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Calorie restriction ( CR) and rapamycin ( RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin ( mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR. Protein abundance and turnover were measured in vivo using 2H3-leucine heavy isotope labeling followed by LC- MS/ MS, and translation was assessed by polysome profiling. We observed 35-60% increased protein half-lives after CR and 15% increased half-lives after RP compared to age-matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions. [ABSTRACT FROM AUTHOR]

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [²H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman's ρ = 0.57; P< 10-4), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions. [ABSTRACT FROM AUTHOR]

Purpose: Goblet cells may represent a potentially successful adaptive response to acid and bile by producing a thick mucous barrier that protects against cancer development in Barrett's esophagus (BE). The aim of this study was to determine the relationship between goblet cells (GC) and risk of progression to adenocarcinoma, and DNA content flow cytometric abnormalities, in BE patients. Experimental Design: Baseline mucosal biopsies (N=2988) from 213 patients, 32 of whom developed cancer during the follow up period, enrolled in a prospective dynamic cohort of BE patients were scored in a blinded fashion, for the total number (#) of GC, mean # of GC/crypt (GC density), # of crypts with ≥ 1 GC, and the proportion of crypts with ≥1 GC, in both dysplastic and non-dysplastic epithelium separately. The relationship between these four GC parameters and DNA content flow cytometric abnormalities and adenocarcinoma outcome was compared, after adjustment for age, gender, and BE segment length. Results: High GC parameters were inversely associated with DNA content flow cytometric abnormalities, such as aneuploidy, ploidy >2.7N, and an elevated 4N fraction > 6%, and with risk of adenocarcinoma. However, a Kaplan-Meier analysis showed that the total # of GC and the total # crypts with ≥1 GC were the only significant GC parameters (p<0.001 and 0.003, respectively). Conclusions: The results of this study show, for the first time, an inverse relationship between high GC counts and flow cytometric abnormalities and risk of adenocarcinoma in BE. Further studies are needed to determine if GC depleted foci within esophageal columnar mucosa are more prone to neoplastic progression or whether loss of GC occurs secondary to underlying genetic abnormalities. [ABSTRACT FROM AUTHOR]

Purpose: Colon cancers deficient in mismatch repair (MMR) may exhibit diminished expression of the DNA repair gene, MRE11, as a consequence of contraction of a T11 mononucleotide tract. This study investigated MRE11 status and its association with prognosis, survival and drug response in patients with stage III colon cancer. Patients and Methods: Cancer and Leukemia Group B 89803 (Alliance) randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly adjuvant bolus 5-fluorouracil/leucovorin (FU/LV) or irinotecan+FU/LV (IFL), with 8 year follow-up. Tumors from these patients were analyzed to determine stability of a T11 tract in the MRE11 gene. The primary endpoint was overall survival (OS), and a secondary endpoint was disease-free survival (DFS). Non-proportional hazards were addressed using time-dependent covariates in Cox analyses. Results: Of 625 tumor cases examined, 70 (11.2%) exhibited contraction at the T11 tract in one or both MRE11 alleles and were thus predicted to be deficient in MRE11 (dMRE11). In pooled treatment analyses, dMRE11 patients showed initially reduced DFS and OS but improved long-term DFS and OS compared with patients with an intact MRE11 T11 tract. In the subgroup of dMRE11 patients treated with IFL, an unexplained early increase in mortality but better long-term DFS than IFL-treated pMRE11 patients was observed. Conclusions: Analysis of this relatively small number of patients and events showed that the dMRE11 marker predicts better prognosis independent of treatment in the long-term. In subgroup analyses, dMRE11 patients treated with irinotecan exhibited unexplained short-term mortality. MRE11 status is readily assayed and may therefore prove to be a useful prognostic marker, provided that the results reported here for a relatively small number of patients can be generalized in independent analyses of larger numbers of samples. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Chronic caloric restriction (CR) and rapamycin inhibit the mechanistic target of rapamycin ( mTOR) signaling, thereby regulating metabolism and suppressing protein synthesis. Caloric restriction or rapamycin extends murine lifespan and ameliorates many aging-associated disorders; however, the beneficial effects of shorter treatment on cardiac aging are not as well understood. Using a recently developed deuterated-leucine labeling method, we investigated the effect of short-term (10 weeks) CR or rapamycin on the proteomics turnover and remodeling of the aging mouse heart. Functionally, we observed that short-term CR and rapamycin both reversed the pre-existing age-dependent cardiac hypertrophy and diastolic dysfunction. There was no significant change in the cardiac global proteome (823 proteins) turnover with age, with a median half-life 9.1 days in the 5-month-old hearts and 8.8 days in the 27-month-old hearts. However, proteome half-lives of old hearts significantly increased after short-term CR (30%) or rapamycin (12%). This was accompanied by attenuation of age-dependent protein oxidative damage and ubiquitination. Quantitative proteomics and pathway analysis revealed an age-dependent decreased abundance of proteins involved in mitochondrial function, electron transport chain, citric acid cycle, and fatty acid metabolism as well as increased abundance of proteins involved in glycolysis and oxidative stress response. This age-dependent cardiac proteome remodeling was significantly reversed by short-term CR or rapamycin, demonstrating a concordance with the beneficial effect on cardiac physiology. The metabolic shift induced by rapamycin was confirmed by metabolomic analysis. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases. [ABSTRACT FROM AUTHOR]

