Recent research on anti-oncogenes will certainly provide the strongest support for the hypothesis of negative growth control. However, in discussing normal growing cells vs. nongrowing cells, the authors focus on lines of evidence from cell fusion studies and unique nonproliferating-specific gene expression. A series of studies on heterokaryons, produced by fusion between senescent fibroblasts and their young counterparts or transformed derivatives, suggests that the cessation of proliferation in the former cells is controlled by "dominant factors". This impression is based on the observation that in such hybrids, DNA synthesis in the replicating cells is turned off by factors associated with the nuclei of senescent fibroblasts. Ultrastructural localization by gold labeling shows that statin is specifically localized at the nuclear lamina region, facing the nucleoplasm. By manipulating the concentration of serum, one can regulate cells to stay in or leave the G0 growth block.

INTRODUCTION. POSITIVE CONTROL of CELL PROLIFERATION. The Regulation of Cell Senescence. Expression of Growth-Related Genes in Senescent Human Diploid Fibroblasts. Changes in Gene Expression during Senescence/Immortalization of Mouse Embryo Fibroblasts. Nuclear Responsiveness to Intracellular Signals in Normal and Senescent Lymphocytes. Activation Defects in T Cells from Old Mice. Age-Re-lated Differences in DNA Polymerase Alpha Specific Activity: Potential for Interaction in DNA Repair. Characterization of Proliferating Cell Nuclear Antigen (PCNA), or Cyclin, as a Cellular Marker for S-Phase Cells.. NEGATIVE CONTROL of CELL PROLIFERATION. Cellular Senescence: The Result of a Genetic Program. Inhibitors of DNA Synthesis in Senescent and Quiescent Human Diploid Fibroblasts. Growth Control in Cultured 3T3 Fibroblasts: Molecular Properties of a Growth Regulatory Factor Isolated from Conditioned Medium. Programmed Gene Expressions Suggest Multiple Blocks to Replication during Cell Aging. GENERAL RELATED SUBJECTS. A Potential Role for Interspersed Repetitive Sequence Elements in Negative Growth Regulation. DNA Methylation, Maintenance CpG-Methylase, and Senescence. Finite Proliferative Capacity of Syrian Hamster Fetal and Adult Fibroblasts In Vitro: A Model System for the Analysis of the Cellular Basis of Aging. Cellular Factors Related to Cessation of Proliferation and Differentiation. Control of Muscle Differentiation by Mitogen: A Rationale for Multiple Restriction Points in the Cell Cycle. SUMMARY. Index.

It is well-known that pluripotent human embryonic stem cells (hPSC) can differentiate into any cell type. Recently, we reported that hPSC colonies enter stasis when immersed in an extremely soft hydrogel comprising hydroxyl-functional block copolymer worms (I. Canton, N. J. Warren, A. Chahal, K. Amps, A. Wood, R. Weightman, E. Wang, H. Moore and S. P. Armes, ACS Centr. Sci., 2016, 2, 65-74). The gel modulus and chemical structure of this synthetic hydrogel are similar to that of natural mucins, which are implicated in the mechanism of diapause for mammalian embryos. Does stasis induction occur merely because of the very soft nature of such hydrogels or does chemical functionality also play a role? Herein, we address this key question by designing a new hydrogel of comparable softness in which the PGMA stabilizer chains are replaced with non-hydroxylated poly(ethylene glycol) [PEG]. Immunolabeling studies confirm that hPSC colonies immersed in such PEG-based hydrogels do not enter stasis but instead proliferate (and differentiate if no adhesion substrate is present). However, pluripotency is retained if an appropriate adhesion substrate is provided. Thus, the chemical functionality of the hydrogel clearly plays a decisive role in the stasis induction mechanism.

