Retrospective assessment of metabolic syndrome components in early adult life on vegetarian dietary status.

Background: Metabolic syndrome (MetS) encompasses several clinical presentations that include truncal obesity and insulin resistance at its core. MetS afflicts 23% of the adult US population, increasing their risk of diabetes and cardiovascular disease. Many studies have indicated the importance of a vegetarian diet in improving overall health and more specifically MetS components. Unfortunately, these findings have been inconsistent and cannot be extended to examine effects on MetS incidence in the younger adult population. Objective: This study aimed to conduct a retrospective analysis of a vegetarian vs. non-vegetarian dietary status in young adults (age 18-24) based on MetS components in later adulthood (age 20-30). This study focuses on elucidating any relationship between a vegetarian diet and MetS components of central obesity, hypertension, and hyperlipidemia. Methods: Waves 3 and 4 data were acquired from AddHealth. One-to-one propensity score matched vegetarians to non-vegetarians in a cohort of 535 women and 159 men. Logistical regression assessed the relationship between vegetarian status and MetS components, including truncal obesity (cm), hypertension (normal, pre-HT, HT1, and HT2), and hyperlipidemia (high and low). Results MetS components from ages 20 to 30 are not associated with vegetarian dietary status. Truncal obesity [N = 694; M = 92.82 cm; OR 0.999; p = 0.893; 95% CI (0.980, 1.017)]; hypertension [N = 694; OR 0.949; p = 0.638; 95% CI (0.764, 1.179)]; hyperlipidemia [N = 694; OR 0.840; p = 0.581; 95% CI (0.453, 1.559)]. Conclusion: Current study results were consistent with previous findings suggesting that consumption of a vegetarian diet cannot be directly linked to MetS outcomes. However, further investigation should be completed as MetS is a risk factor for several chronic diseases.

Specific PIWI-Interacting RNAs and Related Small Noncoding RNAs Are Associated With Ovarian Aging in Ames Dwarf (df/df) Mice.

The Ames dwarf (df/df) mouse is a well-established model for delayed aging. MicroRNAs (miRNAs), the most studied small noncoding RNAs (sncRNAs), may regulate ovarian aging to maintain a younger ovarian phenotype in df/df mice. In this study, we profile other types of ovarian sncRNAs, PIWI-interacting RNAs (piRNAs) and piRNA-like RNAs (piLRNAs), in young and aged df/df and normal mice. Half of the piRNAs derive from transfer RNA fragments (tRF-piRNAs). Aging and dwarfism alter the ovarian expression of these novel sncRNAs. Specific tRF-piRNAs that increased with age might target and decrease the expression of the breast cancer antiestrogen resistance protein 3 (BCAR3) gene in the ovaries of old df/df mice. A set of piLRNAs that decreased with age and map to D10Wsu102e mRNA may have trans-regulatory functions. Other piLRNAs that decreased with age potentially target and may de-repress transposable elements, leading to a beneficial impact on ovarian aging in df/df mice. These results identify unique responses in ovarian tissues with regard to aging and dwarfism. Overall, our findings highlight the complexity of the aging effects on gene expression and suggest that, in addition to miRNAs, piRNAs, piLRNAs, tRF-piRNAs, and their potential targets can be central players in the maintenance of a younger ovarian phenotype in df/df mice.

Profiling of tRNA Halves and YRNA Fragments in Serum and Tissue From Oral Squamous Cell Carcinoma Patients Identify Key Role of 5' tRNA-Val-CAC-2-1 Half.

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer and, as indicated by The Oral Cancer Foundation, kills at an alarming rate of roughly one person per hour. With this study, we aimed at better understanding disease mechanisms and identifying minimally invasive disease biomarkers by profiling novel small non-coding RNAs (specifically, tRNA halves and YRNA fragments) in both serum and tumor tissue from humans. Small RNA-Sequencing identified multiple 5' tRNA halves and 5' YRNA fragments that displayed significant differential expression levels in circulation and/or tumor tissue, as compared to control counterparts. In addition, by implementing a modification of weighted gene coexpression network analysis, we identified an upregulated genetic module comprised of 5' tRNA halves and miRNAs (miRNAs were described in previous study using the same samples) with significant association with the cancer trait. By consequently implementing miRNA-overtargeting network analysis, the biological function of the module (and by "guilt by association," the function of the 5' tRNA-Val-CAC-2-1 half) was found to involve the transcriptional targeting of specific genes involved in the negative regulation of the G1/S transition of the mitotic cell cycle. These findings suggest that 5' tRNA-Val-CAC-2-1 half (reduced in serum of OSCC patients and elevated in the tumor tissue) could potentially serve as an OSCC circulating biomarker and/or target for novel anticancer therapies. To our knowledge, this is the first time that the specific molecular function of a 5'-tRNA half is specifically pinpointed in OSCC.