Abstract: BAC arrays were used to evaluate genomic instability along the colon of patients with ulcerative colitis (UC). Genomic instability increases with disease progression and biopsies more proximal to dysplasia showed increased instability. Pan-colonic field copy number gain or loss involving small (<1Mb) regions were detected in most patients and were particularly apparent in the UC progressor patients who had dysplasia or cancer. Chromosomal copy gains or losses affecting large regions were mainly restricted to dysplastic biopsies. Areas of significant chromosomal losses were detected in the UC progressors on chromosomes 2q36, 3q25, 3p21, 4q34, 4p16.2, 15q22, and 16p13 (p-value⩽0.04). These results extend our understanding of the dynamic nature of pan-colonic genomic instability in this disease. [Copyright &y& Elsevier]

Objectives: We investigated the effect of reducing mitochondrial oxidative stress by the mitochondrial-targeted antioxidant peptide SS-31 in hypertensive cardiomyopathy. Background: Oxidative stress has been implicated in hypertensive cardiovascular diseases. Mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase have been proposed as primary sites of reactive oxygen species (ROS) generation. Methods: The mitochondrial targeted antioxidant peptide SS-31 was used to determine the role of mitochondrial oxidative stress in angiotensin II (Ang)-induced cardiomyopathy as well as in Gαq overexpressing mice with heart failure. Results: Ang induces mitochondrial ROS in neonatal cardiomyocytes, which is prevented by SS-31, but not the nontargeted antioxidant N-acetyl cysteine (NAC). Continuous administration of Ang for 4 weeks in mice significantly increased both systolic and diastolic blood pressure, and this was not affected by SS-31 treatment. Ang was associated with up-regulation of NADPH oxidase 4 (NOX4) expression and increased cardiac mitochondrial protein oxidative damage, and induced the signaling for mitochondrial biogenesis. Reducing mitochondrial ROS by SS-31 substantially attenuated Ang-induced NOX4 up-regulation, mitochondrial oxidative damage, up-regulation of mitochondrial biogenesis, and phosphorylation of p38 mitogen-activated protein kinase and prevented apoptosis, concomitant with amelioration of Ang-induced cardiac hypertrophy, diastolic dysfunction, and fibrosis, despite the absence of blood pressure-lowering effect. The NAC did not show any beneficial effect. The SS-31 administration for 4 weeks also partially rescued the heart failure phenotype of Gαq overexpressing mice. Conclusions: Mitochondrial targeted peptide SS-31 ameliorates cardiomyopathy resulting from prolonged Ang stimulation as well as Gαq overexpression, suggesting its potential clinical application for target organ protection in hypertensive cardiovascular diseases. [Copyright &y& Elsevier]

Objective Mitochondrial complex I deficits have long been associated with Parkinson disease (PD). However, it remains unknown whether such defects represent a primary event in dopaminergic neurodegeneration. Methods Apoptosis-inducing factor (AIF) is a mitochondrial protein that, independently of its proapoptotic properties, plays an essential physiologic role in maintaining a fully functional complex I. We used AIF-deficient harlequin (Hq) mice, which exhibit structural deficits in assembled complex I, to determine whether primary complex I defects linked to AIF depletion may cause dopaminergic neurodegeneration. Results Despite marked reductions in mitochondrial complex I protein levels, Hq mice did not display apparent alterations in the dopaminergic nigrostriatal system. However, these animals were much more susceptible to exogenous parkinsonian complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Subtoxic doses of MPTP, unable to cause damage to wild-type animals, produced marked nigrostriatal dopaminergic degeneration in Hq mice. This effect was associated with exacerbated complex I inhibition and increased production of mitochondrial-derived reactive oxygen species (ROS) in Hq brain mitochondria. The antioxidant superoxide dismutase-mimetic compound tempol was able to reverse the increased susceptibility of Hq mice to MPTP. Supporting an instrumental role for mitochondrial-derived ROS in PD-related neurodegeneration, transgenic mice overexpressing mitochondrially targeted catalase exhibited an attenuation of MPTP-induced mitochondrial ROS and dopaminergic cell death. Interpretation Structural complex I alterations linked to AIF deficiency do not cause dopaminergic neurodegeneration but increase the susceptibility of dopaminergic neurons to exogenous parkinsonian neurotoxins, reinforcing the concept that genetic and environmental factors may interact in a common molecular pathway to trigger PD. ANN NEUROL 2010;68:184-192 [ABSTRACT FROM AUTHOR]