Overweight children and adolescents are at high risk for adult and late life obesity. This report investigates some underlying mechanisms contributing to obesity during early life in an animal model. We generated a strain of transgenic mice, cU2, overexpressing human microRNA 34c, a microRNA functionally implicated in adipogenesis. Male and female cU2 mice exhibit significant weight gain, accompanied by marked increase in abdominal fat mass and metabolic abnormalities, including reduction of both glucose clearance rate and insulin sensitivity, as early as two months of age. Adipogenesis derailment at this early age is suggested by decreased expression of adiponectin, the fat mass and obesity-associated gene, and the adiponectin receptor R1, coupled with a reduction of the brown fat biomarker PAT2 and the adipogenesis inhibitor SIRT1. Notably, adiponectin is an important adipokine and an essential regulator of glucose and fatty acid homeostasis. cU2 mice may provide a crucial animal model for investigating the role of miR-34c in early onset insulin resistance and visceral fat mass increase, contributing to accelerated body weight gain and metabolic disorders. Intervention in this dysregulation may open a new preventive strategy to control early-life weight gain and abnormal insulin resistance, and thus prevalent adult and late life obesity.

Purpose A mouse model of Alzheimer's disease demonstrates reduced beta-amyloid levels in the whole brain, associated with a gain of hippocampal memory, after drinking taurine-enriched water; this suggests that a taurine supplement could be a promising treatment for cognitive deficit. The objective of this study is to establish a methodology for quantifying taurine in the whole brain, taking advantage of the rapid development of non-invasive imaging techniques such as magnetic resonance imaging and magnetic resonance spectroscopy (MRS). Procedures Single-voxel proton MRS was used to obtain quantifiable taurine peaks at 3.25 and 3.43 ppm. Quantitative MRS results were obtained in C57BL/6 mice of various age groups: 4, 11, 18, and 27 months old. Results Compared with the 4-month-old group, taurine levels dropped significantly only at 27 months of age (p = 0.03). However, a significant decrease of N-acetyl-aspartate (NAA) in the brain was observed at both 18 and 27 months (p = 0.03 and p = 0.02). In addition, MRS-measured taurine level is highly correlated with hippocampal volume (r = 0.95). Conclusions These results suggest that decreased taurine levels in the brain could be used as biomarkers for hippocampal changes and are fully translatable into putative cognitive loss in both animal models and human studies without the ex vivo approach.

The occurrence of myocardial infarction (MI) increases appreciably with age. In the Framingham Heart Study, the incidence of MI more than doubles for men and increases more than five‐fold in women (ages 55–64 years compared to 85–94 years). MicroRNAs (miRNAs) quantitatively regulate their target's expression post‐transcriptionally by either silencing action through binding at the 3′UTR domains or degrading the messages at their coding regions. In either case, these regulations affect the cardiac transcriptional output and cardiac function. Among the known cardiac associated miRNA, miRNA‐1, miRNA‐133a, and miRNA‐34a have been shown to induce adverse structural remodeling to impair cardiac contractile function. In the present study, an in vivo model of MI in young (3 month) and old (22 month) mice is used to investigate the possible role whereby these three miRNAs exert negative effects on heart function following MI. Herein we demonstrate that in older mouse heart, all three microRNAs show increased levels of expression, while miRNA‐1 shows a further increase in old mouse heart following MI, which corresponds to left ventricular (LV) wall thinning. These structural changes in cardiac tissue may causes downstream LV dilation and subsequent LV dysfunction. Results presented here suggest that significantly elevated levels of miRNA‐1 in post‐MI old heart could be predictive of cardiac injury in older mice as the high risk biomarker for MI in older individuals.

1Advanced Genomic Technology, LLC, Louisville, Kentucky, USA. 2Department of Neurology & Neurosurgery, McGill University, Montréal, Québec, Canada. 3Memory Clinic, Jewish General Hospital, Montréal, Québec, Canada. 4Bloomfield Centre for Research in Aging, Lady Davis Institute, Montréal, Québec, Canada. 5Department of Clinical Neurosciences, Jewish General Hospital, Montréal, Québec, Canada. 6Division of Geriatric Medicine of the Jewish General Hospital, Montréal, Québec, Canada.