Curriculum mapping as a tool to facilitate curriculum development: a new School of Medicine experience.

BACKGROUND: Every curriculum needs to be reviewed, implemented and evaluated; it must also comply with the regulatory standards. This report demonstrates the value of curriculum mapping (CM), which shows the spatial relationships of a curriculum, in developing and managing an integrated medical curriculum. METHODS: A new medical school developed a clinical presentation driven integrated curriculum that incorporates the active-learning pedagogical practices of many educational institutions worldwide while adhering to the mandated requirements of the accreditation bodies. A centralized CM process was run in parallel as the curriculum was being developed. A searchable database, created after the CM data was uploaded into an electronic curriculum management system, was used to ensure placing, integrating, evaluating and revising the curricular content appropriately. RESULTS: CM facilitated in a) appraising the content integration, b) identifying gaps and redundancies, c) linking learning outcomes across all educational levels (i.e. session to course to program), c) organizing the teaching schedules, instruction methods, and assessment tools and d) documenting compliance with accreditation standards. CONCLUSIONS: CM is an essential tool to develop, review, improve and refine any integrated curriculum however complex. Our experience, with appropriate modifications, should help other medical schools efficiently manage their curricula and fulfill the accreditation requirements at the same time.

Data Mining of Small RNA-Seq Suggests an Association Between Prostate Cancer and Altered Abundance of 5' Transfer RNA Halves in Seminal Fluid and Prostatic Tissues.

Extracellular RNAs are gaining clinical interest as biofluid-based noninvasive markers for diseases, especially cancer. In particular, derivatives of transfer RNA (tRNA) are emerging as a new class of small-noncoding RNAs with high biomarker potential. We and others previously reported alterations in serum levels of specific tRNA halves in disease states including cancer. Here, we explored seminal fluid for tRNA halves as potential markers of prostate cancer. We found that 5' tRNA halves are abundant in seminal fluid and are elevated in prostate cancer relative to noncancer patients. Importantly, most of these tRNA halves are also detectable in prostatic tissues, and a subset were increased in malignant relative to adjacent normal tissue. These findings emphasize the potential of 5' tRNA halves as noninvasive markers for prostate cancer screening and diagnosis and provide leads for future work to elucidate a putative role of the 5' tRNA halves in carcinogenesis.

Organ reserve, excess metabolic capacity, and aging.

"Organ reserve" refers to the ability of an organ to successfully return to its original physiological state following repeated episodes of stress. Clinical evidence shows that organ reserve correlates with the ability of older adults to cope with an added workload or stress, suggesting a role in the process of aging. Although organ reserve is well documented clinically, it is not clearly defined at the molecular level. Interestingly, several metabolic pathways exhibit excess metabolic capacities (e.g., bioenergetics pathway, antioxidants system, plasticity). These pathways comprise molecular components that have an excess of quantity and/or activity than that required for basic physiological demand in vivo (e.g., mitochondrial complex IV or glycolytic enzymes). We propose that the excess in mtDNA copy number and tandem DNA repeats of telomeres are additional examples of intrinsically embedded structural components that could comprise excess capacity. These excess capacities may grant intermediary metabolism the ability to instantly cope with, or manage, added workload or stress. Therefore, excess metabolic capacities could be viewed as an innate mechanism of adaptability that substantiates organ reserve and contributes to the cellular defense systems. If metabolic excess capacities or organ reserves are impaired or exhausted, the ability of the cell to cope with stress is reduced. Under these circumstances cell senescence, transformation, or death occurs. In this review, we discuss excess metabolic and structural capacities as integrated metabolic pathways in relation to organ reserve and cellular aging.

Caloric restriction impacts plasma microRNAs in rhesus monkeys.

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR-regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR-125a-5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR-associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR-induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA-based homeostatic mechanisms to coordinate a program of delayed aging.

MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner.

