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Here we report on the first successful exoplanet transit observation with the Stratospheric Observatory for Infrared Astronomy (SOFIA). We observed a single transit of the hot Jupiter HD 189733 b, obtaining two simultaneous primary transit lightcurves in the B and z' bands as a demonstration of SOFIA's capability to perform absolute transit photometry. We present a detailed description of our data reduction, in particular the correlation of photometric systematics with various in-flight parameters unique to the airborne observing environment. The derived transit depths at B and z' wavelengths confirm a previously reported slope in the optical transmission spectrum of HD 189733 b. Our results give new insights to the current discussion about the source of this Rayleigh scattering in the upper atmosphere and the question of fixed limb darkening coefficients in fitting routines., 40 pages, 7 figures, accepted for publication in JATIS

Motor neurons form a specialized synapse with skeletal muscle known as the neuromuscular junction (NMJ), and degeneration of the NMJ has been implicated in disease and aging. Histone deacetylases mediate NMJ-regulated gene transcription and are involved in neurogenic muscle atrophy, although their role in age-related muscle atrophy is not known. HDAC4 and HDAC5 knockout mice are protected against surgical denervation, and pharmacological inhibition of histone deacetylases is protective in multiple models of neuromuscular disease. In this study, we examined the effect of butyrate, a histone deacetylase inhibitor, on muscle atrophy during sciatic nerve crush and age-related muscle atrophy. We demonstrate that butyrate increases histone acetylation in vivo and protects against the muscle loss induced by sciatic nerve crush and aging. Control-fed mice lost 22% of their gastrocnemius mass while the butyrate-fed mice lost only 11% one week after sciatic nerve crush surgery. Butyrate protects against the loss of cross-sectional area, increases catalase and MnSOD activity, and reduces oxidative damage during nerve crush. Consistently, butyrate protects against age-related muscle atrophy in mice by modulating antioxidant activity, reducing oxidative damage, and increasing mitochondrial biogenesis. We also report improved metabolism in old mice fed butyrate, including improved glucose tolerance and increased whole-body oxygen consumption. Future studies will determine the mechanism by which butyrate protects against age-related muscle atrophy., Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies, are age-related neurodegenerative disorders characterized by pathological a-synuclein inclusions. Synucleinopathies have common motor deficits and affect millions of patients worldwide. The A53T human a-synuclein mutation is linked to both sporadic and familial PD. Rapamycin, an mTOR inhibitor, reduces human a-synuclein accumulation and neurodegenerative phenotype in vitro and in vivo. Long-term feeding of a rapamycin diet extends mouse lifespan and the mechanisms are hypothesized to be mediated via delaying age-related diseases including PD. The aim of the study is to determine whether long-term feeding rapamycin diet at the dose that extends mouse lifespan attenuates motor deficits in neuronal A53T a-synuclein transgenic mice. A diet containing microencapsulated rapamycin (14 ppm in diet; 2.25 mg/kg body weight/day) or the microencapsulation polymer was fed to age-matched wild-type and A53T mice from 13 weeks of age. After 24 weeks of treatment, rapamycin improved performance on forepaw stepping adjustment test, accelerating rotarod test and pole test in both genders. Rapamycin also increased front stride length in male A53T mice. Total human a-synuclein levels in midbrain, striatum, brain stem, cerebellum, and spinal cord were not altered by rapamycin. Oxidative stress plays an important role in pathogenesis of neurodegenerative diseases. The lipid peroxidation product 4-hydroxynonenal has been detected in the postmortem brain of PD patients and is believed to contribute to the neurotoxic effects of a-synuclein. Levels of 4-hydroxynonenal protein adducts were significantly decreased in midbrain, striatum, and spinal cord of both genders of A53T mice as well as brain stem and cerebellum of female A53T mice. In conclusion, long-term rapamycin treatment attenuated motor deficits and reduced 4-hydroxynonenal in the central nervous system regions that subserve motor function and motor coordination in the A53T mice., The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signal transduction cascade called the unfolded protein response (UPR). The most immediate response is the attenuation of protein synthesis, initiated by autophosphorylation of PKR-like ER kinase (PERK). Recently, our group reported that calcineurin (CN) strengthened the UPR by binding to PERK and enhancing its autophosphorylation (Bollo et al. PLoSOne 5 (8): e11925). Here, we report that CN-A beta protects astrocytes from ER stress, likely by enhancing autophosphorylation of PERK. First, we found that levels of phosphorylated-PERK and CN-A beta were significantly increased in astrocytes within 1 hour of oxygen and glucose deprivation (OGD). Second, overexpression of CN-A beta significantly increased the viability of wild-type astrocytes during OGD (1 hr), but not that of PERK−/ − astrocytes. Third, co-immunoprecipitation showed that CN-A beta preferentially interacted with PERK in ER-stressed astrocytes. Fourth, experiments with recombinant proteins demonstrated that PERK autophosphorylation and oligomerization were increased in the presence of CN-A beta. Finally, rapamycin-induced dimerization of CFP-FRB-cytochrome5 (ER anchor) and YFP-FKBP-CN-A beta inside human astrocytes increased PERK phosphorylation. Taken together, we suggest a novel physiological function of the classic phosphatase CN-A beta is to bind PERK and enhance the early UPR., The subventricular zone (SVZ) is the largest reservoir of neural stem cells (NSCs) in the adult brain. Neurogenesis is reduced during aging and this decline in NSC function is thought to contribute to reduced brain function. Here, we propose immune activation and cellular senescence as contributors to age-related declines in NSC function. We observed a sharp decrease in neurogenesis in mice between the ages of 6 and 12 months. Neuroblast number was also reduced with age. We observed the appearance of β-galactosidase-positive cells, a marker of cellular senescence concomitant with reductions in neurogenesis. Senescent cells secrete factors into the extracellular environment, a phenomenon known as the senescence associated secretory phenotype (SASP). The SASP has been associated with upregulated inflammation and immune activation due to proinflammatory factors. Microglia are the prominent immune cells in the brain. Microglial activation results in the release of proinflammatory cytokines. There was no change in the number or percent of microglia cells relative to other cells in the SVZ during aging. However, both morphological changes and increased CD68 expression were observed during aging indicative of microglia becoming more activated. Interestingly, a short-term diet of encapsulated rapamycin, an inhibitor of the mTOR pathway, resulted in reduced cellular senescence in the aged brain compared to the young and an overall increase in NSC proliferation. Our results suggest that during aging there is increased inflammation in the SVZ, which may be an important contributor to declines in neural stem cell function., Increased mitochondrial reactive oxygen species (ROS) is believed to play a key role in cognitive decline associated with aging and Alzheimer's disease, but the mechanism remains unclear. Inflammasomes are multiprotein oligomers that recognize damage-associated molecular patterns (DAMPs) to activate caspase-1, thereby allowing for cleavage and subsequent release of proinflammatory cytokines IL-1β and IL-18. Among the inflammasomes, the NLRP3 inflammasome plays a key role in sensing oxidative stress such as increased mitochondrial ROS. We are interested in studying the role of mitochondrial ROS in cognitive decline associated with aging and Alzheimer's disease using mouse models exposed to paraquat, a mitochondrial ROS inducer. Our results indicate that exposure to paraquat results in exacerbated cognitive decline in both WT mice and APP/PS1 mice. To determine whether paraquat may regulate expression of the NLRP3 inflammasome to impair cognition, we measured the relative mRNA levels of ASC and Nlrp3, two major components of the NLRP3 inflammasome, in brains from paraquat-exposed animals by qPCR. We found that paraquat-exposed WT mice had higher levels of both ASC and Nlrp3 mRNA than control WT mice. Paraquat-exposed APP/PS1 mice also had increased levels of ASC and Nlrp3 mRNA compared with control APP/PS1 mice. Our results further showed that paraquat treatment increased ASC and Nlrp3 mRNA levels in primary astrocytes. Thus, our data suggest that increased expression of NLRP3 inflammasome may play an important role in mediating cognitive decline induced by mitochondrial ROS., The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and metabolism. mTOR assembles with Raptor or Rictor to form mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively. Reduced activity of the TOR pathway extends invertebrate lifespan, and, in mice, pharmacologic reduction of mTOR signaling during adulthood, as well as germline reduction of mTOR or S6K1 extending lifespan. In other models of extended longevity, selective reduction of function in the nervous system is sufficient to extend lifespan. The role of mTORC1 signaling from the nervous system in the control of lifespan and metabolism in mammals, however, has not yet been determined. Because neuronal-specific Raptor null mice die perinatally, to explore this question we generated mice expressing a tamoxifen-inducible Cre recombinase in neurons in combination with individual homozygous floxed alleles of genes in the mTORC1 pathway (mTORfl/fl, Raptorfl/fl, and S6K1fl/fl). Cre recombinase was partially active without tamoxifen stimulation, resulting in the genetic ablation of 20–40% of the target floxed alleles during development. A greater than 20% neuronal knockdown of any of the target genes in the mTORC1 pathway reduced fat content in both sexes. Knockdown of mTOR or Raptor in neurons also significantly reduced body size as well as lower fasting and resting blood glucose levels. These data agree with previously reported data for the germline knockout of S6K1, which also exhibited reduced fat mass, but unlike the germline S6K1−/ − animals, body weight in neuronal-specific S6K1 knockdown mice was unchanged. Only neuronal knockdown of mTOR increased oxygen consumption. Lower glucose and increased lactate in blood were observed in WT and neuronal S6K1 knockdown mice, but not in neuronal mTOR and Raptor knockdown mice challenged with treadmill exercise, indicating altered muscle metabolism as a consequence of neuronal reduction of mTOR or Raptor. Remarkably, S6K1 knockout mice ran farther on the treadmill than WT mice before they reached exhaustion. In contrast, neuronal knockdown of mTOR and Raptor resulted in decreased exercise capacity. Survival to weaning age was reduced but adult mortality was unchanged for Raptor or S6K1 neuronal knockdown mice. Conversely, neuronal mTOR knockdown mice were weaned at the expected Mendelian rates but were short lived, with less than 10% surviving beyond 300 days., Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of nigral dopaminergic neurons leading to motor dysfunction. Several lines of evidence implicate mitochondrial dysfunction, oxidative stress, and protein aggregation in the pathology of PD. Recent evidence, including evidence from our laboratory, suggests that shifts in dopamine and/or aldehyde metabolism lead to aldehyde accumulation, which may contribute to parkinsonism. Aldehyde cytotoxicity may be a result of their long half-life and their ability to cross cell membranes both locally and distant. Aldehyde dehydrogenase (Aldh) is the primary pathway for the detoxification of aldehydes. Mice with mutations for two midbrain isoforms of Aldh, cytosolic Aldh1a1 and mitochondrial Aldh2, exhibit age-dependent neurochemical and behavioral deficits, and respond to levodopa treatment. Aldehyde scavengers such as N-benzylhydroxylamine and hydralazine have been shown to provide cytoprotective effects both in vitro and in vivo. Thus, the detoxification or removal of aldehydes could prove to be effective in PD therapy. The goal of this study is to investigate the protective properties of the FDA-approved anti-hypertensive drug as an aldehyde trapping agent to suppress the parkinsonian phenotype observed in Aldh1a1−/ − X Aldh2−/ − mice. We found that chronic oral administration of hydralazine in drinking water to Aldh1a1−/ − X Aldh2−/ − mice ameliorated both behavioral and neurochemical deficits. In continuance of this investigation, primary cultures isolated from the mesencephalon of Aldh1a1 and/or Aldh2 deficient mice will be subjected to Complex I inhibitors, modeling environmental exposure in PD. Aldehyde load and neuronal death will be assessed in the absence and presence of hydralazine treatment., Alzheimer's disease (AD) is the most prevalent neurodegenerative disease. The majority of AD cases develop sporadically with unknown etiology. Over the past decade, a strong link between type 2 diabetes (T2D) and AD has been established; however, the mechanisms responsible for this relationship remain unknown. The mammalian target of rapamycin (mTOR) plays a key role in maintaining protein homeostasis, regulating energy metabolism, and diabetic insulin resistance. We hypothesized that mTOR may serve as a mechanistic link between AD and T2D. To explore this potential association, we supplemented the drinking water of 3xTg-AD mice, a transgenic mouse model of AD, with 20% sucrose. A second cohort of mice was given sucrose in conjunction with an mTOR inhibitor, rapamycin. All were compared to age and genotype-matched mice on control diet. We found that the sucrose treatment significantly increased fat mass, impaired glucose tolerance, and altered urine concentration all of which are consistent with physiological changes that occur in T2D. Additionally, sucrose exacerbated AD-like cognitive impairment and Aß and tau brain pathology. Impressively, rapamycin mitigated the sucrose-induced increase in pathology and cognitive impairment. Our results provide compelling in vivo evidence that diabetes-associated AD progression occurs through an mTOR-related mechanism. Furthermore, our results indicate that treating diabetic patients with mTOR