Though defective genome maintenance and DNA repair have long been known to promote phenotypes of premature aging, the role protein methylation plays in these processes is only now emerging. We have recently identified the first N-terminal methyltransferase, NRMT1, which regulates protein-DNA interactions and is necessary for both accurate mitotic division and nucleotide excision repair. To demonstrate if complete loss of NRMT1 subsequently resulted in developmental or aging phenotypes, we constructed the first NRMT1 knockout (Nrmt1(-/-)) mouse. The majority of these mice die shortly after birth. However, the ones that survive, exhibit decreased body size, female-specific infertility, kyphosis, decreased mitochondrial function, and early-onset liver degeneration; phenotypes characteristic of other mouse models deficient in DNA repair. The livers from Nrmt1(-/-) mice produce less reactive oxygen species (ROS) than wild type controls, and Nrmt1(-/-) mouse embryonic fibroblasts show a decreased capacity for handling oxidative damage. This indicates that decreased mitochondrial function may benefit Nrmt1(-/-) mice and protect them from excess internal ROS and subsequent DNA damage. These studies position the NRMT1 knockout mouse as a useful new system for studying the effects of genomic instability and defective DNA damage repair on organismal and tissue-specific aging.

BackgroundOver‐expression of the heme‐degrading enzyme, heme oxygenase‐1 (HO‐1) promotes iron deposition, mitochondrial damage, and autophagy in astrocytes and enhances the vulnerability of nearby neuronal constituents to oxidative injury. These neuropathological features and aberrant brain microRNA (miRNA) expression patterns have been implicated in the etiopathogeneses of various neurodevelopmental and aging‐related neurodegenerative disorders.ObjectiveTo correlate glial HO‐1 overexpression with altered miRNA patterns, which have been linked to the aforementioned “core” neuropathological features.MethodsmiRNA microchip assays were performed on HMOX1‐ and sham‐transfected primary rat astroglia and affected miRNAs were further validated by qPCR. The roles of the heme degradation products, carbon monoxide (CO), iron (Fe) and bilirubin on miRNA expression were assessed and salient mRNA targets of the impacted miRNAs were ascertained.ResultsIn HMOX1‐transfected astrocytes, rno‐miR‐140*, rno‐miR‐17, and rno‐miR‐16 were significantly up‐regulated, and rno‐miR‐297, rno‐miR‐206, rno‐miR‐187, rno‐miR‐181a, rno‐miR‐138 and rno‐miR‐29c were down‐regulated, compared to sham‐transfected controls. CO and Fe were implicated in the HMOX1 effects, whereas bilirubin was inert or counteracted the HMOX1‐related changes. mRNA levels of Ngfr, Vglut1, Mapk3, Tnf‐α, and Sirt1, known targets of the down‐regulated miRNAs and abnormal in various human brain disorders, were significantly increased in the HMOX‐1‐transfected astrocytes.ConclusionsIn chronic CNS disorders, altered expression of salient miRNAs and their mRNA targets may contribute to the neural damage accruing from the over‐expression of glial HO‐1. © 2015 Wiley Periodicals, Inc. GLIA 2015;63:1270–1284

Protein targeting to glycogen (PTG) is a ubiquitously expressed scaffolding protein that critically regulates glycogen levels in many tissues, including the liver, muscle and brain. However, its importance in transformed cells has yet to be explored in detail. Since recent studies have demonstrated an important role for glycogen metabolism in cancer cells, we decided to assess the effect of PTG levels on the ability of human hepatocellular carcinoma (HepG2) cells to respond to metabolic stress. Although PTG expression did not significantly affect the proliferation of HepG2 cells under normal culture conditions, we determined that PTG plays an important role during glucose deprivation. Overexpression of PTG protected cells from cell death in the absence of glucose, whereas knocking down PTG further promoted cytotoxicity, as measured by the release of lactate dehydrogenase (LDH) into the media. Additionally, we demonstrated that PTG attenuates glucose deprivation induced haeme oxygenase-1 (HO-1) expression, suggesting that PTG protects against glucose deprivation-induced oxidative stress. Indeed, treating cells with the antioxidant N-acetyl cysteine (NAC) rescued cells from cytotoxicity caused by glucose deprivation. Finally, we showed that loss of PTG resulted in enhanced autophagy. In control cells, glucose deprivation suppressed autophagy as determined by the increase in the levels of p62, an autophagy substrate. However, in knockdown cells, this suppression was relieved. Blockade of autophagy also attenuated cytotoxicity from glucose deprivation in PTG knockdown cells. Taken together, our findings identify a novel role for PTG in protecting hepatocellular carcinoma cells from metabolic stress, in part by regulating oxidative stress and autophagy.