In mammals, extracellular miRNAs circulate in biofluids as stable entities that are secreted by normal and diseased tissues, and can enter cells and regulate gene expression. Drosophila melanogaster is a proven system for the study of human diseases. They have an open circulatory system in which hemolymph (HL) circulates in direct contact with all internal organs, in a manner analogous to vertebrate blood plasma. Here, we show using deep sequencing that Drosophila HL contains RNase-resistant circulating miRNAs (HL-miRNAs). Limited subsets of body tissue miRNAs (BT-miRNAs) accumulated in HL, suggesting that they may be specifically released from cells or particularly stable in HL. Alternatively, they might arise from specific cells, such as hemocytes, that are in intimate contact with HL. Young and old flies accumulated unique populations of HL-miRNAs, suggesting that their accumulation is responsive to the physiological status of the fly. These HL-miRNAs in flies may function similar to the miRNAs circulating in mammalian biofluids. The discovery of these HL-miRNAs will provide a new venue for health and disease-related research in Drosophila.

Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear. Since MB is redox-active, we investigated its effect on the NAD/NADH ratio in IMR90 cells. The transient increase in NAD/NADH observed in MB-treated cells triggered an investigation of the energy regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex-IV biogenesis. Subsequently MB-treated cells exhibited >100% increase in complex-IV activity and a 28% decline in cellular oxidants. The telomeres erosion rate was also significantly lower in MB-treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence. We identified that the anti-senescence activity of MB (transient activator) was 8-times higher than that of AICAR (chronic activator). Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense pathways as a possible key factor in MB's potent anti-senescence activity.

Circulating microRNA signature of genotype-by-age interactions in the long-lived Ames dwarf mouse.

Recent evidence demonstrates that serum levels of specific miRNAs significantly change with age. The ability of circulating sncRNAs to act as signaling molecules and regulate a broad spectrum of cellular functions implicates them as key players in the aging process. To discover circulating sncRNAs that impact aging in the long-lived Ames dwarf mice, we conducted deep sequencing of small RNAs extracted from serum of young and old mice. Our analysis showed genotype-specific changes in the circulating levels of 21 miRNAs during aging [genotype-by-age interaction (GbA)]. Genotype-by-age miRNAs showed four distinct expression patterns and significant overtargeting of transcripts involved in age-related processes. Functional enrichment analysis of putative and validated miRNA targets highlighted cellular processes such as tumor suppression, anti-inflammatory response, and modulation of Wnt, insulin, mTOR, and MAPK signaling pathways, among others. The comparative analysis of circulating GbA miRNAs in Ames mice with circulating miRNAs modulated by calorie restriction (CR) in another long-lived mouse suggests CR-like and CR-independent mechanisms contributing to longevity in the Ames mouse. In conclusion, we showed for the first time a signature of circulating miRNAs modulated by age in the long-lived Ames mouse.

Circulating small non-coding RNA signature in head and neck squamous cell carcinoma.

The Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common human cancer, causing 350,000 individuals die worldwide each year. The overall prognosis in HNSCC patients has not significantly changed for the last decade. Complete understanding of the molecular mechanisms in HNSCC carcinogenesis could allow an earlier diagnosis and the use of more specific and effective therapies. In the present study we used deep sequencing to characterize small non-coding RNAs (sncRNAs) in serum from HNSCC patients and healthy donors. We identified, for the first time, a multi-marker signature of 3 major classes of circulating sncRNAs in HNSCC, revealing the presence of circulating novel and known miRNAs, and tRNA- and YRNA-derived small RNAs that were significantly deregulated in the sera of HNSCC patients compared to healthy controls. By implementing a triple-filtering approach we identified a subset of highly biologically relevant miRNA-mRNA interactions and we demonstrated that the same genes/pathways affected by somatic mutations in cancer are affected by changes in the abundance of miRNAs. Therefore, one important conclusion from our work is that during cancer development, there seems to be a convergence of oncogenic processes driven by somatic mutations and/or miRNA regulation affecting key cellular pathways.

ApoHRP-based assay to measure intracellular regulatory heme.

The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-ß (Aß) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.

Deep Sequencing of Serum Small RNAs Identifies Patterns of 5' tRNA Half and YRNA Fragment Expression Associated with Breast Cancer.

