Among-population variation in telomere regulatory proteins and their potential role as hidden drivers of intraspecific variation in life history.

Biologists aim to explain patterns of growth, reproduction and ageing that characterize life histories, yet we are just beginning to understand the proximate mechanisms that generate this diversity. Existing research in this area has focused on telomeres but has generally overlooked the telomere's most direct mediator, the shelterin protein complex. Shelterin proteins physically interact with the telomere to shape its shortening and repair. They also regulate metabolism and immune function, suggesting a potential role in life history variation in the wild. However, research on shelterin proteins is uncommon outside of biomolecular work. Intraspecific analyses can play an important role in resolving these unknowns because they reveal subtle variation in life history within and among populations. Here, we assessed ecogeographic variation in shelterin protein abundance across eight populations of tree swallow (Tachycineta bicolor) with previously documented variation in environmental and life history traits. Using the blood gene expression of four shelterin proteins in 12-day-old nestlings, we tested the hypothesis that shelterin protein gene expression varies latitudinally and in relation to both telomere length and life history. Shelterin protein gene expression differed among populations and tracked non-linear variation in latitude: nestlings from mid-latitudes expressed nearly double the shelterin mRNA on average than those at more northern and southern sites. However, telomere length was not significantly related to latitude. We next assessed whether telomere length and shelterin protein gene expression correlate with 12-day-old body mass and wing length, two proxies of nestling growth linked to future fecundity and survival. We found that body mass and wing length correlated more strongly (and significantly) with shelterin protein gene expression than with telomere length. These results highlight telomere regulatory shelterin proteins as potential mediators of life history variation among populations. Together with existing research linking shelterin proteins and life history variation within populations, these ecogeographic patterns underscore the need for continued integration of ecology, evolution and telomere biology, which together will advance understanding of the drivers of life history variation in nature.

Effect of long-term caloric restriction on telomere length in healthy adults: CALERIE™ 2 trial analysis.

Caloric restriction (CR) modifies lifespan and aging biology in animal models. The Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE™) 2 trial tested translation of these findings to humans. CALERIE™ randomized healthy, nonobese men and premenopausal women (age 21-50y; BMI 22.0-27.9 kg/m2 ), to 25% CR or ad-libitum (AL) control (2:1) for 2 years. Prior analyses of CALERIE™ participants' blood chemistries, immunology, and epigenetic data suggest the 2-year CR intervention slowed biological aging. Here, we extend these analyses to test effects of CR on telomere length (TL) attrition. TL was quantified in blood samples collected at baseline, 12-, and 24-months by quantitative PCR (absolute TL; aTL) and a published DNA-methylation algorithm (DNAmTL). Intent-to-treat analysis found no significant differences in TL attrition across the first year, although there were trends toward increased attrition in the CR group for both aTL and DNAmTL measurements. When accounting for adherence heterogeneity with an Effect-of-Treatment-on-the-Treated analysis, greater CR dose was associated with increased DNAmTL attrition during the baseline to 12-month weight-loss period. By contrast, both CR group status and increased CR were associated with reduced aTL attrition over the month 12 to month 24 weight maintenance period. No differences were observed when considering TL change across the study duration from baseline to 24-months, leaving it unclear whether CR-related effects reflect long-term detriments to telomere fidelity, a hormesis-like adaptation to decreased energy availability, or measurement error and insufficient statistical power. Unraveling these trends will be a focus of future CALERIE™ analyses and trials.

High-density stable glasses formed on soft substrates.

Enabled by surface-mediated equilibration, physical vapour deposition can create high-density stable glasses comparable with liquid-quenched glasses aged for millions of years. Deposition is often performed at various rates and temperatures on rigid substrates to control the glass properties. Here we demonstrate that on soft, rubbery substrates, surface-mediated equilibration is enhanced up to 170 nm away from the interface, forming stable glasses with densities up to 2.5% higher than liquid-quenched glasses within 2.5 h of deposition. Gaining similar properties on rigid substrates would require 10 million times slower deposition, taking ~3,000 years. Controlling the modulus of the rubbery substrate provides control over the glass structure and density at constant deposition conditions. These results underscore the significance of substrate elasticity in manipulating the properties of the mobile surface layer and thus the glass structure and properties, allowing access to deeper states of the energy landscape without prohibitively slow deposition rates.