modulators may decrease their risk of developing AD., To investigate the effects of pigment epithelium-derived factor (PEDF) peptides on muscle regeneration, a rat myonecrosis model of a single injection of bupivacaine into the soleus muscle was employed. PEDF peptides were delivered by bolus injection of alginate-based sustained release regiment. The muscle fiber proliferation was assayed by the incorporation of BrdU in the proliferating nuclei. The soleus muscle specimens were also stained for satellite cell marker, Pax7, so as to investigate the muscle regeneration activity. Results suggested that the administration enhances the proliferative activities of muscle fibers and/or satellite cells, which in turn may promote the muscle to regenerate. There were higher percentages of muscle fibers containing centrally located nuclei in animals treated with the PEDF peptides than with vehicle. Moreover, on average, diameters of muscle fibers from animals treated with PEDF peptides were larger than those from animals in the blank or bolus control group. These indicated that PEDF peptides stimulate both the proliferation and maturation of muscle stem cells. Gene expression profiling and inhibitor assay on C2C12 cells revealed that the stimulation on muscle progenitor cells by PEDF is mediated by PI3KAKT and mitogen-activated protein kinases (MAPK) p38., Nuclear receptor interaction protein (NRIP) is a calcium-dependent calmodulin (CaM) binding protein (Ca+2/CaM). To investigate the functional role of NRIP in skeletal muscle, we first generated conventional NRIP knock out mice. Here, we demonstrate that NRIP-deficient mice lose muscle strength from the assays of in vitro muscle contraction test with neuromuscular blocker to rule out the nerve effect on muscle contraction; plus NRIP-deficient mice show the susceptibility to fatigue during repetitive contraction. Additionally, the exercise performances using rotarod and treadmill tests, NRIP-deficient mice are shown to be weaker than wild-type (WT) mice. We then investigated the mechanisms of NRIP involved in muscle strength and contraction; the results illustrate that NRIP regulates striated muscle function at least via calcineurin phosphatase dephosphorylating NFATc1 and CaMKII phosphorylation to increase slow myosin gene expression and the influx of internal stored Ca+2 from sarcoplasmic reticulum by measuring caffeine-induced maximal muscle force. To further investigate the NRIP role in regeneration potential, we then subjected NRIP-deficient and WT mice to unilateral cardiotoxin injection into the tibialis anterior. NRIP-deficient mice show lower centralized nuclei, a lower cell-surface recovery, a decreased area of degenerated tissues, and heterogenous myofiber size compared to weight. In conclusion, NRIP regulates skeletal muscle strength via calcineurin-NFATc1 and CaMKII phosphorylation to regulate muscle excitation/contraction; plus it also has function for regeneration capacity from injured muscles., As life expectancy rises in developed countries, sarcopenia and dynapenia pose significant threats for the aged, both physically and economically. Current research methods used to study age-related muscle wasting may include limitations, including model organisms that exhibit muscle growth characterized by a pre-defined growth plateau. The lack of model organisms for sarcopenia which exhibit continual growth potential with age has possibly limited advances in understanding this condition for two reasons: (1) the presence of a growth asymptote prevents the discovery of novel genes and/or regulatory pathways involved in the continuation of muscle fiber recruitment throughout adulthood; and (2) the inherent growth plateau may lead to sarcopenia itself, as sustained muscle growth throughout adulthood predicts the presence of myogenic precursor cells resistant to aging. Therefore, we propose a unique muscle growth model for the study of sarcopenia that takes advantage of natural continued muscle fiber recruitment. Evidence from a comparative approach utilizing the subfamily Danioninae suggests that the indeterminate growth paradigm of many teleosts arises from adult muscle stem cells with greater proliferative capacity, even in spite of smaller progenitor populations. We hypothesize that paired-box transcription factors, Pax3/7, are involved with this enhanced self-renewal and that prolonged expression of these factors may allow some fish species to escape, or at least forestall, sarcopenia/dynapenia. Future research efforts should focus on the experimental validation of these genes as key factors in indeterminate growth, both in the context of muscle stem cell proliferation and in the prevention of skeletal muscle senescence., Sarcopenia, the severe loss of skeletal muscle mass and function, is a serious health problem affecting aged individuals. Sarcopenic individuals are weak and frail, which predisposes them to disability and death. The mechanisms responsible for muscle wasting during aging are poorly understood. Muscle maintenance depends primarily on muscle stem cells (satellite cells) that exit quiescence during regeneration and replicate to generate daughter cells that either differentiates to maintain muscle or return to quiescence for self-renewal. Controversy surrounds whether the satellite cell pool is heterogeneous in regards to self-renewal, replication or differentiation. Furthermore, during aging it is unclear whether there is a decline in satellite cell number, function or whether cell fate is altered for a subpopulation of cells. To examine satellite cell heterogeneity in vivo, we developed a novel multicolor fluorescent lineage system for studying satellite cell clonal expansion in young and aged mouse skeletal muscle. Our system involves an inducible Pax7-Cre driver to express an avian retroviral receptor specifically in satellite cells, permitting retroviral-mediated gene expression of one of three distinct fluorescent proteins. This highly flexible system has advantages over current multicolor recombination-based systems as the timing of receptor expression and infection can be controlled. This improved multicolor lineage system will permit examination of satellite cell heterogeneity via detection of bias in fluorescence in daughter cells. Characterizing satellite cell behavior in vivo will permit us to examine possible defects in cell fates and alterations in subpopulations that may be responsible for the age-related decline in muscle function., Growth of proliferative cells is dependent on regulation of protein synthesis and subsequent cell division. In post-mitotic tissues such as cardiac and skeletal muscle, growth is accomplished by increased protein synthesis and the donation of nuclear DNA by stem cells. Regardless of proliferative capacity, growth and DNA synthesis are associated with increased protein synthesis. Our central hypothesis is that somatic maintenance, which we define as increased protein synthesis in the absence of growth, is associated with slowed aging. To pursue this central hypothesis, we simultaneously measure both protein synthesis and DNA synthesis using deuterium oxide. Using this approach, we have reproducibly measured DNA synthesis in laboratory rodent cardiac tissue. When comparing several long-lived rodent models, we have shown that the rate of new DNA synthesis in the heart is altered and, in some cases, this is an age-dependent or sex-dependent finding. The observed changes in DNA synthesis in cardiac tissue, which is predominantly populated by terminally differentiated, post-mitotic myocytes, suggests that cardiac muscle has either greater proliferative potential than is perhaps appreciated, or that the new DNA synthesized can be accounted for by donation from cardiac progenitor cells. We will present a comparative analysis of DNA synthesis in cardiac tissue from several long-lived models and the current results of ongoing experiments to identify the origin of the new DNA., Traditional aging models are short lived, a useful trait for tracking changes over the course of their lifespan. However, it is within the exceptional long-lived models that we can expect to find the mechanisms necessary to resist aging, as evolution has provided them. To this end, we are utilizing a range of marine bivalve mollusk species, with lifespans ranging from under a decade to over 500 years, in a comparative study to investigate the hypothesis that a long life requires superior proteome stability. These ages can be individually determined by counting growth rings in the shell. This experimental system provides a unique opportunity to study closely related organisms with vastly disparate longevities, including the longest lived animal. We are testing the long-lived bivalve's ability to maintain protein structure and function under various stressors, and identifying the macromolecules responsible for this protection. As protein homeostasis has been implicated in the aging process and age-related diseases, these macromolecules could have dramatic medical value. Preservation of protein function is measured by representative enzyme activity, such as GAPDH, when stressed in vitro. Stressors include both oxidative and unfolding agents. We demonstrate a remarkable persistence in enzyme activity in the long-lived bivalves despite egregious chemical insults. Results also indicate that C57Bl6 exhibit very poor protein stability in comparison to the bivalves. The influence of each species’ metabolite fraction on this stability is also investigated, and an attempt to rescue stability in the short-lived bivalves is attempted. Ultimately, stabilizing compounds will be identified via mass spectrometry. Demonstrating the protection of these proteins and identifying the macromolecules facilitating enhanced proteostasis in the longest lived animal species could be dramatically important in relation to various age-related protein diseases., Caspase-2 is a protein that is well known for its involvement in cellular apoptosis. However, its function in osteoclasts, cells that resorb bone, has not yet been described. In this work, the role of caspase-2 in osteoclast differentiation and apoptosis is evaluated. During the differentiation process from macrophage precursors, caspase-2 levels decrease so that protein expression is low in the mature osteoclast. Interestingly, when caspase-2 is knocked down in the RAW 264.7 macrophage cell line, osteoclast formation is increased as shown by higher levels of the osteoclast marker Cathepsin K. Furthermore, in cells derived from global Casp2 knockout mice, osteoclast differentiation is enhanced as demonstrated by increased osteoclast numbers and TRAP activity. With regard to osteoclast apoptosis, cells exposed to oxidative stressors such as H2O2 and rotenone show increased cleaved caspase-2 protein expression indicating early apoptosis mediated by caspase-2. Furthermore, additional data show that osteoclasts lacking caspase-2 and exposed to oxidative stressors are more resistant to apoptosis compared to wild-type cells. Together, these data suggest that caspase-2 may play a dual role in the osteoclast whereby it modulates osteoclast differentiation and may help regulate osteoclast apoptosis., Genetic manipulations allow controlled interventions into aging progression by changing the genetic composition of model organisms. Such manipulations can include the addition of the ‘anti-aging’ genes, the inactivation of the ‘pro-aging’ genes, or the replacement of the ‘pro-aging’ allelic variants of certain genes with their ‘anti-aging’ counterparts. The genome engineering approaches that can be used to add or replace genes are based on site-specific nucleases and DNA recombinases. Our results indicate that the latter approaches, which currently utilize primarily Flp/FRT and Cre/loxP systems, can accomplish the task both efficiently and precisely if the fine-tuned dual recombinase-mediated cassette exchange is used as a gene delivery/replacement tool. Under optimal conditions, the efficiency of dual RMCE catalyzed by the Flp–Cre pair can reach about 50% of the transfected cells. Dual RMCE depends on the pre-introduction of the recombination target sites into genome before the replacement reaction can be carried out. This dependence can be lifted if variants of site-specific recombinases are evolved to recognize pre-existing target-like sequences that flank a genome region of interest. Here, we present the results of the engineering of the hybrid Flp-TAL and Cre-TAL recombinases that recognize genomic sequences of interest. These task-specific variants of Flp and Cre recombinases can be paired to be used in the dual RMCE approach to replace the desired genome regions. We show that the engineered hybrid Flp-TAL and Cre-TAL recombinases can be used to mimic the replacement of the mutation that causes sickle-cell anemia in the model setting of CHO cells., Induced pluripotent stem (iPS) cells have the potential to improve health with age. IPS cells may be useful for biological teeth engineering. The critical cells in tooth development are neural crest-derived dental mesenchyme. Here, we utilize pharmacological molecules to assess iPS cells derived from the common marmoset (Callithrix jacchus) for the neural tube and neural crest lineage differentiation. Approach: To assess neural differentiation, we utilized an embryoid body generation assay in combination with a cocktail treatment of active small molecules: DMH1 (BMP inhibitor), SB431542 (activin/nodal/TGF-beta inhibitor), BIO (GSK-3 beta inhibitor), Y-27632 (ROCK/Rho inhibitor) and all-trans retinoic acid. Results: With 6 days of treatment with bFGF withdrawal and GSK-3 beta inhibitor, we found significant increases in the mRNA levels of neural markers NCAD (30-fold increase, P=0.0001) and ERBB3 (12-fold increase, P=0.0129). We found that bFGF addition had an inhibitory effect on NCAD/ERBB3 induction. When GSK-3 beta inhibitor (BIO) was included, NCAD mRNA levels further increased (from a 34- to 102-fold increase over baseline, P=0.0001), as was ERBB3 (34-fold, P=0.0035). bFGF withdrawal also increased FOXA2 levels (endoderm marker), but we report that inclusion of the GSK-3 beta inhibitor (BIO) ablated induction from a 15-fold increase to a 3-fold increase, P=0.0318). Conclusions: Results indicate that FGF inhibits neural differentiation, while GSK-3 beta inhibition promotes neuralization as indicated by NCAD and ERBB3 expression. We report that marmoset iPS cells respond to pharmacological inducers of neural differentiation. These studies will accelerate the development of the marmoset as a model for engineered teeth engraftment., The naked mole rat (Heterocephalus glaber; NMR) is the longest lived rodent, living >31 years. Unlike aged humans and