Reduction in metabolic noise reveals rejuvenation following transient severe caloric restriction.

Among land vertebrates, the laying hen stands out due to its great reproductive efficiency: producing an egg daily all year long. This production rate makes the laying hen a special model animal to study the general process of reproduction and aging. One unique aspect of hens is their ability to undergo reproductive plasticity and to rejuvenate their reproductive tract during molting, a standard industrial feed restriction protocol for transiently pausing reproduction, followed by improved laying efficiency almost to peak production. Here we use longitudinal metabolomics, immunology, and physiological assays to show that molting promotes reproduction, compresses morbidity, and restores youthfulness when applied to old hens. We identified circulating metabolic biomarkers that quantitatively predict the reproduction and age of individuals. Lastly, we introduce metabolic noise, a robust, unitless, and quantifiable measure for heterogeneity of the complete metabolome as a general marker that can indicate the rate of aging of a population. Indeed, metabolic noise increased with age in control hens, whereas molted hens exhibited reduced noise following molting, indicating systemic rejuvenation. Our results suggest that metabolic noise can be used as a quick and universal proxy for assessing successful aging treatments, accelerating the timeline for drug development.

A decline in avian cytokine expression with age revealed by commercially available cytokine array.

Cytokines are secreted immunomodulators that are key regulators of the avian immune response. Currently, the most commonly used method to follow cytokine expression is qPCR, which measures cellular levels of mRNA, rather their extracellular circulating levels. Here we present a commercially available cytokine array designed to assay circulating expression levels of multiple cytokines and immunomodulators simultaneously. Upon minor modifications to the manufacturer protocol, background noise was reduced, leading to a significant increase in the sensitivity of the device. Our data indicate that the array is reliable and produce consistent data between biological repeats. We tested the reproducibility of the array in a biologically relevant context by assessing age-related changes in circulating cytokines. While individual features did not show a consistent pattern, our data revealed a consistent decline in the median of all cytokine values, supporting the validity of the array in studying biological processes.

Time-Restricted Feeding in Commercial Layer Chickens Improves Egg Quality in Old Age and Points to Lack of Adipostat Activity in Chickens.

In mammals, time-restricted feeding (TRF) with no caloric restriction provides health benefits and extends longevity, usually with a minor (∼3%) or no reduction in total food consumption. In the current study, a TRF regimen of 6 h free access to food (08:00-14:00 h) was applied to Leghorn chickens from 25 to 86 weeks of age; control birds ate freely during the light hours (06:00-20:00 h). Unexpectedly, the TRF-treated birds consumed, on average, 11.7% less food than the controls. This was manifested by an average reduction of 9.6% in body weight, 2.6-fold in visceral fat accumulation, and 6.5% in egg weight. Hen-housed egg production was reduced by 3.6% in the TRF group compared with the control, along the first 40 weeks of the follow-up (P < 0.05), and changed into a tendency of 0.7% higher egg production thereafter. Several parameters of egg quality showed significant improvement (P < 0.05) in the TRF group compared with the controls. A comparison of diurnal patterns of feed consumption revealed a higher rate of hourly consumption in the TRF group and increased consumption before dark in the control group. In conclusion, the reduced feed intake in response to the TRF treatment and loss in visceral fat accumulation supports the lack of a strong adipostat activity in chickens and different appetite regulation mechanisms compared with mammals. Therefore, future TRF studies in chickens should be adjusted by extending the ad libitum time window. The lower feed intake by the TRF-treated chickens compared with the ad libitum-fed controls seems to reduce the efficiency of egg production. Nevertheless, the improved egg quality and persistence of egg lay at the older age suggest that similarly to mammals, the TRF treatment delayed at least some of the negative impacts associated with advanced age.

Metabolomic Changes Are Predictive of Aging in Laying Hens.

Aging in vertebrates is an extremely complex process that is still poorly understood. One confining factor to studying vertebrate aging is the lack of appropriate models. The laying hen is a good model to study vertebrate aging, as it can be maintained under standard housing conditions, its breeds are genetically well defined and it exhibits significant aging phenotypes at around 18 months of age. Furthermore, laying hens are maintained in a challenging realistic environment and possess a fully functional immune system. Here we used, for the first time, metabolomic profiling of laying hens' blood for identifying biomarkers of aging. Random forest classifier was used to quantify the quality of the markers and found that the markers can predict the correct age group of individuals with 90% accuracy. Animals under time-restricted feeding, a condition known to increase health span, appeared younger under the markers, indicating that the aging biomarkers can also predict the effectiveness of environmental treatments. Additionally, we found that noise, defined as the ratio between the standard deviation and the mean, is an exceptionally robust and universal biomarker of aging, as metabolomic noise increases significantly with age in laying hens, humans, and mice. Our study suggests the laying hen as a useful model to study aging in vertebrates and establishes metabolomic noise as a novel, universal biomarker of aging.

Increased LOH due to Defective Sister Chromatid Cohesion Is due Primarily to Chromosomal Aneuploidy and not Recombination.