Cross-tissue comparison of telomere length and quality metrics of DNA among individuals aged 8 to 70 years.

Telomere length (TL) is an important biomarker of cellular aging, yet its links with health outcomes may be complicated by use of different tissues. We evaluated within- and between-individual variability in TL and quality metrics of DNA across five tissues using a cross-sectional dataset ranging from 8 to 70 years (N = 197). DNA was extracted from all tissue cells using the Gentra Puregene DNA Extraction Kit. Absolute TL (aTL) in kilobase pairs was measured in buccal epithelial cells, saliva, dried blood spots (DBS), buffy coat, and peripheral blood mononuclear cells (PBMCs) using qPCR. aTL significantly shortened with age for all tissues except saliva and buffy coat, although buffy coat was available for a restricted age range (8 to 15 years). aTL did not significantly differ across blood-based tissues (DBS, buffy coat, PBMC), which had significantly longer aTL than buccal cells and saliva. Additionally, aTL was significantly correlated for the majority of tissue pairs, with partial Spearman's correlations controlling for age and sex ranging from ⍴ = 0.18 to 0.51. We also measured quality metrics of DNA including integrity, purity, and quantity of extracted DNA from all tissues and explored whether controlling for DNA metrics improved predictions of aTL. We found significant tissue variation: DNA from blood-based tissues had high DNA integrity, more acceptable A260/280 and A260/230 values, and greater extracted DNA concentrations compared to buccal cells and saliva. Longer aTL was associated with lower DNA integrity, higher extracted DNA concentrations, and higher A260/230, particularly for saliva. Model comparisons suggested that incorporation of quality DNA metrics improves models of TL, although relevant metrics vary by tissue. These findings highlight the merits of using blood-based tissues and suggest that incorporation of quality DNA metrics as control variables in population-based studies can improve TL predictions, especially for more variable tissues like buccal and saliva.

Telomere length and chronological age across the human lifespan: A systematic review and meta-analysis of 414 study samples including 743,019 individuals.

Telomere attrition is a proposed hallmark of aging. To evaluate the association of telomere length (TL) with chronological age across the human lifespan, we conducted a systematic review and meta-analysis of 414 study samples comprising 743,019 individuals aged 0-112 years. We examined both cross-sectional and longitudinal data, and evaluated the impact of various biological and methodological factors including sex, health status, tissue types, DNA extraction procedures, and TL measurement methods. The pooled corrected correlation between TL and age from cross-sectional samples was -0.19 (95%CI: -0.22 to -0.15), which weakened with increased chronological age (ß = 0.003, p < 0.001). Z-score change rates of TL across the lifespan showed a gradual decrease in shortening rate until around age 50 and remained at a relatively stable rate towards the elderly period. A greater attrition rate was observed in longitudinal than cross-sectional evaluations. For TL measured in base pairs, the median change rate of TL was -23 bp/year in cross-sectional samples and -38 bp/year in longitudinal samples. Methodological factors including TL measurement methods and tissue types impacted the TL-age correlation, while sex or disease status did not. This meta-analysis revealed the non-linear shortening trend of TL across the human lifespan and provides a reference value for future studies. Results also highlight the importance of methodological considerations when using TL as an aging biomarker.

The shelterin protein expansion of telomere dynamics: Linking early life adversity, life history, and the hallmarks of aging.

Aging is characterized by functional decline occurring alongside changes to several hallmarks of aging. One of the hallmarks includes attrition of repeated DNA sequences found at the ends of chromosomes called telomeres. While telomere attrition is linked to morbidity and mortality, whether and how it causally contributes to lifelong rates of functional decline is unclear. In this review, we propose the shelterin-telomere hypothesis of life history, in which telomere-binding shelterin proteins translate telomere attrition into a range of physiological outcomes, the extent of which may be modulated by currently understudied variation in shelterin protein levels. Shelterin proteins may expand the breadth and timing of consequences of telomere attrition, e.g., by translating early life adversity into acceleration of the aging process. We consider how the pleiotropic roles of shelterin proteins provide novel insights into natural variation in physiology, life history, and lifespan. We highlight key open questions that encourage the integrative, organismal study of shelterin proteins that enhances our understanding of the contribution of the telomere system to aging.