mice that exhibit large declines in diastolic function, the NMR maintains both systolic and diastolic function for at least 66% of its long lifespan. We have further investigated NMR cardiac function using young (2–4 years) NMRs and have compared them to physiologically age-matched C57BL/6 mice (3–5 months). Surprisingly, we have found that NMRs have low heart rates compared to mice (256±8 vs. 704±11 bpm). NMR heart rates are also about half that predicted for their body size. NMRs display significantly lower cardiac fractional shortening (∼28%) than mice (∼39%). Invasive blood pressure measurements revealed that peak intraventricular pressure and ventricular contractility were much lower in the NMR (p, The concept that mitochondrial function declines during aging has been a basic tenet of the biology of aging for many years. However, it has been shown recently, first in invertebrates and later in mice, that lifespan is increased in response to genetic manipulations of some mitochondrial electron transport chain (ETC) complexes. For example, inhibition of complex IV (cytochrome c oxidase, COX), an essential transmembrane protein complex in the mitochondrial respiratory electron chain, has been shown to extend lifespan in worms and flies. Similarly, mice lacking the COX assembly protein Surf1 show increased longevity associated with decreased adiposity and enhanced insulin sensitivity, despite 50–70% reduction in COX activity. Here, we asked whether a mouse model of cytochrome c oxidase deficiency due to a mutation in the Sco2 gene, a copper chaperone that is required for the activity of COX, would have a similar metabolic phenotype as Surf1 −/ − mice. A complete knockout of the Sco2 gene in mice is embryonic lethal, however mice harboring a Sco2 knockout allele and a mutated Sco2 knockin allele (KI/KO) are viable, and have a 30–60% reduction in COX activity. We found that Sco2 KI/KO mice have increased fat mass associated with a reduction in whole white adipose tissue oxygen consumption. The Sco2 KI/KO mice have increased hepatosteatosis, elevated serum triglyceride (32%) and cholesterol levels (32%), and changes in circulating adipokine levels compared to wild-type controls. Interestingly, these alterations are associated with the development of insulin resistance in the Sco2 KI/KO mice. These findings counter to the metabolic phenotype of Surf1 −/− mice, illuminating the complex nature of mitochondrial dysfunction on physiology. Results from this study will further enhance our understanding of the role of complex IV in physiological outcomes due to mitochondrial dysfunction., Animal models of extended longevity have a shared characteristic of enhanced cytotoxin resistance. The longest lived rodent, the naked mole rat (∼31 years), is extremely resistant to cancer and a wide array of xenobiotics in vitro, and exhibits no age-related physiological or molecular declines during this extraordinarily long lifespan. We hypothesized that this profound broad-based stress resistance was due to enhanced signaling of the nuclear factor-erythroid 2-related factor-2 (Nrf2) cytoprotective pathway. The transcription factor Nrf2 is highly conserved in eukaryotes and ubiquitously expressed in all tissues. Nrf2 levels are regulated by kelch-like ECH-associated protein 1 (Keap1), which targets Nrf2 for ubiquitination and subsequent degradation via the proteasome. After a stressful insult (i.e. toxins, ROS), interactions between Nrf2 and Keap1 are inhibited and Nrf2 is able to translocate into the nucleus, bind to the antioxidant response element (ARE) and thereby activate the transcription of greater than 600 cytoprotective molecules, including those involved in detoxification, glutathione metabolism, molecular chaperones, and proteasome subunits. Commonly studied with regard to cancer, Nrf2 has also been shown to interact with p53 and p21, playing a role in modulation of the cell cycle and cancer progression. We found that Nrf2 signaling activity showed a positive correlation with maximum lifespan in rodents with varying longevity. Interestingly, this was largely due to inverse correlations with several negative regulators of Nrf2, including Keap1, which inhibited degradation of Nrf2. This Nrf2 signaling activity appears to be conserved with aging across 10 different rodent species and the type of regulation may have an impact on positive health span and longevity., In proliferative tissues, growth is accomplished with a doubling of cellular machinery by protein synthesis and subsequent cellular replication. In post-mitotic tissues, growth is accomplished by increased protein synthesis followed by DNA donation from stem cells to keep the cyto-nuclear domain constant. In both cases of growth, increased DNA synthesis is associated with increased protein synthesis. Our working hypothesis is that increased somatic maintenance, which we define as increased protein synthesis in the absence of growth, slows down aging. To this end, we have employed methods utilizing deuterium oxide (D2O) that simultaneously measure both protein synthesis and DNA synthesis in a variety of tissue types. We have repeatedly demonstrated DNA synthesis in skeletal muscle tissue in human and rodent models. Furthermore, in rodent models of slowed aging (caloric restricted, rapamycin treated, Snell, and crowded litter), we have demonstrated that this rate of new DNA synthesis in skeletal muscle can change based on the model or stage of life. The observable and changeable DNA synthesis in post-mitotic skeletal muscle indicates that skeletal muscle indeed has some replicative potential (as all tissues in mice are telomerase-positive) or that the new DNA comes from stem cells that have replicated and donated DNA to the myocyte. We will present changes in skeletal muscle DNA synthesis in models of slowed aging, and results of studies striving to determine the origin of the new DNA. It is hoped that these studies will provide further insight into how to maximize somatic maintenance for slowed aging., Over the last few decades, there was a 30% increase in the number of fathers over the age of 35 years. At this age, a father has the potential to transmit twice as many mutations to his child as a 20-year-old father. Paternal mutations continue to double approximately every 16 years, a phenomenon known as the paternal age effect. The driving force of the paternal age effect is mutagenesis. Base excision repair activity is essential for maintaining a low mutant frequency in the male germline. However, spermatogenic cells isolated from old mice display a 50% decrease in base excision repair activity, with a concomitant increase in mutant frequency, as compared to cells prepared from young mice. Reduced base excision repair activity appears to be mediated by reduced AP endonuclease 1 (APE1), a key base excision repair protein. Mice heterozygous for Ape1 show an increased germline mutant frequency as young adults, while APE1 transgenic mice are protected from age-dependent increases in spontaneous mutagenesis. Our objective is to delineate the mechanism/s mediating reduced APE1 abundance in spermatogenic cells with increasing age. In pachytene spermatocytes and round spermatids obtained from old mice, there is a significant 35 and 25% reduction, respectively, in APE1 abundance as compared to young mice. The age-related decrease in APE1 abundance is not accompanied by a reduction in Ape1 transcript abundance, thereby suggesting that post-transcriptional or post-translational regulation is involved. In somatic cells, p53 plays a role in regulating Ape1 abundance. There is a significant increase in p53 activation in spermatogenic cell populations obtained from old mice. APE1 expression is reduced by 40% in spermatogenic populations obtained from p53 null mice, relative to wild-type mice. Combined, these results indicate a strong relationship between APE1 abundance, germline mutagenesis, and p53 activation contributing to the paternal age effect., Nearly two decades have passed since the initial discovery that disruption of the mitochondrial electron transport chain can unexpectedly increase lifespan. Much research has been done to find the mechanisms behind this seeming paradox, but while many factors have been shown to be required, none has proven sufficient. The mitochondrial unfolded protein response (UPRmt) under the control of ATFS-1 has received much attention for its role in Mit mutant longevity, but it is not the only retrograde response induced by mitochondrial dysfunction. We show that the ATFS-1 pathway itself divaricates into separable pathways, one being the UPRmt and the other being the activation of SKN-1. Moreover, we have found a novel retrograde pathway independent of ATFS-1. This MAPK signaling cascade acts in a parallel and compensatory manner with the ATFS-1 network, such that if one pathway is deactivated, the other is turned up to compensate., Aging is associated with progressive loss of muscle mass, quality, and strength. Moderate (40–50%) calorie restriction is known to reduce the rate of muscle loss in animal models; however, too high of a calorie-restrictive diet can induce loss of bone mass. In this study, we tested the hypothesis that mild caloric restriction (CR) would still attenuate age-associated loss of muscle mass without any further deleterious effect on bone mass. To determine the long-term effect of mild CR (20% restriction of calorie without reducing vitamins and minerals relative to baseline intake of ad libitum (AL)) on musculoskeletal health during aging, 6-month-old C57BL/6 female mice were fed AIN93 diet AL or CR for 8 or 16 months. Mice were scanned with dual energy x-ray absorptiometry (DXA) at 6 months (baseline), 14 months (middle age), and 22 months (aging) and analyzed for the bone and muscle mass. Higher levels of muscle mass are maintained in both 14 and 22 months of age in 20% CR groups as compared to AL groups as measured by DXA, and wet weight at sacrifice. Better muscle strength is also maintained during aging in 20% CR groups as compared to AL groups as measured by rotarod performance test and endurance stress test. Interestingly, bone mass in different bone regions as measured by DXA was not reduced in 20% CR groups as compared to AL groups. These data indicate that 20% CR without compromising vitamin and minerals could be a natural, cost effective, and safe optimal dietary regimen that would improve muscle mass and strength without any deleterious effect on bone., Adipose tissue has dynamic endocrine and secretory functions that play significant roles in the regulation of metabolism and health span. There is increasing evidence that different adipose depots may have different, and often contradictory, effects on this regulation. For example, visceral white adipose is largely thought to be detrimental to metabolism whereas subcutaneous white adipose has been shown to have several beneficial effects. Furthermore, aging is associated with a loss of subcutaneous adipose tissue, accumulation of fat in intra-abdominal visceral adipose tissue and ectopic accumulation of fat in other tissues such as muscle and liver. Adipocyte precursor cells (pre-adipocytes) play a significant role in the homeostatic regulation of adipose tissue. In this study, we tested whether differences in mitochondrial function among the pre-adipocyte pools of each adipose depot might be a determining factor in depot-specific functional differences. Pre-adipocytes from visceral and subcutaneous adipose depots were isolated and sub-cultured from young C57BL/6J mice. Using the Seahorse bioanalyzer, we found that mitochondrial respiration rate is high and reserve capacity is low in visceral depot-derived pre-adipocytes relative to those from subcutaneous depots. In addition, we found that pre-adipocytes from visceral depots are significantly more sensitive to oxidative stress in vitro than those from subcutaneous depots. These data then suggest that even modest increases in oxidative stress may dramatically alter the function of adipose precursor cells to inhibit adipogenesis and stimulate lipolysis in visceral depots, which could be a mechanism for increased adipose dysfunction with age. Future studies will address whether alleviation of oxidative stress can preserve healthy functional adipose tissue with age., Every year, millions of people are diagnosed with traumatic brain injury (TBI). Of those, many have repetitive TBI (rTBI). Athletes and soldiers are at particular risk for rTBI, and those with rTBI commonly exhibit psychological symptoms including depression, anxiety, poor impulse control, and suicidal ideation. Some also exhibit signs of neuro-degeneration. Currently, treatments to stem the long-term consequences of TBI have been unsuccessful. Astrocytes play a central role in neuronal support and are excellent targets for therapy after TBI. We previously showed that the purinergic agonist 2-methylthioadenosinediphosphate (2MeSADP) effectively reduces astrocyte edema following TBI in mice. We hypothesize that 2MeSADP can similarly reduce the potential long-term effects of repetitive TBI. Here, we report our preliminary findings of a novel rTBI mouse model. Three-month-old mice were subjected to five closed-skull cortical impacts over 5 days. One year after rTBI, behavior was analyzed using a variety of tests. We found that control rTBI mice exhibited more anxiety-like phenotypes compared to 2MeSADP and sham-treated mice in the open field test and the three chambered social anxiety test. Fewer control TBI mice were able to complete the vertical pole test, suggesting an impairment of motor coordination. Continuing experiments will involve histological analysis of brain tissue, and further elucidation of the behavioral phenotype in a second cohort of mice. To our knowledge, this is the first report of a long-term analysis of rTBI in mice., Autologous cell therapies have been proposed as the cure for a wide variety of human pathologies and injuries including diabetes, myocardial infarction, and spinal cord injuries. One obstacle that is often overlooked is that donor age could be a barrier to the derivation of induced pluripotent stem cells (iPSCs) from somatic cells. This is important, as the group for which cell therapy holds the most benefit would be the middle aged to elderly population. It has also been well documented that, during aging, mitochondrial mutations accumulate (due to ROS damage or low-fidelity replication) and oxidative capacity decreases. What, if any, effect these changes would have on the quality of iPSCs derived has yet to be addressed. It has also been shown that in a mouse model carrying a mutation that increases the accumulation of mtDNA mutations, a premature aging phenotype has been