Circulating microRNAs, present either in the cellular component, peripheral blood mononuclear cells (PBMC), or in cell-free plasma, have emerged as biomarkers for age-dependent systemic, disease-associated changes in many organs. Previously, we have shown that microRNA (miR)-34a is increased in circulating PBMC of Alzheimer’s disease (AD) patients. In the present study, we show that this microRNA’s sister, miR-34c, exhibits even greater increase in both cellular and plasma components of AD circulating blood samples, compared to normal age-matched controls. Statistical analysis shows the accuracy of levels of miR-34c assayed by receiver operating characteristic (ROC) analysis: the area under the curve is 0.99 (p < 0.0001) and the 95% confidence level extends from 0.97 to 1. Pearson correlation between miR-34c levels and mild and moderate AD, as defined by the mini-mental state examination (MMSE), shows an r-value of -0.7, suggesting a relatively strong inverse relationship between the two parameters. These data show that plasma levels of microRNA 34c are much more prominent in AD than those of its sister, miR-34a, or than its own level in PBMC. Transfection studies show that miR-34c, as does its sister miR-34a, represses the expression of several selected genes involved in cell survival and oxidative defense pathways, such as Bcl2, SIRT1 and others, in cultured cells. Taken together, our results indicate that increased levels of miR-34c in both PBMC and plasma may reflect changes in circulating blood samples in AD patients, compared to age-matched normal controls.

Burns are a significant health challenge and healing can result in scar formation. Chitosan, a derivative of chitin, has been used to promote wound healing. In this study we used gene expression profiling in a mouse model of full thickness cutaneous burn to assess the benefits of treating with a chitosan lactate dressing. Three days after wounding mice treated with chitosan showed increased expression of genes associated with formation of granulation tissue. At a later time point, seven days after wounding, genes that initially showed increased expression were now down-regulated, and there was increased expression of genes involved in remodeling suggesting that the chitosan treatment results in accelerated healing. Quantitative RT-PCR showed modulated mRNA levels for TGFβ1 by the chitosan dressing. TGFβ1 initially promotes healing but extended activity can result in scarring. Importantly we found that expression was elevated at day three, but decreased at day seven suggesting that chitosan treatment will not result in scar formation, and may even be beneficial in preventing scar formation. Additionally, the biphasic regulation of expression of TGFβ1 could be a powerful biomarker for future studies of the wound-healing potential of chitosan based and other treatments for burn wounds.

Complexity in higher animals derives in part from various modalities of protein-coding gene expression regulation, including microRNA repression by binding to 3'-untranslated regions (UTRs) of specific genes. Reporter constructs containing candidate microRNA target sites are a popular approach of functional studies, and full-length 3'-UTR sequences are preferred because they contain all regulatory elements and preserve higher order structure as much as possible. However, this approach is often handicapped by the extreme length of the 3'-UTR. Here, we present a rapid and accurate cloning procedure to generate full-length 3'-UTR reporter constructs by recombinogenic engineering (recombineering) in vivo cloning. The approach includes making retrieval constructs by sequence- and ligation-independent cloning (SLIC) and retrieving the full-length 3'-UTR in one exon to the retrieval construct from a bacterial artificial chromosome (BAC) by recombineering to generate the final full-length 3'-UTR reporter construct for the gene of interest. This method is successfully implemented with mouse full-length 3'-UTRs of Igf1 (6.5 kb), Igf1r (7.5 kb), and Sp1 (5.5 kb). Expansion of this method is adaptable to retrieve 3'-UTRs encoded in more than one exon by removing the introns from the BAC first with recombineering. This method will advance functional studies of regulation of gene expression at the post-transcriptional level through microRNA suppression.