Abstract: Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn −/− mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn −/− mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts. These analyses confirmed previously reported cellular phenotypes of WRN-mutant and WRN-deficient human fibroblasts, and demonstrated that the human WRN-deficient cellular phenotype can be detected in cells grown in 5% or in 20% oxygen. In contrast, we did not identify prominent cellular phenotypes present in WRN-deficient human cells in Wrn −/− mouse fibroblasts. Our results indicate that human and mouse fibroblasts have different functional requirements for WRN protein, and that the absence of a strong cellular phenotype may in part explain the failure of Wrn −/− mice to develop an organismal phenotype resembling Werner syndrome. [Copyright &y& Elsevier]

Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer. [ABSTRACT FROM AUTHOR]

Age-related hearing loss (AHL), known as presbycusis. is a universal feature of mammalian aging and is the most common sensory disorder in the elderly population. The molecular mechanisms underlying AHL are unknown, and currently there is no treatment for the disorder. Here we report that C57B116J mice with a deletion of the mitochondrial pro-apoptotic gene Bak exhibit reduced age-related apoptotic cell death of spiral ganglion neurons and hair cells in the cochlea, and prevention of AHL. Oxidative stress induces Bak expression in primary cochlear cells, and Bak deficiency prevents apoptotic cell death. Furthermore, a mitochondrially targeted catalase transgene suppresses Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL. Oral supplementation with the mitochondrial antioxidants α-lipoic acid and coenzyme Q10 also suppresses Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL. Thus, induction of a Bak-dependent mitochondrial apoptosis program in response to oxidative stress is a key mechanism of AHL in C57BL/6J mice. [ABSTRACT FROM AUTHOR]

Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIb, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIβ-/-) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIβ-/- male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging. [ABSTRACT FROM AUTHOR]

Oxidative and nitrosative damage are major contributors to cone cell death in retinitis pigmentosa (RP). In this study, we explored the effects of augmenting components of the endogenous antioxidant defense system in models of RP, rd1, and rd10 mice. Unexpectedly, overexpression of superoxide dismutase 1 (SOD1) in rd1 mice increased oxidative damage and accelerated cone cell death. With an elaborate mating scheme, genetically engineered rd10 mice with either inducible expression of SOD2, Catalase, or both in photoreceptor mitochondria were generated. Littermates with the same genetic background that did not have increased expression of SOD2 nor Catalase provided ideal controls. Coexpression of SOD2 and Catalase, but not either alone, significantly reduced oxidative damage in the retinas of postnatal day (P) 50 rd10 mice as measured by protein carbonyl content. Cone density was significantly greater in P50 rd10 mice with coexpression of SOD2 and Catalase together than rd10 mice that expressed SOD2 or Catalase alone, or expressed neither. Coexpression of SOD2 and Catalase in rd10 mice did not slow rod cell death. These data support the concept of bolstering the endogenous antioxidant defense system as a gene-based treatment strategy for RP, and also indicate that coexpression of multiple components may be needed.Molecular Therapy (2009) 17 5, 778–786 doi:10.1038/mt.2009.47 [ABSTRACT FROM AUTHOR]

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. [Copyright &y& Elsevier]

Sustained caloric restriction (CR) extends lifespan in animal models but the mechanism and primary tissue target(s) have not been identified. Gene expression changes with aging and CR were examined in both heart and white adipose tissue (WAT) of Fischer 344 (F344) male rats using Affymetrix® RAE 230 arrays and validated by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) on 18 genes. As expected, age had a substantial effect on transcription on both tissues, although only 21% of cardiac age-associated genes were also altered in WAT. Gene set enrichment analysis revealed coordinated small magnitude changes in ribosomal, proteasomal, and mitochondrial genes with similarities in aging between heart and WAT. CR had very different effects on these two tissues at the transcriptional level. In heart, very few age-associated expression changes were affected by CR, while in WAT, CR suppressed a substantial subset of the age-associated changes. Genes unaltered by aging but altered by CR were identified in WAT but not heart. Most interestingly, we identified a gene expression signature associated with mammalian target of rapamycin (mTOR) activity that was down-regulated with age but preserved by CR in both WAT and heart. In addition, lipid metabolism genes, particularly those associated with peroxisome proliferator-activated receptor γ (PPARγ)-mediated adipogenesis were reduced with age but preserved with CR in WAT. These results highlight tissue-specific differences in the gene expression response to CR and support a role for CR-mediated preservation of mTOR activity and adipogenesis in aging WAT. [ABSTRACT FROM AUTHOR]