shown. Recently, publications have implicated the balance of mitochondrial oxidation to glycolysis as crucial to reprogramming efficiency and have reported that cells acquire high mitochondrial membrane potential during reprogramming. Conflicting reports that this is maintained and leads to increased oxidative capacity after redifferentiation led us to hypothesize that these age-related changes in mitochondrial metabolism play a controlling role in the efficiency of derivation of iPSCs. The current aim is the creation of a polycistronic, floxed retroviral vector to allow for reprogramming with a single virus, which will then be used to investigate effects of donor age on the quality of iPS cells, derived skin samples of marmosets of different ages., Participation in physical activity can effectively prevent muscle loss and functional decline that occurs with age. Thus, understanding the mechanistic basis for preservation of skeletal muscle health with exercise may provide clues for developing therapeutic tools to combat age-related disabilities. We have demonstrated that mesenchymal stem cells (MSCs) accumulate in the muscle of young mice in response to a single bout of eccentric exercise and indirectly facilitate muscle repair and vessel growth via paracrine factor release. The observation that mechanical strain can potently influence growth factor release from freshly isolated, muscle-derived MSCs (mMSCs) provided the impetus to utilize physiological preconditioning as a method to improve mMSC viability and function in the aged muscle microenvironment. In this study, mMSCs isolated from young mouse muscle were preconditioned (10% multiaxial strain, 5 hr) and immediately transplanted into the gastrocnemius/soleus complex of 24-month-old mice. Separate groups of mice receiving saline or non-strained mMSCs served as controls. Although satellite cell number and fiber size were unchanged in all groups, vessel size and number of NMJs were greater in mice injected with preconditioned mMSCs versus controls one week post-injection (P < 0.05), resulting in a trend toward an increase in muscle function. Additionally, in vitro experiments suggest that preconditioned mMSCs are able to overcome a stiff, collagen-rich microenvironment that mimics aged skeletal muscle, showing increased survival and proliferation as compared to non-strained mMSCs. Overall, MSC preconditioning prior to transplantation may provide a novel method to prevent or reverse age-related impairments in muscle health and function., It is known that an increase in caloric intake leads to an increase in plasma growth hormone (GH) level that subsequently induces secretion of insulin-like growth factor-1 (IGF-1) from the liver, what then leads to accelerated aging (Nature 2010;464:504). However, caloric restriction and a resulting decrease in plasma IGF-1 level have the opposite effect and extend lifespan. It is also known that adult tissues contain a population of pluripotent very small embryonic-like stem cells (VSELs) that play, as postulated, an important role in the rejuvenation of long-term hematopoietic stem cells (LT-HSCs) in bone marrow (BM) Leukemia 2011; doi:10.1038/leu.2011.73, Exp. Hematology 2011;39:225–237). As we observed previously, the number of these cells in murine BM decreases with age and VSELs are kept quiescent in BM and protected from premature depletion by the erasure of the somatic imprint in differentially methylated regions (DMRs) of some paternally imprinted genes involved in insulin/insulin growth factors signaling (IIS) such as, for example, Igf2-H19 and RasGRF1 (Leukemia 2009;23:2042). Hypothesis: To explain and connect these phenomena together, we hypothesized that prolonged insulin/insulin growth factor signaling (IIS) prematurely depletes VSELs from the adult tissues and in BM may negatively impact on a population of HSCs. Material and methods: The number of VSELs and HSCs in long-living murine strains with inborn low levels of circulating IGF-1 (Laron- and Ames-dwarfs) as well as in short-living mice with high levels of circulating IGF-1 (e.g. transgenic mice that overexpress bovine growth hormone; bGH) was evaluated by FACS. VSELs were isolated and the epigenetic status of genes regulating pluripotency (e.g. Oct 4) as well as imprinted genes regulating IIS was evaluated by employing bisulfate modification of DNA followed by sequencing and by COBRE assay. We also challenged long-living mice with low IGF-1 plasma levels by daily injections of recombinant GH or IGF-1. Results: We found that the number of VSELs and HSCs residing in BM inversely correlates with plasma GH/IGF-1 level. To support this, mice with low circulating plasma IGF-1 levels (Laron- and Ames-dwarf mice) have higher numbers of VSELs and HSCs in BM that, in contrast to age-matched normal litter mates, are maintained at high levels even into advanced age. The analysis of molecular signature of VSELs in these animals revealed prolonged retention of hypomethylation in the DMRs within the Igf2-H19 and RasGRF1 loci, which attenuates IIS signaling in these cells. The number of VSELs, however, decreased in these animals after prolonged treatment with GH or recombinant IGF-I. Conversely, mice with elevated IGF-I level in plasma due to expression of the GH transgene or normal wild-type mice injected for a sustained period with recombinant GH both exhibit significant decreases in the number of VSELs and HSCs in BM compared to control animals. These decreases were paralleled by epigenetic changes in Igf2-H19 and RasGRF1 loci in which DMRs became hypermethylated over time. These changes in methylation lead to increases in IGF-2 and RasGRF1 expression and may explain why GH transgenic mice have an increase in IIS that leads to a shortening of life span in these animals. Conclusions: Our data shed new light on the relationships between senescence, GH/IGF-1 level, prolonged IIS, and number of VSELs and LT-HSCs. Accordingly, we propose a new paradigm in which a decrease in IIS (e.g. due to caloric restriction that lowers plasma IGF-1 level) may delay the age-dependent elimination of VSELs from adult tissues. In contrast, chronic IIS (e.g. due to chronic high caloric intake and the resulting elevated GH and IGF-1 levels) prematurely depletes VSELs residing in adult organs, which for example in BM leads to a decrease in the number of LT-HSCs. This study also indicates that GH-based anti-aging therapies need careful re-evaluation of their potentially uncontrolled stimulation of VSELs in BM that may lead to development of hematological malignancies. In support of this, elevated GH and IIS lead to hematological malignancies, while, in contrast, Laron-dwarf mice and Laron-dwarf patients, which have low plasma IGF-1 levels, do not develop leukemia., Parkinson's disease is a neurodegenerative condition affecting every sixth person older than 70 years in the United States. Current therapeutic approaches provide succour from symptoms, but not a cure. Stem cells, with their propensity to regenerate the dopaminergic neurons (which are actually depleted in this condition) are offering new hope. Steady improvement in the ‘Off’/‘On’ unified Parkinson's disease rating scale (UPDRS), Hoehn & Yahr scores, Schwab and England scores, absence of tumorigenic growth on MRI as well as the absence of any AE's/SAE's following stereotactic transplantation of bone marrow derived mesenchymal stem cells (BM-MSC's) in the sub-lateral ventricular zone (in our earlier study) have infused tremendous optimism and galvanized us to undertake the present, open-labeled, non-randomized, single-center study to evaluate further on the positive outcomes on a larger scale. The current study plans to track the administered BM-MSCs, employing in vivo MRI imaging after labeling them with super paramagnetic iron oxide (SPIO), which might help us in confirming and quantifying the success of the transplantation. Also, it might provide early insights on possible mechanisms of any intercellular crosstalk between transplanted BM-MSCs and resident stem cells in the subventricular zone. Finally, this knowledge will help us modify, in future studies, the route of administration, optimize dosage, determine total duration/frequency of transplantation, thus enhancing the therapeutic efficacy. Three of the subjects have shown an 11% improvement in their UPDRS scores ‘Off’/‘On’. There is a marginal improvement in H&Y, as well as H&S scores, in at least five patients. The analysis report of the imaging studies is awaited., In mammals, the small intestines are lined by single-layered epithelium organized into self-renewing crypt-villus units. Dividing stem cells line the valley-like crypts, and these differentiate upward to become quiescent, functional cell types covering the finger-like villi. All of the crypt epithelial cells produce the thyroid-stimulating hormone (TSH) and the cytokine interleukin-7 (IL-7), while villus epithelial cells do not. In the crypts, TSH- and IL-7-expressing cells are layered into three compartments. This layering is not present in knockout mice that do not have intestinal T-cells, suggesting that T-cells are targets for activation via TSH and IL-7, through their surface receptors for both (TSHR and IL-7R). In healthy mice, TSH and IL-7 production are generally limited to the crypts, as noted above, but long contiguous blocks of dividing cells, expressing TSH/IL-7, are present on the villi in intestines of mice infected with enteric pathogens. These reactive cells may be a reserve stem cell population or may have dedifferentiated for T-cell signaling and barrier defense. Dividing cells in the intestinal crypts are recognized targets of agents that cause neoplastic transformation (e.g. ionizing irradiation). We suggest here that dividing, back-differentiated villus cells on the villi may also generate tumors, especially when activating conditions persist, maintaining large numbers of dividing cells on the villi, e.g. in inflammatory bowel syndromes. Targeted delivery of TSH or IL-7 agonists or antagonists may have therapeutic effects for these conditions, via their effects on intestinal T-cells, and may prevent tumor initiation in the intestinal epithelium.

The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, USA, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location, and common areas are ideal for a small conference (80–100 participants), where copious informal intellectual interchange supplements that of the formal sessions. The 2011 meeting, part of an annual series sponsored by the University of Texas Health Science Center San Antonio, TX, USA, and the Nathan Shock Center of Excellence in the Biology of Aging, addressed the causes of age-associated inflammation and its effect on age-associated diseases., Emerging evidence has implicated the gastrointestinal flora in the pathogenesis of insulin resistance by causing chronic, low-grade endotoxemia. The goals of this study were to determine: (1) whether obese non-diabetic and obese diabetic subjects have abnormal endotoxin [lipopolysaccharide (LPS)] concentrations in plasma; (2) whether LPS promotes an inflammatory response and induces insulin resistance in human muscle, employing a primary muscle cell culture system. We measured plasma LPS concentrations and insulin-stimulated glucose metabolism (M) with the euglycemic insulin clamp (160 mU/m2.min) in 13 lean [BMI=25±1 kg/m2, age=39±2 years, fasting plasma glucose (FPG)=92±3 mg/dl, M=10.4±0.7 mg/kg.min], 9 obese non-diabetic (BMI=32±1, age=48±3, FPG=92±2, M=8.5±0.7), and 11 obese diabetic (BMI=34±1, age=48±3, FPG=151±13, M=4.2±0.5) subjects. Obese non-diabetic and diabetic subjects had elevated LPS concentrations in plasma when compared with lean healthy controls. There was an inverse correlation between LPS concentrations and insulin sensitivity (M value). Incubation of human myotubes derived from lean non-diabetic subjects with LPS caused an activation of NFκB signaling in a time-dependent manner, as evidenced by a significant increase in IL-6 and MCP1 gene expressions and phosphorylations of p38 MAPK and Jun N-terminal kinase (JNK). We, then, determined the metabolic consequence of the inflammatory response initiated by LPS in the human myotubes. We found that LPS induced a significant reduction of Akt, AS160, and GSK3 phosphorylations that were correlated with a diminished insulin-stimulated glucose uptake. Interestingly, knocking down toll-like receptor (TLR4), a receptor for LPS, reduced the LPS-induced increases in p38 and JNK phosphorylation as well as IL-6 and MCP1 mRNA expression. Summary (1) Obese non-diabetic and obese diabetic subjects have increased concentrations of endotoxin in plasma, and this could play a role in the pathogenesis of insulin resistance; (2) LPS directly causes an inflammatory response that leads to insulin resistance in human muscle; and (3) downregulation of TLR4 blunts the LPS-induced inflammatory response in human muscle, suggesting that pharmacologic blockade of TLR4 could be a useful strategy for the treatment of insulin resistance., Inflammation and oxidative stress/damage exist in a cellular yin and yang; the pro-inflammatory state promotes oxidative stress, whereas oxidative damage can induce inflammation at the same time. The interaction of these interconnected, complementary processes may promote many aspects of aging and age-related diseases. Protein oxidation may play a central role in this process as oxidized proteins are potent inducers of inflammation. Oxidation of proteins can also reduce their function, promote their aggregation, and lead to cellular senescence or apoptosis. One way cells reduce these effects is by repairing oxidized proteins with enzymes such as methionine sulfoxide reductases (Msr), which catalyze the reduction of oxidized methionine residues. MsrA is the primary Msr isoform in mammals and is ubiquitously expressed with cellular content of MsrA localized, approximately 25/75 to the mitochondria/cytosol. In this study, we tested the effects of modulation of MsrA on aging processes using transgenic mice lacking MsrA (MsrA -/-), mice overexpressing MsrA in the mitochondria (TgMito_MsrA), and mice overexpressing MsrA in the cytosol (TgCyto_MsrA). Using several different markers, we have determined that modulation of MsrA can have profound effects on physiological processes thought to represent healthspan. For example, modulation of MsrA can significantly alter cellular and whole animal resistance to oxidative stress and can affect the mitochondrial generation of oxidative stress. Our data also suggest that modulation of MsrA affects muscle physiology, as we have found significant differences among transgenic mice in their ability to perform forced treadmill running. Perhaps most directly related to