MicroRNAs in blood samples have been identified as an important class of biomarkers, which can reflect physiological changes from cancer to brain dysfunction. In this report we identify concordant increases in levels of expression of miR-34a in brain and two components of mouse blood samples, peripheral blood mononuclear cells (PBMCs) and plasma, from 2 day old neonates through young adulthood and mid-life to old age at 25 months. Levels of this microRNA's prime target, silent information regulator 1 (SIRT1), in brain and the two blood-derived specimens decrease with age inversely to miR-34a, starting as early as 4 months old, when appreciable tissue aging has not yet begun. Our results suggest that: 1. Increased miR-34a and the reciprocal decrease of its target, SIRT1, in blood specimens are the accessible biomarkers for age-dependent changes in brain; and 2. these changes are predictors of impending decline in brain function, as early as in young adult mice.

Alzheimer's disease (AD) is a primary degenerative brain disorder that also affects peripheral tissues such as peripheral blood mononuclear cells (PBMC). Several hypotheses for the pathogenesis of AD are intrinsically related to mechanisms of aging and include excitotoxicity, neuroinflammation, mitochondrial dysfunction, and enhanced oxidative stress. Delineation of biochemical alterations in AD blood components may provide insights into the pathogenesis of sporadic AD and facilitate the development of diagnostic and prognostic biomarkers. Here we describe our published experimental approaches and PBMC gene expression data in sporadic AD, with emphasis on the role of altered redox homeostasis in the development of this common affliction.

Long-lived mutant mice, both Ames dwarf and growth hormone receptor gene-disrupted or knockout strains, exhibit heightened cognitive robustness and altered IGF1 signaling in the brain. Here, we report, in both these long-lived mice, that three up-regulated lead microRNAs, miR-470, miR-669b, and miR-681, are involved in posttranscriptional regulation of genes pertinent to growth hormone/IGF1 signaling. All three are most prominently localized in the hippocampus and correspond to reduced expression of key IGF1 signaling genes: IGF1, IGF1R, and PI3 kinase. The decline in these genes' expression translates into decreased phosphorylation of downstream molecules AKT and FoxO3a. Cultures transfected with either miR-470, miR-669b, or miR-681 show repressed endogenous expression of all three genes of the IGF1 signaling axis, most significantly IGF1R, while other similarly up-regulated microRNAs, including let-7g and miR-509, do not induce the same levels of repression. Transduction study in IGF1-responsive cell cultures shows significantly reduced IGF1R expression, and AKT to some extent, most notably by miR-681. This is accompanied by decreased levels of downstream phosphorylated forms of AKT and FoxO3a upon IGF1 stimulation. Suppression of IGF1R by the three microRNAs is further validated by IGF1R 3'UTR reporter assays. Taken together, our results suggest that miR-470, miR-669b, and miR-681 are all functionally able to suppress IGF1R and AKT, two upstream genes controlling FoxO3a phosphorylation status. Their up-regulation in growth hormone signaling-deficient mutant mouse brain suggests reduced IGF1 signaling at the posttranscriptional level, for numerous gains of neuronal function in these long-lived mice.

The decline in cognitive robustness with aging can be attributed to complex genetic pathways involving many cellular dysfunctions, cumulative over time, precipitating in frailty and loss of wellness in the elderly brain. The size and health of the neuronal cell population determines cognitive robustness in mammals. A transgenic mouse model over-expressing Bcl-2 has been shown to rescue neurons from naturally occurring cell death (NOCD). Here we show that in the brain of calorie-restricted (CR) mice, there is an age-dependent decreased expression of microRNAs mmu-miR-181a-1*, mmu-miR-30e and mmu-miR-34a, with a corresponding gain in Bcl-2 expression, and decreases in pro-apoptosis genes such as Bax and cleavage of Caspases. Functional characterization shows that these miRNAs repress Bcl-2 expression by the 3'UTR reporter assays, accompanied by loss of this gene's endogenous expression, and a gain in pro-apoptosome-specific proteins. Over-expression of these miRNAs increases the rate of apoptosis, accompanied by a decline in Bcl-2 expression in miRNA-transfected mouse and human cell lines. We report here that down-regulation of miR-34a, -30e, and -181a permits their shared target gene expression (Bcl-2) to remain at a high level without post-transcriptional repression, accompanied by concomitant low levels of Bax expression and Caspase cleaving; this chain event may be a part of the underlying mechanism contributing to the gain in neuronal survival in long-lived CR-fed mice.