Loss of heterozygosity (LOH) is an important factor in cancer, pathogenic fungi, and adaptation to changing environments. The sister chromatid cohesion process (SCC) suppresses aneuploidy and therefore whole chromosome LOH. SCC is also important to channel recombinational repair to sister chromatids, thereby preventing LOH mediated by allelic recombination. There is, however, insufficient information about the relative roles that the SCC pathway plays in the different modes of LOH. Here, we found that the cohesin mutation mcd1-1, and other mutations in SCC, differentially affect the various types of LOH. The greatest effect, by three orders of magnitude, was on whole chromosome loss (CL). In contrast, there was little increase in recombination-mediated LOH, even for telomeric markers. Some of the LOH events that were increased by SCC mutations were complex, i.e., they were the result of several chromosome transactions. Although these events were independent of POL32, the most parsimonious way to explain the formation of at least some of them was break-induced replication through the centromere. Interestingly, the mcd1-1 pol32Δ double mutant showed a significant reduction in the rate of CL in comparison with the mcd1-1 single mutant. Our results show that defects in SCC allow the formation of complex LOH events that, in turn, can promote drug or pesticide resistance in diploid microbes that are pathogenic to humans or plants.

The addition of a developmental factor, unc-62, to already long-lived worms increases lifespan and healthspan.

Aging is a complex trait that is affected by multiple genetic pathways. A relatively unexplored approach is to manipulate multiple independent aging pathways simultaneously in order to observe their cumulative effect on lifespan. Here, we report the phenotypic characterization of a strain with changes in five aging pathways: (1) mitochondrial reactive oxygen species (ROS) production, (2) innate immunity, (3) stress response, (4) metabolic control and (5) developmental regulation in old age. The quintuply modified strain has a lifespan that is 160% longer than the transgenic control strain. Additionally, the quintuply modified strain maintains several physiological markers of aging for a longer time than the transgenic control. Our results support a modular approach as a general scheme to study how multiple pathways interact to achieve extreme longevity.

Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes.

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species.

An engineering approach to extending lifespan in C. elegans.

We have taken an engineering approach to extending the lifespan of Caenorhabditis elegans. Aging stands out as a complex trait, because events that occur in old animals are not under strong natural selection. As a result, lifespan can be lengthened rationally using bioengineering to modulate gene expression or to add exogenous components. Here, we engineered longer lifespan by expressing genes from zebrafish encoding molecular functions not normally present in worms. Additionally, we extended lifespan by increasing the activity of four endogenous worm aging pathways. Next, we used a modular approach to extend lifespan by combining components. Finally, we used cell- and worm-based assays to analyze changes in cell physiology and as a rapid means to evaluate whether multi-component transgenic lines were likely to have extended longevity. Using engineering to add novel functions and to tune endogenous functions provides a new framework for lifespan extension that goes beyond the constraints of the worm genome.

High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity.

Homologous recombination plays a key role in generating genetic diversity, while maintaining protein functionality. The mechanisms by which RecA enables a single-stranded segment of DNA to recognize a homologous tract within a whole genome are poorly understood. The scale by which homology recognition takes place is of a few tens of base pairs, after which the quest for homology is over. To study the mechanism of homology recognition, RecA-promoted homologous recombination between short DNA oligomers with different degrees of heterology was studied in vitro, using fluorescence resonant energy transfer. RecA can detect single mismatches at the initial stages of recombination, and the efficiency of recombination is strongly dependent on the location and distribution of mismatches. Mismatches near the 5' end of the incoming strand have a minute effect, whereas mismatches near the 3' end hinder strand exchange dramatically. There is a characteristic DNA length above which the sensitivity to heterology decreases sharply. Experiments with competitor sequences with varying degrees of homology yield information about the process of homology search and synapse lifetime. The exquisite sensitivity to mismatches and the directionality in the exchange process support a mechanism for homology recognition that can be modeled as a kinetic proofreading cascade.

Modulation of DNA conformations through the formation of alternative high-order HU-DNA complexes.

HU is an abundant, highly conserved protein associated with the bacterial chromosome. It belongs to a small class of proteins that includes the eukaryotic proteins TBP, SRY, HMG-I and LEF-I, which bind to DNA non-specifically at the minor groove. HU plays important roles as an accessory architectural factor in a variety of bacterial cellular processes such as DNA compaction, replication, transposition, recombination and gene regulation. In an attempt to unravel the role this protein plays in shaping nucleoid structure, we have carried out fluorescence resonance energy transfer measurements of HU-DNA oligonucleotide complexes, both at the ensemble and single-pair levels. Our results provide direct experimental evidence for concerted DNA bending by HU, and the abrogation of this effect at HU to DNA ratios above about one HU dimer per 10-12 bp. These findings support a model in which a number of HU molecules form an ordered helical scaffold with DNA lying in the periphery. The abrogation of these nucleosome-like structures for high HU to DNA ratios suggests a unique role for HU in the dynamic modulation of bacterial nucleoid structure.