inflammatory processes, transgenic MsrA mice also show significantly different physiological responses to high fat diet-induced obesity. However, genetic modulation of MsrA does not seem to affect the maximum lifespan of mice. These data, then, support an important role for MsrA in regulating mammalian healthspan and the promotion of healthy aging., Preservation of mitochondrial function and reduced generation of reactive oxygen species are correlated with increased lifespan and healthspan. Recent studies in invertebrates and rodents have challenged this paradigm by demonstrating that several mitochondrial electron chain alterations are associated with increased longevity. For example, mice lacking Surfeit locus protein 1 (SURF1), a 30 kDa inner mitochondrial membrane protein essential for the assembly of electron transport chain complex IV (Cytochrome c oxidase, COX), have a 50%–80% decrease in COX activity compared to control littermates, yet increased longevity. Maintenance of mitochondrial function is essential for insulin sensitivity, which is one important correlate of longevity. In this study, we asked whether SURF1 null mice have alterations in insulin sensitivity that might contribute to the increased longevity in these mice. Compared to wild-type littermates, SURF1 null mice displayed reduced body weight that could be attributed to decreased fat mass. This reduction in adipose mass in SURF1 null mice is not due to impairments in lipid storage, rather due to increased lipolysis and fatty acid oxidation as indicated by increased phosphorylation levels of hormone sensitive lipase and acetyl CoA carboxylase. PGC1α and its downstream targets NRF1 and Tfam were elevated in the adipose tissue of SURF1 null mice, indicative of increased mitochondrial biogenesis. Increases in fatty acid oxidation and mitochondrial biogenesis are important correlates of insulin sensitivity. Consistent with this notion, a significant improvement in insulin sensitivity was observed in SURF1 null mice; however, their glucose clearance capacity remains unaffected. A strong upregulation of mitochondrial proteases Lon, ClpP, and mitochondrial Hsp60 (components of the mitochondrial unfolded protein response) was observed in the adipose tissue of SURF1 null mice suggesting the involvement of additional components in insulin sensitivity or longevity. Together, these data demonstrate a novel crosstalk between reduced complex IV activity and mitochondrial biogenesis that causes increased fatty acid oxidation and insulin sensitivity and suggest increased insulin sensitivity as a potential mechanism for increased longevity in these mice., The accumulation of oxidative damage is a proposed mechanism regulating the aging process and the development of disease. Proteins are sensitive to such oxidative stress that can cause them to accumulate, altering conformational structure, and thus the function, of cellular proteins. Methionine sulfoxide reductase A (MsrA) plays an important role in the antioxidant defense but is unique in that it repairs protein oxidative damage. MsrA reduces methionine sulfoxide residues to non-oxidized methionine, thus participating in the antioxidant defense system of cells specifically by protecting proteins from oxidative stress. We have found that mice that lack MsrA (MsrA -/-) and mice that over express MsrA (MsrATg) are phenotypically similar to wild-type (WT) mice under normal conditions, but that MsrA levels can regulate susceptibility to oxidative stress. Because these mice are grossly normal, this suggests that excess methionine oxidation may not occur under these physiological conditions. In vivo, increasing adiposity has been associated with increases in oxidative stress, altered redox signaling, and increased oxidative damage to cellular macromolecules in several disease models, including obesity-induced metabolic diseases. When placed on a high fat (HF) diet, MsrA-/- mice become more insulin resistant than WT mice, whereas MsrATg mice are protected from development of insulin resistance. The increase in insulin resistance in MsrA -/- mice fed on HF diets correlated with reduced insulin-stimulated signaling in the insulin signaling pathway. We found that HF-fed MsrA -/- mice had reduced phosphorylation of both insulin receptor and Akt with administration of insulin under HF fed conditions. Also, increased insulin sensitivity seen in the HF-fed MsrATg mice correlated with an increase in insulin-stimulated signaling in the insulin signaling pathway. These results suggest that oxidative damage, specifically to proteins, may play an important role in obesity-induced insulin resistance. To address how protein oxidation may cause insulin resistance, we have utilized ex vivo studies to test the effect of MsrA on oxidative stress-induced insulin resistance. By utilizing these models, this study will test the hypothesis that MsrA can regulate the development of insulin resistance by repairing oxidative damage in proteins involved in the insulin signaling pathway in vitro. Insulin resistance can be induced ex vivo by various forms of oxidative stress (H2O2 and palmitate). In this study, skeletal muscle isolated from MsrA -/-, MsrATG, and WT mice was tested for resistance to oxidative stress under these conditions. Insulin-signaling protein phosphorylation correlates with in vivo signaling observations, determined by western blot after insulin stimulation. Our hypothesis is that the level of protein oxidation can be correlated with the degree of insulin resistance in this system. Because oxidation of proteins can lead to a decline in their function, future studies will focus on both function of the insulin-signaling proteins isolated from these models as well as oxidation status of these proteins., Recent evidence suggests that the lipopolysaccharide (LPS) receptor, toll-like receptor-4 (TLR4), and signaling pathways downstream of TLR4 play a role in skeletal muscle insulin resistance. Much data implicate saturated free fatty acids (FFA), whose circulating levels are often elevated in individuals with insulin resistance, as ligands for TLR4. Our lab recently demonstrated elevated concentrations of LPS in plasma of patients with type 2 diabetes (T2D), providing another mechanism for elevated TLR4-mediated inflammation in these individuals. TAK-242, a small-molecule antisepsis agent, binds selectively to TLR4 and inhibits its downstream signaling events. The purpose of the present study was to investigate the effect of TAK-242 on LPS and FFA-induced inflammatory pathways in skeletal muscle. L6 myotubes were preincubated with/without TAK-242 (1µM) for 1 h, prior to stimulation with 100 ng/ml of LPS for 1 h, or 400 uM stearic acid for 1 and 6 h. LPS caused an inflammatory response, as evidenced by increased phosphorylation of Jun N-terminal kinase (JNK) (2.1-fold), p38 MAPK (5.4-fold), IKBα (3.9-fold) and p65 NFkB (8.4-fold), and reduced IKBα protein levels (5.3-fold). TAK-242 completely inhibited the phosphorylation of these proteins in response to LPS. Stearic acid treatment for 1 h increased phosphorylation of JNK (1.5-fold), IKBα (74%), and p65 NFkB (94%), whereas TAK-242 partially reduced the increase in the phosphorylation of the inflammatory proteins by 22, 62, and 84%, respectively. Stearic acid treatment for 6 h increased phosphorylation of JNK (3.6-fold), p38 (6.6 fold), and p65 NFkB (1.2-fold), and TAK-242 reduced the stearic acid-mediated increases in JNK (by 38%) and p38 (by 51%), although p65 NFkB phosphorylation was not affected. Summary (1) TAK-242 reduces LPS- and lipid-induced inflammation in muscle cells; (2) LPS induces an inflammatory response primarily by activating TLR4; and (3) saturated FFA also works through TLR4 to induce an inflammatory response, although other mechanisms likely are involved in this process. Conclusion TAK-242 may represent a novel therapeutic approach to reduce inflammation and improve insulin action in conditions associated with insulin resistance such as obesity, aging, and T2D., Dysfunctional mitochondria can lead to disruptions in ATP production and increase the reactive oxygen species related to aging. Previously, it was thought that dysfunctional mitochondria would lead to a decrease in lifespan; however, mutations or knockdowns resulting in a decrease in the activity of the mitochondrial electron transport chain have been shown to increase longevity in Caenorhabditis elegans, drosophila, and recently mice. However, in C. elegans, disruption of the mitochondrial unfolded protein response (mtUPR) reverses this lifespan extension. The mtUPR is a retrograde signaling response resulting in an increase in mitochondrial specific antioxidants, chaperones, and proteases aimed at refolding misfolded proteins and degrading damaged proteins. Here, we explore a novel conserved mechanism of longevity in the mitochondrial complex IV-deficient Surf1 knockout mouse. Knockout of Surf1, which codes for a complex IV assembly factor, results in a 50%–80% decline in complex IV activity and a 20% increase in median lifespan. This dysfunction results in tissue-specific differences in superoxide and hydrogen peroxide production, membrane potential, and ATP production. Interestingly, these fibroblasts are more resistant to the superoxide generator paraquat. Here, we show that these mice have an increase in mitochondrial specific chaperones (HSP60) and proteases (Lon and CLPP) that have been linked to mtUPR providing a possible mechanism for the increased resistance to cellular stresses. Taken together, the Surf1 knockout mouse could have enhanced longevity due to a novel mechanism conserved from invertebrates to mammals., Introduction Muscle maintenance is a lifelong process in which myofibers are removed and replaced, a response that is increased after muscle injury. Sarcopenia or the loss of muscle mass occurs with aging. One theory suggests that muscle regenerative capacity declines with age to the point that muscle regeneration cannot keep pace with injury that occurs through daily living activities. Although multiple studies have quantitated sarcopenia in animal models of aging, few reports document recovery from injury in aged animals. We hypothesized that aged mice would exhibit impaired muscle regeneration defined as smaller fiber size and increased adipocyte accumulation as compared to young mice. Thus, the present study examined myofiber size and adipocyte accumulation within regenerated muscle in a murine model of aging. Methods Three age groups of wild-type mice (C57Bl/6J) were studied: young (4–6 months old), middle (12–14 months), and old (25+ months) and included males and females (n=5–13/group). Mice were obtained at 8 weeks of age from Jackson Laboratories. Cardiotoxin (CTX) was injected into the right hind limb muscles to cause muscle injury. After 14 days, the hind limb anterior muscle compartment was harvested, formalin fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Untreated animals (baseline) served as a control. Light microscopic histomorphometry was used to quantitate tibialis anterior myofiber size (cross-sectional area, µm2) and intramuscular adipocyte content (percentage of regenerated muscle area). Results At baseline, female mice had smaller myofiber size at all ages as compared to male mice of the corresponding age (see Table 1). Although young and middle-aged mice had similar baseline myofiber size, myofibers were considerably smaller in old animals of both sexes. After injury, regenerated myofiber size was proportionally comparable to that of baseline myofibers in male mice at all ages. Note that at 14 days post-injury, myofiber size did not return to baseline size. Similar patterns of myofiber regeneration were observed in female mice following CTX-induced injury. Regardless of age, no intramuscular adipocytes were present at baseline in either male or female mice. Following injury, young, middle, and old male mice had comparable increases in adipocyte accumulation within the areas of regenerated muscle (0.99±0.24%, 1.1±0.34%, and 0.95±0.38%, respectively). Females shared a similar pattern of fat accumulation with increased fat as compared to corresponding aged males, i.e. 2.9±0.65%, 4.4±0.49%, and 2.1±0.54%, respectively. Conclusion In an age-independent manner, female mice accumulated more adipose tissue within the area of muscle regeneration as compared to males. Nevertheless and despite sarcopenia, aging did not have a significant impact on myofiber regenerative capacity in either male or female mice., Osteoarthritis (OA) is a leading cause of disability in the United States, and it afflicts more than 30 million Americans who are past middle age. Currently, there are no FDA approved drugs specific for the treatment of this debilitating age-associated degenerative joint disease. Although not a classical inflammatory arthropathy, OA is frequently associated with signs and symptoms of inflammation, including joint pain, swelling/stiffness that may lead to functional impairment. Furthermore, increased levels of inflammatory cytokines such as IL-1β, either locally in the cartilage or in the synovial fluid during OA pathogenesis, can repress the synthesis of type II collagen (COL2A1) and aggrecan (ACAN) and upregulate proteinases such as matrix metalloproteinases and the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of aggrecanases, thereby ultimately favoring cartilage degradation. We have previously demonstrated that mice lacking the matrilin-3 gene develop OA very early in life. Elucidating the biological mechanism by which MATN3 can delay OA is a critical first step in understanding its potential as a novel OA therapy. In our efforts to do so, we discovered that recombinant human matrilin-3 (rhMATN3) protein can inhibit several catabolic effects of IL-1β (including its downregulation of COL2A1 and ACAN and its stimulation of the major catabolic proteinase, ADAMTS-5). Furthermore, we have also recently obtained strong evidence suggesting that matrilin-3's regulation of these targets is dependent on its ability to stimulate interleukin-1 receptor antagonist (IL-1Ra): a potent IL-1β pathway inhibitor. Future studies will focus on characterizing the signal transduction pathway involved in matrilin-3 induced stimulation of IL-1Ra., Introduction Synovial fluid (SF) contains hyaluronan (HA), an important lubricant for joint articulation at high-molecular weight (Mr) and concentration. After injury and in osteoarthritis, SF HA concentration and Mr decrease, although the biophysical mechanisms for this are unclear. Hypothesis HA efflux from SF is higher after injury in a Mr-dependent manner. Methods The residence time of HA for various Mr was determined in n=12 rabbit knees at days 7 (n=4), 14 (n=4), and 28 (n=4) after unilateral