Organismal effects of heat in a fixed ecological niche: Implications on the role of behavioral buffering in our changing world.

Increasingly frequent and intense heatwaves generate new challenges for many organisms. Our understanding of the ecological predictors of thermal vulnerability is improving, yet, at least in endotherms, we are still only beginning to understand one critical component of predicting resilience: exactly how do wild animals cope with sub-lethal heat? In wild endotherms, most prior work focuses on one or a few traits, leaving uncertainty about organismal consequences of heatwaves. Here, we experimentally generated a 2.8°C heatwave for free-living nestling tree swallows (Tachycineta bicolor). Over a week-long period coinciding with the peak of post-natal growth, we quantified a suite of traits to test the hypotheses that (a) behavioral or (b) physiological responses may be sufficient for coping with inescapable heat. Heat-exposed nestlings increased panting and decreased huddling, but treatment effects on panting dissipated over time, even though heat-induced temperatures remained elevated. Physiologically, we found no effects of heat on: gene expression of three heat shock proteins in blood, muscle, and three brain regions; secretion of circulating corticosterone at baseline or in response to handling; and telomere length. Moreover, heat had a positive effect on growth and a marginal, but not significant, positive effect on subsequent recruitment. These results suggest that nestlings were generally buffered from deleterious effects of heat, with one exception: heat-exposed nestlings exhibited lower gene expression for superoxide dismutase, a key antioxidant defense. Despite this one apparent cost, our thorough organismal investigation indicates general resilience to a heatwave that may, in part, stem from behavioral buffering and acclimation. Our approach provides a mechanistic framework that we hope will improve understanding of species persistence in the face of climate change.

Experimental ectoparasite removal has a sex-specific effect on nestling telomere length.

Parasites are a strong selective force that can influence fitness-related traits. The length of chromosome-capping telomeres can be used to assess the long-term costs of parasitism, as telomere loss accelerates in response to environmental stressors and often precedes poorer survival prospects. Here, we explored the sex-specific effects of ectoparasite removal on morphology and telomere length in nestling tree swallows (Tachycineta bicolor). To do so, we experimentally removed blow fly (Protocalliphora spp.) larvae from nests using Permethrin, a broad-spectrum insecticide. Compared to water-treated controls, insecticide treatment of nests had a sex-biased effect on blood telomere length: ectoparasite removal resulted in significantly longer telomeres in males but not females. While this treatment did not influence nestling body mass, it was associated with reduced feather development regardless of sex. This may reflect a relaxed pressure to fledge quickly in the absence of parasites, or alternatively, could be a negative side effect of permethrin on morphology. Exploring robust sex-specific telomere dynamics in response to early-life environmental pressures such as parasitism will shed light on sexual dimorphism in adult life histories and aging.

The role of intramolecular relaxations on the structure and stability of vapor-deposited glasses.

Stable glasses (SGs) are formed through surface-mediated equilibration (SME) during physical vapor deposition (PVD). Unlike intermolecular interactions, the role of intramolecular degrees of freedom in this process remains unexplored. Here, using experiments and coarse-grained molecular dynamics simulations, we demonstrate that varying dihedral rotation barriers of even a single bond, in otherwise isomeric molecules, can strongly influence the structure and stability of PVD glasses. These effects arise from variations in the degree of surface mobility, mobility gradients, and mobility anisotropy, at a given deposition temperature (Tdep). At high Tdep, flexible molecules have access to more configurations, which enhances the rate of SME, forming isotropic SGs. At low Tdep, stability is achieved by out of equilibrium aging of the surface layer. Here, the poor packing of rigid molecules enhances the rate of surface-mediated aging, producing stable glasses with layered structures in a broad range of Tdep. In contrast, the dynamics of flexible molecules couple more efficiently to the glass layers underneath, resulting in reduced mobility and weaker mobility gradients, producing unstable glasses. Independent of stability, the flattened shape of flexible molecules can also promote in-plane orientational order at low Tdep. These results indicate that small changes in intramolecular relaxation barriers can be used as an approach to independently tune the structure and mobility profiles of the surface layer and, thus, the stability and structure of PVD glasses.