Small noncoding RNAs circulating in the blood may serve as signaling molecules because of their ability to carry out a variety of cellular functions. We have previously described tRNA- and YRNA-derived small RNAs circulating as components of larger complexes in the blood of humans and mice; the characteristics of these small RNAs imply specific processing, secretion, and physiological regulation. In this study, we have asked if changes in the serum abundance of these tRNA and YRNA fragments are associated with a diagnosis of cancer. We used deep sequencing and informatics analysis to catalog small RNAs in the sera of breast cancer cases and normal controls. 5' tRNA halves and YRNA fragments are abundant in both groups, but we found that a breast cancer diagnosis is associated with changes in levels of specific subtypes. This prompted us to look at existing sequence datasets of serum small RNAs from 42 breast cancer cases, taken at the time of diagnosis. We find significant changes in the levels of specific 5' tRNA halves and YRNA fragments associated with clinicopathologic characteristics of the cancer. Although these findings do not establish causality, they suggest that circulating 5' tRNA halves and YRNA fragments with known cellular functions may participate in breast cancer syndromes and have potential as circulating biomarkers. Larger studies with multiple types of cancer are needed to adequately evaluate their potential use for the development of noninvasive cancer screening.

Acarbose, 17-α-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males.

Four agents--acarbose (ACA), 17-α-estradiol (EST), nordihydroguaiaretic acid (NDGA), and methylene blue (MB)--were evaluated for lifespan effects in genetically heterogeneous mice tested at three sites. Acarbose increased male median lifespan by 22% (P < 0.0001), but increased female median lifespan by only 5% (P = 0.01). This sexual dimorphism in ACA lifespan effect could not be explained by differences in effects on weight. Maximum lifespan (90th percentile) increased 11% (P < 0.001) in males and 9% (P = 0.001) in females. EST increased male median lifespan by 12% (P = 0.002), but did not lead to a significant effect on maximum lifespan. The benefits of EST were much stronger at one test site than at the other two and were not explained by effects on body weight. EST did not alter female lifespan. NDGA increased male median lifespan by 8-10% at three different doses, with P-values ranging from 0.04 to 0.005. Females did not show a lifespan benefit from NDGA, even at a dose that produced blood levels similar to those in males, which did show a strong lifespan benefit. MB did not alter median lifespan of males or females, but did produce a small, statistically significant (6%, P = 0.004) increase in female maximum lifespan. These results provide new pharmacological models for exploring processes that regulate the timing of aging and late-life diseases, and in particular for testing hypotheses about sexual dimorphism in aging and health.

5'-YRNA fragments derived by processing of transcripts from specific YRNA genes and pseudogenes are abundant in human serum and plasma.

Small noncoding RNAs carry out a variety of functions in eukaryotic cells, and in multiple species they can travel between cells, thus serving as signaling molecules. In mammals multiple small RNAs have been found to circulate in the blood, although in most cases the targets of these RNAs, and even their functions, are not well understood. YRNAs are small (84-112 nt) RNAs with poorly characterized functions, best known because they make up part of the Ro ribonucleoprotein autoantigens in connective tissue diseases. In surveying small RNAs present in the serum of healthy adult humans, we have found YRNA fragments of lengths 27 nt and 30-33 nt, derived from the 5'-ends of specific YRNAs and generated by cleavage within a predicted internal loop. Many of the YRNAs from which these fragments are derived were previously annotated only as pseudogenes, or predicted informatically. These 5'-YRNA fragments make up a large proportion of all small RNAs (including miRNAs) present in human serum. They are also present in plasma, are not present in exosomes or microvesicles, and circulate as part of a complex with a mass between 100 and 300 kDa. Mouse serum contains far fewer 5'-YRNA fragments, possibly reflecting the much greater copy number of YRNA genes and pseudogenes in humans. The function of the 5'-YRNA fragments is at present unknown, but the processing and secretion of specific YRNAs to produce 5'-end fragments that circulate in stable complexes are consistent with a signaling function.

5' tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction.

BACKGROUND: Small RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30-33 nt RNAs that are processed derivatives of the 5' ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5' tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells. RESULTS: In sequencing small RNAs extracted from mouse serum, we identified abundant 5' tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5' tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100-300 kDa. The size of these particles suggest that the 5' tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5' tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5' tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5' tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction. CONCLUSIONS: We demonstrate that 5' tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

Deep sequencing identifies circulating mouse miRNAs that are functionally implicated in manifestations of aging and responsive to calorie restriction.