ACLT, with IACUC approval, by collecting SF, lavaging with saline, injecting bilaterally with saline or HA (n=1 or 3 at each time point), and sampling at 1, 3, and 8 hr. Joint fluids were analyzed for HA concentration and Mr., and time constants (τ) were calculated. Data are expressed as mean±SEM. Results After saline injection, high Mr HA accumulated over time, whereas after HA injection, [HA] was lower (1) with decreasing Mr, (2) for ACLT compared to Non-Op knees, and (3) at day 7 compared to 28 (Figures 1 and 2). HA residence time in the joint was lower in ACLT than Non-Op control joints at day 7, but not at day 28 (Fig. 3). Discussion The decreased HA residence time in the joint at early times after ACLT may be due to the inflammatory response in synovium following surgery and ACLT injury. This study identifies increased efflux of high-Mr HA as a biophysical mechanism of diminished HA concentration after injury and during joint inflammation, as often occurs in osteoarthritis., Dynamic events in injured skeletal muscle lead to a robust regenerative response that is critically dependent on macrophages; however, the regulatory role of these inflammatory cells remains to be established. Given the important role of microRNA (miRNA) in macrophage biology, it seems likely that these small, non-coding RNAs are involved in macrophage-specific biological events. The present study was designed to explore this possibility by exploiting the known defect in macrophage recruitment and activation in CCR2-/- mice. Thus, the temporal expression of macrophage-related miRNA in muscle regenerative events in vivo was examined in cardiotoxin-injured muscle derived from wild type (WT, C57Bl/6J) or CCR2-/- mice. Total RNA was extracted from muscle and miRNA expression determined by qRT-PCR in TaqMan assays (Applied Biosystems). As compared to baseline (uninjured) muscle, log fold change in miRNA expression at 1, 3, or 7 days following injury (n=4 mice/group/timepoint) was determined. In a separate group of animals studied at similar timepoints, monocyte/macrophage numbers in injured muscle were determined by flow cytometry and confirmed a significant reduction in macrophage infiltration in CCR2-/- animals. In both mouse strains, considerable changes in diverse miRNAs were observed following injury. But, most importantly, in comparisons of the temporal expression patterns of miRNA from WT and CCR2-/- mice, cluster analysis revealed 60 miRNAs with different patterns of expression, including miRNAs that have been reported to be highly expressed in macrophages. Interestingly, 15 miRNAs with no known relationship to macrophages were matched with the temporal expression patterns of macrophage-related miRNAs. Thus, it is conceivable that this latter group of miRNAs may be unique, macrophage-related miRNA that regulate the muscle regenerative response. Given the parallel development of sarcopenia and altered macrophage biology during aging, the miRNAs identified in the present study may be useful in the elucidation of new targets for therapeutic intervention of sarcopenia., Aging-associated bone mineral density (BMD) loss is becoming a significant, worldwide social and financial health care problem. In this study, we demonstrated the beneficial role of FDA-approved prescription omega-3 fatty acids, Lovaza® against BMD loss during aging using 12-month-old C57BL/6 mice. Mice were fed with 1 and 4% of Lovaza® and placebo diets including a group with 4% regular fish oil (18/12 (EPA/DHA)) diet for 12 months. DXA scan was performed once before starting the diet at 12 month of age and again after 12 months of feeding. After analyzing the BMD of different bone regions, we found that 4% Lovaza® prevents age-associated BMD loss, while 1% Lovaza® offers mild protection. However, 1% Lovaza® showed better protection than that of 4% regular fish oil. We also measured the effect of LPS-stimulated cytokines production in bone marrow (BM) cells collected from mice fed with different experimental diets. Interestingly, 4% Lovaza® showed decreased production of pro-inflammatory cytokines, TNF-α, and IL-6 and increased production of anti-inflammatory cytokines IL-10 and IFN-γ as compared to that of other groups. We also analyzed RANKL-stimulated activation of inflammatory and pro-osteoclastogenic signaling molecules p38 MAPK and JNK in BM cells using Fast Activated Cell-based ELISA (FACE) assay system. Interestingly, both 1 and 4% Lovaza® showed reduced activation of both p38 MAPK and JNK. Furthermore, we analyzed the components of bone remodeling in serum samples for TRAP5b, PINP, RANKL, and OPG. Bone resorption marker, TRAP5b level was dramatically reduced in 4% Lovaza® treated mice, whereas bone formation marker, PINP level was unaffected. Moreover, the osteoclast stimulating factor, RANKL was significantly reduced in 4% Lovaza® treated mice, whereas the decoy receptor for RANKL, OPG level was unaffected. These data indicate that Lovaza® protects age-associated bone loss in C57BL/6 mice by modulating inflammatory signaling molecules and related bone resorption., Age-related sarco-osteopenia involves declines in mass, quality, and strength of both muscle and bone. Because the protein deacetylase Sirtuin1 (Sirt1) has been associated with improvements in the healthspan in mice involving both muscle and bone, Sirt1 may be an excellent target for study for its potential in preventing and/or ameliorating sarco-osteopenia. We are currently exploring the effects of resveratrol (RSV), a putative activator of Sirt1, on bone. Our preliminary data demonstrate that in vivo supplementation with RSV dramatically improves bone quality in aged mice, stimulates osteoblastogenesis, and inhibits osteoclastogenesis in young mice, and enhances resistance to oxidative stress in bone marrow cells. Our data suggest a possible mechanism involving downregulation of Rac1 activity and decreased phosphorylation of c-Src, resulting in altered actin polymerization. Given the importance of Rac1-dependent actin reorganization in muscle, we have begun to examine the impacts of RSV and Sirt1 on skeletal muscle mass and performance during aging. To elucidate potential mechanisms of action, we will analyze protein and mRNA transcriptional levels of Sirt1 and proteins involved in inflammatory pathways influencing sarco-osteopenia because Sirt1 appears to play a role in suppressing NF-κB activity. In addition, we will assess the impacts on oxidative stress. Taken together, these studies will shed new light on the interrelatedness of sarco-osteopenia leading to the development of novel strategies for prevention., Normal bone homeostasis is maintained by balancing osteoclast-mediated bone resorption with osteoblast-induced bone formation. If this delicate balance is chronically tipped toward excess bone resorption, osteoporosis occurs. Age-related osteoporosis in men is of particular importance because mortality following fracture is higher in men than in women. Our lab has previously shown that old male mice deficient in caspase-2 (casp-2), a cysteine protease involved in apoptosis, exhibit severe age-related osteoporosis. Specifically, micro-computed tomography (µCT) and dual energy X-ray absorptiometry analyses of long bones from old Casp-2 -/- mice demonstrated decreased bone mineral density. Molecular biological analyses revealed that cleaved caspase-2 decreases as a function of age, directly correlating to a decrease in bone density seen in old wild type (WT) mice. In addition to its role in regulating cellular apoptosis, caspase-2 also plays a role in mesenchymal stromal/stem cell (MSC) differentiation to osteoblasts. Indeed, MSCs from Casp-2 +/- and Casp-2 -/- mice have decreased mineralization activity compared to WT. In addition, cultured MSC lysates and whole bone lysates from Casp-2 -/-mice expressed higher levels of CSF-1, a cytokine that enhances osteoclast survival. Together, these data indicate that the function of Casp-2 -/-osteoblast lineage cells is altered such that bone resorption is favored. Immunohistochemistry of bone sections as well as in vitro culture of osteoclast precursors indicates that caspase-2 is not present in osteoclasts at a basal level but is upregulated in response to mitochondrial oxidative stressors such as rotenone and antimycin A. Therefore, caspase-2 may play a role in osteoclast apoptosis during conditions of mitochondrial oxidative stress, as has been demonstrated in neurons and fibroblasts. Because caspase-2 staining in murine long bone sections and human bone sections is similar, our data are relevant to human studies. Elucidating the function of caspase-2 in bone under normal conditions as well as in age-dependent osteoporosis will lead to novel anti-osteoporosis therapies to aid millions affected by this debilitating disease., Advanced age is associated with aberrant inflammation and innate immune cell activation that contribute to refractory responses to trauma, tissue injury, and infection. Despite clinical observations that aging impairs wound healing, few studies have examined how the innate immune response contributes to resolution of cutaneous wound infection. Using an excisional wound injury model, young (3–4 months) and aged (18–20 months), BALB/c mice received 102–3 CFU/mL per wound of Staphylococus aureus (S. aureus), a common dermatopathogen with a predilection for elderly patients. Over 7 days, aged mice exhibited increased bacterial colonization at early time points with incomplete resolution at day 7 as compared to young mice (two-way ANOVA, F=0.1617; Age, p=0.0003; Time, p=0.0376). To determine the contribution of age-related alterations in the innate immune response to the heightened bacterial colonization seen in aged mice, we examined wound neutrophil and macrophage phagocytosis, as well as antimicrobial peptide expression. Following isolation of wound neutrophils and macrophages, these leukocytes were examined via flow cytometry for phagocytosis using the pHrodo bioparticle system. Although no difference in neutrophil or macrophage phagocytosis was seen between cells from young and aged animals, it was noted that neutrophil expression of FcgRIII (CD16) was decreased following phagocytosis in latter group (p, Neutrophils comprise the first line of host defense, killing invading microbes by generating reactive oxygen species (ROS) through the activable NADPH oxidase (NOX2) system. This respiratory burst oxidase is genetically absent in patients with chronic granulomatous disease, resulting in recurring severe infections with high morbidity and mortality. The phagocyte oxidase features membrane-bound catalytic components (gp91phox/p22phox) and cytosolic cofactors (p47phox, p67phox, Rac1/2) that undergo stimulus-dependent translocation during assembly of the active NADPH oxidase. Given the importance of H2O2 as a cellular signaling molecule and our recent finding demonstrating a positive feedback regulation of the NOX5 isoform by H2O2, we explored the possibility that the phagocytic NOX2 system is regulated by H2O2. Our data show that H2O2 induces superoxide production in human blood neutrophils and monocytes, as well as in K562 leukemia cells overexpressing NOX2 system components. We demonstrated that H2O2 stimulates NOX2-mediated superoxide generation in both neutrophils and K562/NOX2 cells via a signaling pathway involving Ca2+ influx and c-Abl tyrosine kinase acting upstream of PKCδ. Relevant to the oxidative stress associated with aging, we found recently that this pathway is dysregulated in human neutrophils from healthy older individuals. Among subjects age 20–65, there was a trend for an age-related increase in basal superoxide generation, whereas basal rates in those older than 65 were decreased relative to the younger age groups. In addition, one way ANOVA analysis across all age groups (20–87 years) demonstrated significant age-related declines in the activation levels of NOX2 by H2O2, especially for subjects older than 65. Thus, dysregulation of phagocyte ROS production over time (i.e. failure to produce appropriate amounts of ROS in the right place at the right time) may contribute to the aging process. Identification of the signaling proteins responsible for abnormal NOX2 regulation by H2O2 will help to explore mechanisms of inflammation-associated aging phenotypes such as increased susceptibility to infection and autoimmunity, delayed phagocyte apoptosis, and chronic inflammation., T cell function declines with age and impairs the ability to fight new infections and control persistent infection. We previously showed that regulatory T cells (Treg), an immune suppressive CD4+ T cell subset, accumulate dramatically with age in both humans and mice. Furthermore, Treg accrual in aged mice prevented control of persistent infection. Moreover, we found that decreased expression of the pro-apoptotic molecule Bim in aged Treg contributes to enhanced survival and subsequent Treg accrual. Given the role of IL-2 in Treg homeostasis in young animals, we investigated the role of IL-2 on modulating Treg survival and Bim expression in aging. We found that IL-2 is sufficient to promote accumulation of Bimlo Treg in young mice. Also, in aged mice we found that IL-2 neutralization resulted in a partial loss of Treg, but selected for CD25lo Treg. Interestingly, the remaining CD25lo Treg population had increased expression of CD122 (IL-2/IL-15Rβ). Using aged IL-15 deficient mice, we found that IL-15 partially contributed to Treg accrual. Interestingly, we found that the additional loss of Bim (IL-15-/-Bim-/-) rescued the loss of Treg in aged IL-15-deficient mice. Mechanistically, we explored the role of FOXO transcription factors in control of Bim expression in Treg. Notably, we found that deletion of FOXO1/3a/4 in vivo led to significantly decreased Bim expression in Treg. Together, our data suggest that IL-2 and IL-15 contribute to Treg accrual in age and may also contribute to the suppression of Bim expression in aged Treg by signaling through FOXO family members., The thymus is the primary organ for thymopoiesis and the production of naïve T cells from early T cell progenitors (ETP). With age the thymus involutes, resulting in a decline in the production of naïve T cells, which contributes to a compromised immune system in the elderly leaving them more susceptible to illnesses and infection. A mechanism for thymic involution and the decline in naïve T cell production with age is not well elucidated. Previously, we showed that a decline in expression of the transcription factor Foxn1 with age correlated with reduced thymopoiesis as determined by the decline in thymocyte number. Using a transgenic mouse model in which Foxn1 is overexpressed under the human keratin 14 promoter (Foxn1Tg), we found that aged