Role of Molecular Layering in the Enhanced Mechanical Properties of Stable Glasses.

The density, degree of molecular orientation, and molecular layering of vapor-deposited stable glasses (SGs) vary with substrate temperature (Tdep) below the glass-transition temperature (Tg). Density and orientation have been suggested to be factors influencing the mechanical properties of SGs. We perform nanoindentation on two molecules which differ by only a single substituent, allowing one molecule to adopt an in-plane orientation at low Tdep. The reduced elastic modulus and hardness of both molecules show similar Tdep dependences, with enhancements of 15-20% in reduced modulus and 30-45% in hardness at Tdep ≈ 0.8Tg, where the density of vapor-deposited films is enhanced by ∼1.4% compared to that of the liquid-quenched glass. At Tdep < 0.8Tg, one of the molecules produces highly unstable glasses with in-plane orientation. However, both systems show enhanced mechanics. Both the modulus and hardness correlate with the degree of layering, which is similar in both systems despite their variable stability. We suggest that nanoindentation performed normal to the film's surface is influenced by the tighter packing of the molecules in this direction.

Testing hormonal responses to real and simulated social challenges in a competitive female bird.

Competitive interactions often occur in series; therefore animals may respond to social challenges in ways that prepare them for success in future conflict. Changes in the production of the steroid hormone testosterone (T) are thought to mediate phenotypic responses to competition, but research over the past few decades has yielded mixed results, leading to several potential explanations as to why T does not always elevate following a social challenge. Here, we measured T levels in tree swallows (Tachycineta bicolor), a system in which females compete for limited nesting cavities and female aggression is at least partially mediated by T. We experimentally induced social challenges in two ways: (1) using decoys to simulate territorial intrusions and (2) removing subsets of nesting cavities to increase competition among displaced and territory-holding females. Critically, these experiments occurred pre-laying, when females are physiologically capable of rapidly increasing circulating T levels. However, despite marked aggression in both experiments, T did not elevate following real or simulated social challenges, and in some cases, socially challenged females had lower T levels than controls. Likewise, the degree of aggression was negatively correlated with T levels following a simulated territorial intrusion. Though not in line with the idea that social challenges prompt T elevation in preparation for future challenges, these patterns nevertheless connect T to territorial aggression in females. Coupled with past work showing that T promotes aggression, these results suggest that T may act rapidly to allow animals to adaptively respond to the urgent demands of a competitive event.

A multi-tissue view on telomere dynamics and postnatal growth.

Trade-offs between growth and self-maintenance are common in nature, such that early-life effects on growth can generate lasting consequences on survival and longevity. Telomeres-putative biomarkers of self-maintenance-may link early growth with these later phenotypic effects, but evidence for growth-telomere trade-offs is mixed. Null or even positive relationships between growth and telomeres may be driven by heterogeneity in resource availability or invariable allocation towards telomere maintenance within a population. We used nestling tree swallows (Tachycineta bicolor) to assess the directionality and timing of relationships between growth and telomere length in several tissues. We focused on two important phases of growth: first, the peak of postnatal growth occurring around 6 days old when nestlings grow by ~33% in a single day, and subsequently, the later phase of growth occurring as body mass plateaus near adult size at 12 days old. We quantified telomere attrition in blood during postnatal growth, as well as telomere length in the blood, brain, adrenals, and liver at 12 days old. Growth was unrelated to telomere length in the liver and telomere dynamics in blood. However, brain telomere length was positively correlated with peak growth, and adrenal telomere length was positively related to later growth, particularly for chicks that had experienced a temporary stressor. These observations suggest that variation in resource availability may mask trade-offs, generating positive correlations between growth and telomere length at the population level. They also provide insights into complex relationships between growth and self-maintenance that can be revealed by looking in multiple tissues.

The telomere regulatory gene POT1 responds to stress and predicts performance in nature: Implications for telomeres and life history evolution.