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate. Here, we use a new, highly sensitive assay to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single–base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice. [ABSTRACT FROM AUTHOR]

Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN-deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40-transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell phenotypes that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expression of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest. [ABSTRACT FROM AUTHOR]

Background Somatic genetic CDKN2A, TP53, and DNA content abnormalities are common in many human cancers and their precursors, including esophageal adenocarcinoma (EA) and Barrett's esophagus (BE), conditions for which aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as possible chemopreventive agents; however, little is known about the ability of a biomarker panel to predict progression to cancer nor how NSAID use may modulate progression. We aimed to evaluate somatic genetic abnormalities with NSAIDs as predictors of EA in a prospective cohort study of patients with BE. Methods and Findings Esophageal biopsies from 243 patients with BE were evaluated at baseline for TP53 and CDKN2A (p16) alterations, tetraploidy, and aneuploidy using sequencing; loss of heterozygosity (LOH); methylation-specific PCR; and flow cytometry. At 10 y, all abnormalities, except CDKN2A mutation and methylation, contributed to EA risk significantly by univariate analysis, ranging from 17p LOH (relative risk [RR] = 10.6; 95% confidence interval [CI] 5.2–21.3, p < 0.001) to 9p LOH (RR = 2.6; 95% CI 1.1–6.0, p = 0.03). A panel of abnormalities including 17p LOH, DNA content tetraploidy and aneuploidy, and 9p LOH was the best predictor of EA (RR = 38.7; 95% CI 10.8–138.5, p < 0.001). Patients with no baseline abnormality had a 12% 10-y cumulative EA incidence, whereas patients with 17p LOH, DNA content abnormalities, and 9p LOH had at least a 79.1% 10-y EA incidence. In patients with zero, one, two, or three baseline panel abnormalities, there was a significant trend toward EA risk reduction among NSAID users compared to nonusers (p=0.01). The strongest protective effect was seen in participants with multiple genetic abnormalities, with NSAID nonusers having an observed 10-y EA risk of 79% compared to 30% for NSAID users (p < 0.001). Conclusions A combination of 17p LOH, 9p LOH, and DNA content abnormalities provided better EA risk prediction than any single TP53, CDKN2A, or DNA content lesion alone. NSAIDs are associated with reduced EA risk, especially in patients with multiple high-risk molecular abnormalities. [ABSTRACT FROM AUTHOR]

Neoplasms are thought to progress to cancer through genetic instability generating cellular diversity and clonal expansions driven by selection for mutations in cancer genes. Despite advances in the study of molecular biology of cancer genes, relatively little is known about evolutionary mechanisms that drive neoplastic progression. It is unknown, for example, which may be more predictive of future progression of a neoplasm: genetic homogenization of the neoplasm, possibly caused by a clonal expansion, or the accumulation of clonal diversity. Here, in a prospective study, we show that clonal diversity measures adapted from ecology and evolution can predict progression to adenocarcinoma in the premalignant condition known as Barrett's esophagus, even when controlling for established genetic risk factors, including lesions in TP53 (p53; ref. 6) and ploidy abnormalities. Progression to cancer through accumulation of clonal diversity, on which natural selection acts, may be a fundamental principle of neoplasia with important clinical implications. [ABSTRACT FROM AUTHOR]