MicroRNAs (miRNAs) function to modulate gene expression, and through this property they regulate a broad spectrum of cellular processes. They can circulate in blood and thereby mediate cell-to-cell communication. Aging involves changes in many cellular processes that are potentially regulated by miRNAs, and some evidence has implicated circulating miRNAs in the aging process. In order to initiate a comprehensive assessment of the role of circulating miRNAs in aging, we have used deep sequencing to characterize circulating miRNAs in the serum of young mice, old mice, and old mice maintained on calorie restriction (CR). Deep sequencing identifies a set of novel miRNAs, and also accurately measures all known miRNAs present in serum. This analysis demonstrates that the levels of many miRNAs circulating in the mouse are increased with age, and that the increases can be antagonized by CR. The genes targeted by this set of age-modulated miRNAs are predicted to regulate biological processes directly relevant to the manifestations of aging including metabolic changes, and the miRNAs themselves have been linked to diseases associated with old age. This finding implicates circulating miRNAs in the aging process, raising questions about their tissues of origin, their cellular targets, and their functional role in metabolic changes that occur with aging.

Mitochondrial pharmacology: electron transport chain bypass as strategies to treat mitochondrial dysfunction.

Mitochondrial dysfunction (primary or secondary) is detrimental to intermediary metabolism. Therapeutic strategies to treat/prevent mitochondrial dysfunction could be valuable for managing metabolic and age-related disorders. Here, we review strategies proposed to treat mitochondrial impairment. We then concentrate on redox-active agents, with mild-redox potential, who shuttle electrons among specific cytosolic or mitochondrial redox-centers. We propose that specific redox agents with mild redox potential (-0.1 V; 0.1 V) improve mitochondrial function because they can readily donate or accept electrons in biological systems, thus they enhance metabolic activity and prevent reactive oxygen species (ROS) production. These agents are likely to lack toxic effects because they lack the risk of inhibiting electron transfer in redox centers. This is different from redox agents with strong negative (-0.4 V; -0.2 V) or positive (0.2 V; 0.4 V) redox potentials who alter the redox status of redox-centers (i.e., become permanently reduced or oxidized). This view has been demonstrated by testing the effect of several redox active agents on cellular senescence. Methylene blue (MB, redox potential ≅10 mV) appears to readily cycle between the oxidized and reduced forms using specific mitochondrial and cytosolic redox centers. MB is most effective in delaying cell senescence and enhancing mitochondrial function in vivo and in vitro. Mild-redox agents can alter the biochemical activity of specific mitochondrial components, which then in response alters the expression of nuclear and mitochondrial genes. We present the concept of mitochondrial electron-carrier bypass as a potential result of mild-redox agents, a method to prevent ROS production, improve mitochondrial function, and delay cellular aging. Thus, mild-redox agents may prevent/delay mitochondria-driven disorders.

mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction.

Sarcopenia is an age-associated loss of skeletal muscle mass and strength that increases the risk of disability. Calorie restriction (CR), the consumption of fewer calories while maintaining adequate nutrition, mitigates sarcopenia and many other age-related diseases. To identify potential mechanisms by which CR preserves skeletal muscle integrity during aging, we used mRNA-Seq for deep characterization of gene regulation and mRNA abundance in skeletal muscle of old mice compared with old mice subjected to CR. mRNA-Seq revealed complex CR-associated changes in expression of mRNA isoforms, many of which occur without a change in total message abundance and thus would not be detected by methods other than mRNA-Seq. Functional annotation of differentially expressed genes reveals CR-associated upregulation of pathways involved in energy metabolism and lipid biosynthesis, and downregulation of pathways mediating protein breakdown and oxidative stress, consistent with earlier microarray-based studies. CR-associated changes not noted in previous studies involved downregulation of genes controlling actin cytoskeletal structures and muscle development. These CR-associated changes reflect generally healthier muscle, consistent with CR's mitigation of sarcopenia. mRNA-Seq generates a rich picture of the changes in gene expression associated with CR, and may facilitate identification of genes that are primary mediators of CR's effects.

Deep sequencing reveals novel microRNAs and regulation of microRNA expression during cell senescence.

In cell senescence, cultured cells cease proliferating and acquire aberrant gene expression patterns. MicroRNAs (miRNAs) modulate gene expression through translational repression or mRNA degradation and have been implicated in senescence. We used deep sequencing to carry out a comprehensive survey of miRNA expression and involvement in cell senescence. Informatic analysis of small RNA sequence datasets from young and senescent IMR90 human fibroblasts identifies many miRNAs that are regulated (either up or down) with cell senescence. Comparison with mRNA expression profiles reveals potential mRNA targets of these senescence-regulated miRNAs. The target mRNAs are enriched for genes involved in biological processes associated with cell senescence. This result greatly extends existing information on the role of miRNAs in cell senescence and is consistent with miRNAs having a causal role in the process.