Telomeres are emerging as correlates of fitness-related traits and may be important mediators of ecologically relevant variation in life history strategies. Growing evidence suggests that telomere dynamics can be more predictive of performance than length itself, but very little work considers how telomere regulatory mechanisms respond to environmental challenges or influence performance in nature. Here, we combine observational and experimental data sets from free-living tree swallows (Tachycineta bicolor) to assess how performance is predicted by the telomere regulatory gene POT1, which encodes a shelterin protein that sterically blocks telomerase from repairing the telomere. First, we show that lower POT1 gene expression in the blood was associated with higher female quality, that is, earlier breeding and heavier body mass. We next challenged mothers with an immune stressor (lipopolysaccharide injection) that led to "sickness" in mothers and 24 h of food restriction in their offspring. While POT1 did not respond to maternal injection, females with lower constitutive POT1 gene expression were better able to maintain feeding rates following treatment. Maternal injection also generated a 1-day stressor for chicks, which responded with lower POT1 gene expression and elongated telomeres. Other putatively stress-responsive mechanisms (i.e., glucocorticoids, antioxidants) showed marginal responses in stress-exposed chicks. Model comparisons indicated that POT1 mRNA abundance was a largely better predictor of performance than telomere dynamics, indicating that telomere regulators may be powerful modulators of variation in life history strategies.

Design of a homologous series of molecular glassformers.

We design and synthesize a set of homologous organic molecules by taking advantage of facile and tailorable Suzuki cross coupling reactions to produce triarylbenzene derivatives. By adjusting the number and the arrangement of conjugated rings, the identity of heteroatoms, lengths of fluorinated alkyl chains, and other interaction parameters, we create a library of glassformers with a wide range of properties. Measurements of the glass transition temperature (Tg) show a power-law relationship between Tg and molecular weight (MW), with of the molecules, with an exponent of 0.3 ± 0.1, for Tg values spanning a range of 300-450 K. The trends in indices of refraction and expansion coefficients indicate a general increase in the glass density with MW, consistent with the trends observed in Tg variations. A notable exception to these trends was observed with the addition of alkyl and fluorinated alkyl groups, which significantly reduced Tg and increased the dynamical fragility (which is otherwise insensitive to MW). This is an indication of reduced density and increased packing frustrations in these systems, which is also corroborated by the observations of the decreasing index of refraction with increasing length of these groups. These data were used to launch a new database for glassforming materials, glass.apps.sas.upenn.edu.

Glasses denser than the supercooled liquid.

When aged below the glass transition temperature, [Formula: see text], the density of a glass cannot exceed that of the metastable supercooled liquid (SCL) state, unless crystals are nucleated. The only exception is when another polyamorphic SCL state exists, with a density higher than that of the ordinary SCL. Experimentally, such polyamorphic states and their corresponding liquid-liquid phase transitions have only been observed in network-forming systems or those with polymorphic crystalline states. In otherwise simple liquids, such phase transitions have not been observed, either in aged or vapor-deposited stable glasses, even near the Kauzmann temperature. Here, we report that the density of thin vapor-deposited films of N,N'-bis(3-methylphenyl)-N,N'-diphenylbenzidine (TPD) can exceed their corresponding SCL density by as much as 3.5% and can even exceed the crystal density under certain deposition conditions. We identify a previously unidentified high-density supercooled liquid (HD-SCL) phase with a liquid-liquid phase transition temperature ([Formula: see text]) ∼35 K below the nominal glass transition temperature of the ordinary SCL. The HD-SCL state is observed in glasses deposited in the thickness range of 25 to 55 nm, where thin films of the ordinary SCL have exceptionally enhanced surface mobility with large mobility gradients. The enhanced mobility enables vapor-deposited thin films to overcome kinetic barriers for relaxation and access the HD-SCL state. The HD-SCL state is only thermodynamically favored in thin films and transforms rapidly to the ordinary SCL when the vapor deposition is continued to form films with thicknesses more than 60 nm.

Experimental competition induces immediate and lasting effects on the neurogenome in free-living female birds.