Foxn1Tg mice had a higher number of thymocytes compared to Wt. Overexpression of Foxn1 prevented the decline in ETP frequency, and aged Foxn1Tg had a 3-fold higher number of ETP compared to aged Wt. Overexpression of Foxn1 also diminished age-associated changes in thymic epithelial cell (TEC) composition. Foxn1Tg had a higher number of EpCAMpos medullary TEC (mTEC) as well as of the MHCIIhi mTEC; these cells express the highest level of Foxn1 and are identified as transient amplifying cells that are responsible for maintaining the mTEC pool. While the percentage of EpCAMpos MHCIIhi mTEC that is Ki67pos declined with age in Wt, a higher percentage of Ki67pos EpCAMpos MHCIIhi mTEC was found in aged Foxn1Tg. Furthermore, aged Foxn1Tg had more peripheral naïve T cells compared to Wt, and the age-associated expansion of CD4 memory T cells was prevented. Taken together, our data suggest that maintaining high expression of Foxn1 in the thymic environment can prevent age-associated changes in TEC composition leading to improving thymopoiesis and production of naïve T cell with age., Parkinson's disease (PD) is the most prevalent neurodegenerative movement disorder affecting up to 5% of the population aged 65–85 years. PD is characterized by progressive dopaminergic neurodegeneration in the nigrostriatal pathway leading to profound dopamine depletion in the substantia nigra and striatum. Evidence from different labs suggests that oxidative stress plays a role in pathogenesis of PD. Previous studies have shown that expression of ALDH1 and GPX1, which are important for clearance of aldehydes andH2O2, respectively, are reduced in the substantia nigra of PD patients. To determine the contribution of deficiency in these two genes to the pathogenesis of PD, our lab has generated mice with simultaneous deletion of Aldh1a1 and Gpx1 genes (Aldh1a1/Gpx1 KO). Young adult Aldh1a1/Gpx1 KO mice showed a significantly increased latency to fall in the automated accelerating rotarod test and increased reversal time in the pole test compared to age-matched wild type control mice. There were no significant effects on levels of dopamine and its metabolites DOPAC and HVA, dopamine transporter or vesicular monoamine transporter 2 protein in midbrain and striatum. However, levels of 4-hydroxynonenal, an end product of lipid peroxidation, were increased in the midbrain and striatum of Aldh1a1/Gpx1 KO. In summary, simultaneous deletion of Gpx1 and Aldh1a1 genes was not associated with altered measures of dopaminergic function but was associated with motor deficits in young adult mice. In addition, deficiency of Gpx1 and Aldh1a1 genes was associated with increased lipid peroxidation. Future experiments will include determining behavioral phenotypes in mice at middle and old ages, evaluating the number of dopaminergic neurons in the substantia nigra. Also, because Gpx1 is predominantly expressed in microglia and neuroinflammation has been suggested to be important for pathogenesis of PD, levels of the microglia marker Iba-1 in substantia nigra and striatum, as well as the number of Iba-1 positive cells in brain, will be determined., Alzheimer's disease (AD), one of aging-associated diseases, is the most common dementia affecting millions of people around the world. Epidemiological studies indicate that exposure to environmental toxins, such as pesticides, is a risk factor of AD. However, little is known about how pesticide exposure may promote AD pathogenesis. In this study, we investigated the effects of paraquat exposure on cognition and amyloidogenesis in AD mouse models. Our results indicated that paraquat exposure increased the generation of H2O2 by brain mitochondria, which was directly correlated with increased mitochondrial oxidative damage and reduced mitochondrial function. Moreover, APP transgenic mice exposed to paraquat showed exacerbated cognitive impairment and elevated Aβ levels. Peroxiredoxin 3 (Prdx3) is a key enzyme for mitochondrial H2O2 removal. Our results showed that overexpression of Prdx3 prevented paraquat-induced increase in mitochondrial H2O2 generation and mitochondrial damage. Importantly, both cognitive impairment and elevation of Aβ levels induced by paraquat exposure were significantly attenuated in APP transgenic mice with Prdx3 overexpression. Therefore, our results demonstrate that increased mitochondrial H2O2 generation is a key mechanism by which pesticide exposure exacerbates AD pathogenesis., Despite more than a century of research, the primary mechanisms involved in the pathogenesis of the most common age-related neurodegenerative disease, Alzheimer's disease (AD), remain unclear. Laboratory mice and rats do not develop this disease, and this is attributed to their short lifespan. We propose an extraordinarily long-lived rodent, the naked mole-rat as a novel, natural model for the study of AD. We assessed common risk factors for AD in this species and surprisingly found that young (2 year) naked mole-rats (NMRs) exhibited many of these risk factors: We found high levels of lipid peroxidation in NMR brains. High oxidative damage is associated with neuronal damage in human AD. We also found that levels of both soluble and insoluble Aß in the brains of NMRs were comparable or greater than levels found in a genetically manipulated common AD mouse model (3XTg). These findings prompted us to evaluate if specific brain regions commonly associated with AD (cortex, hippocampus) are better protected against these harmful affects than the relatively unaffected cerebellum. Despite their exceptional longevity, young NMRs exhibited high levels of Aß particularly in the hippocampus and this was coupled with low antioxidant defense and low proteasome activity. These factors might render the NMR hippocampus particularly susceptible to neuronal damage. In addition, we report the unique key properties of NMR Aß (sequence, aggregation propensity and toxicity) that suggest NMRs may be an important addition to the sporadic AD models studied to date., Increasing evidence suggests that oxidative stress plays an important role in neuronal function, and proper neuronal communication is important for muscle physiology. We have previously shown that mice lacking CuZnSOD (Sod1 -/-) have an accelerated muscle atrophy phenotype and high levels of oxidative stress and that calorie restriction protects against this loss in both Sod1 -/-) and wild-type (Wt) mice. We also find a significant decrease in nerve conduction velocity (NCV) in Sod1 -/- mice at all ages compared to Wt mice, and calorie restriction (CR) attenuates this loss in the old Wt animals. We hypothesized that oxidative stress plays a role in loss of NCV through demyelination by increases in inflammation and metalloproteinases that converge on myelin function. To test this hypothesis, we used two groups of mice to measure spinal cord and sciatic nerve-dependent biochemical changes: (1) ad libitum-fed C57Bl6 Wt mice at 6, 21, and 29 months of age and calorie restricted (40% restricted) mice at 29 months of age and (2) Wt and Sod1 -/- mice at 6 and 21 months of age when significant muscle atrophy is present. We found a significant decrease in sciatic NCV of Sod1 -/- mice at all ages, and in 29-month-old Wt mice this reduction was reversed by CR. Myelin compactness and neuronal size are lower in sciatic nerves from mice with increased oxidative stress (Sod1 -/- and 29 month Wt) and were reversed by CR at 29 months of age. The observed differences in myelin compactness may support the reduced insulation of neurons and loss of NCV. One potential mediator of this phenotype is inflammation. Sod1 -/- mice have elevated MCP1 protein expression as a percentage of the Wt littermate control value ((6 month 187±14–*) and (20 month 206±13–*)). This level of MCP-1 expression is equivalent to the level in 29-month-old Wt mice (191±19–*) and is attenuated in 29 month CR group (130 ±12–*). Another marker of inflammation, matrix metalloproteinase-9 (MMP9), expressed as a percentage of the littermate control value, is elevated at all ages in the Sod1 -/- mice (6 month (118±5–*) and 20 month (189±13–*)) and is dramatically elevated in the 29-month-old AL mice (438±100–*) and is attenuated by CR (120±8–*). ERK signaling, a known regulator of MCP-1 and MMP-9, expressed as a percentage of the littermate control value is significantly elevated at all ages in the Sod1 -/- mice ((6 month 137±6–*) and (20 month 143±12–*)) and in the 29 month AL (142±13–*). Altogether, these data suggest that a reduction in NCV and myelin may be in part regulated by inflammatory processes related to oxidative stress. Associated with this reduction in NCV and myelin compactness is a concomitant increase in both MCP1 and MMP-9 protein expression in the old wt mice, where oxidative stress is known to peak, and at all ages in the Sod1 -/- mice. Further support is a reduction in MCP-1 and MMP-9 with CR and a concomitant attenuation of the age-dependent loss in NCV and myelin compactness. Future studies with MCP1 and MMP-9 knockout mice will be aimed to determine the role of both of these proteins involvement in loss of NCV, myelin compactness, and muscle atrophy with age., We recently showed that inhibition of mTOR by systemic, long-term rapamycin treatment blocks AD-like cognitive impairments and Aß accumulation in mice modeling AD. To determine the mechanisms by which long-term inhibition of mTOR modulates cognitive function, we examined the complete proteome of brains of control- and rapamycin-treated PDAPP mice. Members of the chaperone/heat shock response (HSR) family were overrepresented among the proteins whose abundance was increased by rapamycin treatment in transgenic brains. In agreement with the observed upregulation of heat shock proteins (HSPs), we found that HSF1 was activated, and that eIF4E-biding protein (4E-BP, which participates in shutoff of protein synthesis during heat shock) was activated, setting up conditions for the preferential translation of chaperone mRNAs in rapamycin-treated brains. Overexpression of HSF1 mimicked the effects of rapamycin, resulting in chaperone upregulation and preservation of cognitive function in PDAPP mice. Conversely, inhibition of the chaperone HSP90 strongly increased Aß levels in transgenic brain slice cultures. Taken together, our results indicate that activation of the chaperone network is sufficient to lower Aß levels and block cognitive impairments in PDAPP mice and suggest that long-term inhibition of the mTOR pathway may decrease Aß levels and preserve cognitive function by increasing the levels of chaperones in transgenic mouse brains. Maintenance of proteostasis may, therefore, be sufficient to preserve cognitive function in AD-like neurodegeneration. Our data suggest that rapamycin, already used in clinical settings, may have potential for the treatment of AD., Decline in sensory acuity is a general hallmark of aging, which in humans decreases quality of life. We report here refinement of a sensory acuity assay in mice. Three features of the assay merit attention. First, as mice are primarily nocturnal in nature, olfaction is an important sensory modality for them. Second, our assay instead of using artificial olfactory cues employs major urinary proteins that are important in both intrasexual and intersexual communication of mice in nature. Third, the assay can be performed in the mouse's home cage, thus avoiding artifacts from distracting, novel environments. Procedurally, the assay uses serial dilutions of urine and preference for the urinary odor relative to water control to olfactory acuity. Age-related changes in olfactory acuity have not previously been reported in mice. We have refined the assay that compares time spent sniffing a sample relative to time spent at a distilled water control, numerous times, and the most recent refinement appears to be sensitive, repeatable, and encompasses particularly informative urinary dilution ranges. Specifically, previous testing revealed that of any age, mice usually cannot distinguish urine from water at a dilution of 1:10,000 (Rendón, unpublished data). The range of experimental dilutions that have been narrowed down through successive modifications of the assay is between 1:10,000 and 1:5,000. Sampling in this range allows us to pinpoint and compare discriminatory ability and to more accurately assess the influence of factors, such as age, or drugs, on mouse olfactory acuity., Parkinson's disease (PD) is an age-associated neurodegenerative disorder affecting 2%–5% of the population 65–85 years of age. PD is characterized by progressive motor dysfunction resulting primarily from death of substantia nigra dopaminergic neurons. Numerous studies indicated that the accumulation of inflammatory and oxidative insults during aging contributes to PD pathogenesis. Biogenic aldehydes can be triggered by inflammation and are particularly toxic due to their reactivity, relatively long half-life, and the fact that they can easily cross cell membranes, acting as second toxic messengers. The accumulation of biogenic aldehydes in brains of deceased PD patients has been extensively reported. Aldehyde dehydrogenases (ALDH) play a major role in detoxification of biogenic aldehydes in the brain. Previously, our lab has shown that mice null for two Aldh isoforms known to be expressed in midbrain dopamine neurons, Aldh1a1 and Aldh2, exhibited age-related deficits in motor performance on the accelerating rotarod and in gait analysis. The deficits were rescued by L-DOPA administration. Consistent with the loss of ALDH, biogenic aldehydes were elevated in the midbrain of the Aldh1a1 -/- ×Aldh2 -/- mice. The lipid peroxidation product, 4HNE, was elevated by 30%–60% in midbrains of Aldh1a1 -/- ×Aldh2 -/- mice. We also found significant (2–6-fold) accumulation of DOPAL, the aldehyde product of dopamine metabolism, in midbrain and striatum of Aldh1a1 -/- ×Aldh2 -/- mice. An increasing number of reports indicate that aldehyde trapping agents may be cytoprotective in conditions of increased ‘aldehyde load.’ Therefore, we hypothesized that aldehyde trapping agents may be beneficial in PD. To test the hypothesis, we first used a PC12 cell culture model and found that Hydralazine prevented 4-HNE-induced cell death. We then delivered Hydralazine (250 mg/L) in drinking water to the Aldh1a1 -/- ×Aldh2 -/- mice. Long-term delivery of Hydralazine (∼ 40 mg/kg/day for 9 months) rescued motor performance deficits in Aldh1a1 -/- ×Aldh2 -/- mice. Reduced midbrain 4-HNE level in Aldh1a1 -/- ×Aldh2 -/ mice after long-term Hydralazine treatment supported our hypothesis that aldehyde trapping agent (i.e. Hydralazine) scavenges accumulating aldehydes and may provide a new therapeutic approach to PD. As a currently FDA-approved antihypertensive treatment, Hydralazine may be readily approved as a neuroprotective agent in PD., Sleep fragmentation is a common health issue in aging populations. Most commonly studied in relation to sleep apnea, it has been demonstrated in humans that sleep fragmentation is associated with disruption of many body functions ranging from glucose metabolism to cognitive function. In an effort to assess the effects of rapamycin on healthspan in C57BL/6 mice, we used a non-invasive assay to monitor animal activity. The Pack Laboratory developed a high-throughput model for phenotyping sleep in mice. which validates the use of activity/inactivity assessments as a measure of sleep, with sleep defined as any bout of inactivity of ≥40 seconds. Based on their results, we measured activity across a 24-h light/dark cycle to assess sleep fragmentation. We report here that C57BL/6 mice that were started on rapamycin at 19 months of age show significant sex differences in sleep fragmentation unrelated to aging. Sex differences in sleep fragmentation are also found in aging humans; the mechanism underlying this sex difference warrants further investigation., Inhibition of the TOR signaling pathway by rapamycin has been shown to extend measures of lifespan in genetically heterogeneous mice and C57BL/6 mice. To determine whether rapamycin treatment delays age-associated declines in healthspan, we performed a single-blinded study using C57BL/6 mice fed with encapsulated rapamycin or control diet starting at 19 months of age. We tested these mice with a series of putative healthspan assays chosen for their similarity to measures of aging and frailty in humans. Several of the assays showed significant differences between rapamycin-treated animals and controls. In some cases, rapamycin-treated animals performed significantly better than age-matched controls, whereas in other assays there were no differences. In addition to treatment effects, we also found several useful indicators of age-related decline in mice comparable to assays used to measure human frailty., Resistance to toxins and other stressors has been shown to impact healthspan and longevity in multiple models of aging, including worms, flies, and rodents. The naked mole-rat, the longest-living rodent with a maximum lifespan of >31 years, demonstrates a profound resistance to a variety of toxins in vitro. Throughout this very long lifespan, naked mole-rats experience very little decline on physiological and biochemical levels with age, and cancer frequency is incredibly rare, with one case ever reported. We hypothesize that the nuclear factor-erythroid 2-related factor-2 [Nrf2] signaling cytoprotective pathway plays a larger role in this profound cytotoxin and cancer resistance. Nrf2 is a transcription factor ubiquitously expressed in all tissues and conserved from worms to humans. Nrf2 degradation is regulated by kelch-like ECH-associated protein 1 [Keap1] that targets Nrf2 for ubiquitinaton and subsequent degradation via the proteasome. After a stressful insult to the cell (i.e. toxin, ROS), interactions between Nrf2 and Keap1 are inhibited and free molecules of Nrf2 are able to move into the nucleus to bind to the antioxidant response element (ARE) to activate the transcription of over 600 cytoprotective molecules, including those involved in detoxification, glutathione metabolism, heat shock factors, as well as subunits of the proteasome. Largely studied with regard to cancer, Nrf2 has also been shown to interact with p53 and p21, playing a role in modulation of the cell cycle and cancer progression. We find that naked mole-rats, in addition to other naturally long-living species, have constitutively elevated levels of Nrf2 signaling in addition to a profound resistance to toxins and carcinogens in vivo demonstrating smaller changes in body temperature, rapid Nrf2 signaling, and very few signs of acute damage compared to shorter-lived rodents. Nrf2 upregulation may be a highly conserved mechanism that contributes to prolonged healthspan and longevity., A 30% increase in the number of older fathers during the last three decades has made the paternal age affect an increasingly important topic in aging reproductive health. Using lacI transgenic C57Bl/6 male mice, in conjunction with mice harboring inactivated alleles of various base excision repair genes, we have determined that normal base excision repair activity is essential in maintaining a low mutant frequency in spermatogenic cells. Comparisons of base excision repair activity in nuclear extracts prepared from spermatogenic cells obtained from young, middle-aged, and old mice revealed that repair activity is reduced by 50% in extracts prepared from old mice. This decreased base excision repair activity appears to be mediated by reduced abundance of a key base excision repair protein, AP endonuclease 1 (APEN1). Our objective is to delineate the mechanisms mediating reduced base excision repair in spermatogenic cells with increasing age by identifying the molecular changes that result in reduced APEN1 abundance and activity. TRP53 has previously been shown to play a role in regulating APEN1 expression within the liver, but this role has yet to be identified in spermatogenesis or with regard to aging. In pachytene spermatocytes obtained from older aged animals, there is an 88 and 80% increase in the phosphorylation of Serine 15 and Serine 20, respectively, on TRP53. Both of these phosphorylation signals are required to activate TRP53 and induce its regulatory function within the cell. Immunohistochemistry data demonstrate that there is reduced APEN1 staining within the pachytene spermatocyte population, suggesting a possible role for TRP53 in male reproductive aging. However, Northern blot analysis revealed that the abundance of the Apex1 transcript remains constant with increasing age. This suggests that the age-related change in APEN1 abundance is regulated at the translational or post-translational level. Spermatogenic cells isolated from Apex1 heterozygous mice, also carrying the lacI mutation reporter, demonstrated an accelerated increase in germ cell mutagenesis. Conversely, transgenic mice that overexpress APEN1 were protected from the age-related increase in mutant frequency. Combined, these results indicate a strong relationship between APEN1 abundance and germline mutagenesis and suggest that TRP53 may be a regulator of APEN1 abundance in spermatogenic cells with increasing age., Evolutionary theory predicts that selection pressure declines with age. On the molecular level, one consequence of this may be that proteins needed for long life show loss-of-function point mutations in short-lived species. We used multiple regression, controlling for both body mass and shared ancestry, to find these lifespan-associated point mutations in several thousand high-quality protein alignments from 33 mammalian species. Overall, we found many more mutations in our data than would be expected by chance. We present examples of proteins that may be relevant to mammalian aging., Measures of health span offer a more unified index of overall aging than measuring lifespan or longevity alone, as health span includes assays on a range of functional systems within the individual. The objectives of this study were to identify robust and reproducible functional measures of healthspan and to determine the interrelationship of these measures, and perhaps measures predictive of mortality. The study uses 16 different assays (e.g. body composition, metabolic rate, gait analysis, nerve conduction, kidney function) to evaluate overall health span in C57BL/6 male mice of four different ages (4, 20, 28, and 32 months of age) in a cross-sectional design. Many of these assays show significant consistent changes with age. By identifying age-related changes in these functional patterns, we can begin to develop biomarkers of aging that will help guide future directions of aging research in animal models and clinical work. For example, geriatric frailty involves the interrelationship of multiple variables – involuntary weight loss, exhaustion, lack of spontaneous activity, slow walking speed, and reduced grip strength. By measuring these variables in aging rodents, we can develop models that explore the physiology and etiology of such complex phenomena and provide critical insights into the mechanisms associated with age-related decline., Rapamycin (rapa) has recently been found to extend lifespan in Saccharomyces cerevisiae, Drosophila, and mice. Base on epistasis studies in invertebrates, it has been hypothesized that rapa is a dietary restriction (DR) mimetic. To test the hypothesis, we used male C57BL6 mice fed ad libitum (AL), DR mice fed with 60% of the ad libitum diet, rapa mice fed ad libitum supplemented with encapsulated rapa (14 ppm; the same dose used by the studies which showed that rapa increased the lifespan of mice) in the food, and DR+Rapa mice fed with 60% of the diet consumed by ad libitum mice supplemented with encapsulated rapa (14 ppm). Microarray analyses were conducted to compare the similarities or differences in genes expression and signaling pathways associated with DR or rapa interventions for 6 months, starting at 2 months of age. Our previous data indicated that there were no differences in body composition, with exception in fat mass, in which only DR treatment had a significant decrease in fat mass, and both treatment showed significantly reduced phosphorylation of S6 (60%), which indicates that both treatments affected mTOR activity similarly. Also, both treatment increased levels of autophagy when measured by the ratio of LC3II/LC3I. From our microarray analysis of the liver, we observed that DR has a more dramatic effect on genes than rapa. Our principal component analysis, which provides a broad overview of changes, showed that there was no overlap between rapa and DR, but there were some shared overlap of components between AL and rapa and also between DR and DR+rapa. The gene analysis showed that DR and DR+rapa share a large overlap of genes, whereas overlap between DR and rapa is fewer. We found similar results in our pathway analysis as well. Our preliminary data indicate that although DR and rapa may have similar effects on mTOR activity and autophagy, there are still major differences at the gene and pathway level and that the differences observed among both treatments suggest that rapamycin is not a DR mimetic., Maintenance of protein structure and function has been implicated in the aging process in a variety of model organisms. We are utilizing a range of bivalve mollusk species, with lifespans ranging from under a decade to over 500 years, in a comparative study to investigate the hypothesis that long life requires superior proteome stability. These ages can be individually determined by counting growth rings in the shell. This experimental system provides a unique opportunity to study closely related organisms with vastly disparate longevities and their relative proteome stabilities. Specifically, we are testing their ability to maintain structure and function under various stressors. The apolar, fluorescent BisANS probe binds to hydrophobic regions of proteins exposed as they unfold, incorporation being directly related to compromised tertiary structure. Protein aggregation and preservation of GAPDH function under stress was also measured. Stressors included TBHP as an oxidative stress, and urea as an unfolding agent. To date, we find that stress-induced protein unfolding decreases with increasing lifespan. This stability corroborates with superior GAPDH function and decreased protein aggregation under the same stressors. Taken together, these data support the hypothesis that proteome stability is a determinant of longevity.

If $\fA$ is a unital weak-$*$ closed algebra of multiplication operators on a reproducing kernel Hilbert space which has the property $\bA_1(1)$, then the cyclic invariant subspaces index a Nevanlinna-Pick family of kernels. This yields an NP interpolation theorem for a wide class of algebras. In particular, it applies to many function spaces over the unit disk including Bergman space. We also show that the multiplier algebra of a complete NP space has $\bA_1(1)$, and thus this result applies to all of its subalgebras. A matrix version of this result is also established. It applies, in particular, to all unital weak-$*$ closed subalgebras of $H^\infty$ acting on Hardy space or on Bergman space., 26 pages; minor revisions; to appear in Integral Equations and Operator Theory

To examine harassment and abusive encounters between family physicians and their patients or colleagues in the workplace.Qualitative case study using semistructured interviews.Province of New Brunswick.Forty-eight family physicians from across the province.A collective case-study approach was developed, with 24 cases of 2 individuals per case. Cases were selected based on sex, location (urban or rural), language (French or English), and number of years since medical school graduation (10 years, 10 to 20 years, or20 years). Physicians were interviewed in either French or English. Participants were recruited using the College of Physicians and Surgeons of New Brunswick's physician directory. Based on the rates of response and participation, some cases were overrepresented, while others were not completed. All interviews were audiotaped, transcribed verbatim, and analyzed thematically using a categorical aggregation approach. A coding scheme for the thematic analysis was developed by the research team before the interviews were transcribed.Although the original intent of this study was to examine the work environment of family physicians in light of the increasing number of women entering the profession, harassment and abusive encounters in the workplace emerged as a main theme. These encounters ranged from minor to severe. Minor abusive encounters included disrespectful behaviour and verbal threats by patients, their families, and occasionally colleagues. More severe forms of harassment involved physical threats, physical encounters, and stalking. Demanding patients, such as heavy drug users, were often seen as threatening. Location of practice, years in practice, and sex of the physician seemed to affect abusive encounters--young, female, rural physicians appeared to experience such encounters most often.Abusive encounters in the workplace are concerning. It is essential to address these issues of workplace harassment and abuse in order to protect physician safety and avoid workplace dissatisfaction. Abusive encounters might push family physicians to leave clinical practice prematurely or refuse to work in higher-risk environments, such as emergency departments or rural areas.