKA substrates that had not been uncovered by other methods. Moreover, these data show how this approach could be generally used to identify the physiologically relevant occurrences of any protein motif identified in a eukaryotic proteome.

The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae.

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.

The C-terminal domain of the largest subunit of RNA polymerase II is required for stationary phase entry and functionally interacts with the Ras/PKA signaling pathway.

The Saccharomyces cerevisiae Ras proteins control cell growth by regulating the activity of the cAMP-dependent protein kinase (PKA). In this study, a genetic approach was used to identify cellular processes that were regulated by Ras/PKA signaling activity. Interestingly, we found that mutations affecting the C-terminal domain (CTD), of Rpb1p, the largest subunit of RNA polymerase II, were very sensitive to changes in Ras signaling activity. The Rpb1p CTD is a highly conserved, repetitive structure that is a key site of control during the production of a mature mRNA molecule. We found that mutations compromising the CTD were synthetically lethal with alterations that led to elevated levels of Ras/PKA signaling. Altogether, the data suggested that Ras/PKA activity was negatively regulating a protein that functioned in concert with the CTD during RNA pol II transcription. Consistent with this prediction, we found that elevated levels of Ras signaling caused growth and transcription defects that were very similar to those observed in mutants encoding an Rpb1p with a truncated CTD. In all, these data suggested that S. cerevisiae growth control and RNA pol II transcription might be coupled by using the Ras pathway to regulate CTD function.

The C terminus of the Vps34p phosphoinositide 3-kinase is necessary and sufficient for the interaction with the Vps15p protein kinase.

Vps34p is a phosphatidylinositol 3-kinase that is part of a membrane-associated complex with the Vps15p protein kinase. This kinase complex is required for the delivery of soluble proteins to the lysosomal/vacuolar compartment of eukaryotic cells. This study examined the Vps34p-Vps15p association and identified the domains within each protein that were important for this interaction. Using several different approaches, the interaction domain within Vps34p was mapped to a 28-amino acid element near its C terminus. This Vps34p motif was both necessary and sufficient for the interaction with Vps15p. Two-hybrid mapping experiments indicated that two separate regions of Vps15p were required for the association with Vps34p; they are the N-terminal protein kinase domain and a set of three tandem repeats of about 39 amino acids each. Neither domain alone was sufficient for the interaction. These Vps15p repeat elements are similar in sequence to the HEAT motifs that have been implicated in protein interactions in other proteins, including the Huntingtin protein. Finally, these studies identified a novel motif at the very C terminus of Vps34p that was required for phosphatidylinositol 3-kinase activity. This domain is highly conserved specifically in all Vps34p-like phosphatidylinositol 3-kinases but is not required for the interaction with Vps15p. This study thus represents a first step toward a better understanding of how this Vps15p.Vps34p kinase complex is assembled and regulated in vivo.