Periods of social instability can elicit adaptive phenotypic plasticity to promote success in future competition. However, the underlying molecular mechanisms have primarily been studied in captive and laboratory-reared animals, leaving uncertainty as to how natural competition among free-living animals affects gene activity. Here, we experimentally generated social competition among wild, cavity-nesting female birds (tree swallows, Tachycineta bicolor). After territorial settlement, we reduced the availability of key breeding resources (i.e., nest boxes), generating heightened competition; within 24 h we reversed the manipulation, causing aggressive interactions to subside. We sampled females during the peak of competition and 48 h after it ended, along with date-matched controls. We measured transcriptomic and epigenomic responses to competition in two socially relevant brain regions (hypothalamus and ventromedial telencephalon). Gene network analyses suggest that processes related to energy mobilization and aggression (e.g., dopamine synthesis) were up-regulated during competition, the latter of which persisted 2 d after competition had ended. Cellular maintenance processes were also down-regulated after competition. Competition additionally altered methylation patterns, particularly in pathways related to hormonal signaling, suggesting those genes were transcriptionally poised to respond to future competition. Thus, experimental competition among free-living animals shifts gene expression in ways that may facilitate the demands of competition at the expense of self-maintenance. Further, some of these effects persisted after competition ended, demonstrating the potential for epigenetic biological embedding of the social environment in ways that may prime individuals for success in future social instability.

A novel TUFM homozygous variant in a child with mitochondrial cardiomyopathy expands the phenotype of combined oxidative phosphorylation deficiency 4.

Translation of mitochondrial-specific DNA is required for proper mitochondrial function and energy production. For this purpose, an elaborate network of dedicated molecular machinery including initiation, elongation and termination factors exists. We describe a patient with an unusual phenotype and a novel homozygous missense variant in TUFM (c.344A>C; p.His115Pro), encoding mtDNA translation elongating factor Tu (EFTu). To date, only four patients have been reported with bi-allelic mutations in TUFM, leading to combined oxidative phosphorylation deficiency 4 (COXPD4) characterized by severe early-onset lactic acidosis and progressive fatal infantile encephalopathy. The patient presented here expands the phenotypic features of TUFM-related disease, exhibiting lactic acidosis and dilated cardiomyopathy without progressive encephalopathy. This warrants the inclusion of TUFM in differential diagnosis of metabolic cardiomyopathy. Cases that further refine genotype-phenotype associations and characterize the molecular basis of mitochondrial disorders allow clinicians to predict disease prognosis, greatly impacting patient care, as well as provide families with reproductive planning options.

Methylmercury Exposure Reduces the Auditory Brainstem Response of Zebra Finches (Taeniopygia guttata ).

Mercury contamination from mining and fossil fuel combustion causes damage to humans and animals worldwide. Mercury exposure has been implicated in mammalian hearing impairment, but its effect on avian hearing is unknown. In this study, we examined whether lifetime dietary mercury exposure affected hearing in domestic zebra finches (Taeniopygia guttata) by studying their auditory brainstem responses (ABRs). Zebra finches exposed to mercury exhibited elevated hearing thresholds, decreased amplitudes, and longer latencies in the ABR, the first evidence of mercury-induced hearing impairment in birds. Birds are a more appropriate model for the human auditory spectrum than most mammals because of similarities in frequency discrimination, vocal learning, and communication behavior. When mercury is considered in combination with other anthropogenic stressors such as noise pollution and habitat alteration, the hearing impairments we document here could substantially degrade avian auditory communication in wild birds.

Widespread gene conversion in centromere cores.

Centromeres are the most dynamic regions of the genome, yet they are typified by little or no crossing over, making it difficult to explain the origin of this diversity. To address this question, we developed a novel CENH3 ChIP display method that maps kinetochore footprints over transposon-rich areas of centromere cores. A high level of polymorphism made it possible to map a total of 238 within-centromere markers using maize recombinant inbred lines. Over half of the markers were shown to interact directly with kinetochores (CENH3) by chromatin immunoprecipitation. Although classical crossing over is fully suppressed across CENH3 domains, two gene conversion events (i.e., non-crossover marker exchanges) were identified in a mapping population. A population genetic analysis of 53 diverse inbreds suggests that historical gene conversion is widespread in maize centromeres, occurring at a rate >1x10(-5)/marker/generation. We conclude that gene conversion accelerates centromere evolution by facilitating sequence exchange among chromosomes.