Summary: Background: Aspirin and other non-steroidal anti-inflammatory drugs (NSAID) probably decrease the risk of colorectal neoplasia; however their effect on development of oesophageal adenocarcinoma is less clear. We aimed to assess the role of NSAID in the development of oesophageal adenocarcinoma and precursor lesions in people with Barrett''s oesophagus—a metaplastic disorder that confers a high risk of oesophageal adenocarcinoma. Methods: We did a prospective study of the relation between duration, frequency, and recency of NSAID use and the risk of oesophageal adenocarcinoma, aneuploidy, and tetraploidy in a cohort of 350 people with Barrett''s oesophagus followed for 20 770 person-months. We used proportional-hazards regression to calculate hazard ratios (HR) adjusted for age, sex, cigarette use, and anthropometric measurements. Findings: Median follow-up was 65·5 months (range 3·1–106·9). Compared with never users, HR for oesophageal adenocarcinoma (n=37 cases) in current NSAID users was 0·32 (95% CI 0·14–0·76), and in former users was 0·70 (0·31–1·58). 5-year cumulative incidence of oesophageal adenocarcinoma was 14·3% (95% CI 9·3–21·6) for never users, 9·7% (4·5–20·5) for former users, and 6·6% (3·1–13·6) for current NSAID users. When changes in NSAID use during follow up were taken into account, the associations were strengthened: HR for oesophageal adenocarcinoma for current users at baseline or afterwards was 0·20 (95% CI 0·10–0·41) compared with never users. Compared with never users, current NSAID users (at baseline and follow-up) had less aneuploidy (n=35 cases; 0·25 [0·12–0·54]) and tetraploidy (n=45 cases; 0·44 [0·22–0·87]). Interpretation: NSAID use might be an effective chemopreventive strategy, reducing the risk of neoplastic progression in Barrett''s oesophagus. [Copyright &y& Elsevier]

Abstract: Ulcerative colitis (UC) is an inflammatory disease of the colon that is associated with increased risk of colorectal cancer associated with genomic instability. We have previously demonstrated that genomic instability is present in UC patients with colonic neoplasia, and hypothesized that the chromosomal alterations may be taking place in regions that are susceptible to mutation or that provide a growth advantage to a cell undergoing neoplastic transformation. In this study, we used two polymerase chain reaction (PCR)-based DNA fingerprinting techniques (arbitrarily primed PCR and inter-simple-sequence-repeat PCR) to study the process of genomic instability. The two techniques of DNA fingerprinting cross-validate the instability observed in these studies. We analyzed the molecular basis of 10 commonly altered DNA bands obtained from DNA fingerprints of biopsies from various histologic grades of UC patients with dysplasia or cancer (UC Progressors). We determined that the band changes in the fingerprint truly represent changes in DNA sequence, and that the fingerprinting provides highly reproducible results. Furthermore, our investigation revealed that 40% of alterations involve repetitive sequences. Two frequently deleted sequences in 6q27 and 2q14 were studied further because they were frequently abnormal in the dysplastic and nondysplastic tissue of UC Progressors. The losses from 6q27 and 2q14 were confirmed by loss of heterozygosity and real-time PCR analysis. Both of these regions in chromosomes 6 and 2 are surrounded by highly repetitive and mobile LINE-1 elements, possibly making the region susceptible to mutational change. These regions were affected (lost) in UC Progressors but not in UC patients who were neoplasia free. Loss of heterozygosity at 6q27 has been described in ovarian and other cancers, while the 2q14 region has been implicated in prostate and sporadic colon cancers. Both regions are likely to contain tumor-suppressor genes. In conclusion, the genomic instability in UC Progressors can occur in regions that are susceptible to change and are locations of putative tumor-suppressor genes. [Copyright &y& Elsevier]

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-β-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway. [Copyright &y& Elsevier]

The Werner syndrome (WS) protein (WRN), a DNA helicase/exonuclease, is required for genomic stability and avoidance of cancer. Current evidence suggests that WRN is involved in the resolution of stalled and/or collapsed replication forks. This function is indicated, in part, by replication defects in WS cells and by hypersensitivity to agents causing major structural aberrations in DNA that block replication. We show here that antisense suppression of WRN in two human glioma cell lines reproduces hallmarks of the drug cytotoxicity profile of WS cells, namely, hypersensitivity to 4-nitroquinoline 1-oxide, camptothecin and hydroxyurea. We also show that antisense-treated cells are hypersensitive to methyl-lexitropsin, a site-specific alkylating agent that produces mainly N3-methyladenine, a cytotoxic and replication-blocking lesion. Antisense-treated cells are hypersensitive to O6-methylguanine adducts as well, but only when repair by O6-methylguanine–DNA methyltransferase is lacking. Our results illustrate the drug sensitivity caused by deficiency of WRN in a uniform genetic background. They extend the WRN DNA damage sensitivity spectrum to methyl base adducts that can result in blocked replication, and suggest that WRN may be required for resumption of processive replication when incomplete repair of DNA damage leaves blocking lesions at forks. The evidence that highly disparate lesions fall within the purview of WRN, and that abrogating DNA repair can reveal dependence on WRN, suggests that WRN may protect the genome from the lethal, mutagenic and carcinogenic effects of widely diverse DNA damage arising from endogenous processes and environmental agents. [Copyright &y& Elsevier]

Background: Persons with Barrett's esophagus have a substantially greater risk of esophageal adenocarcinoma than the general population. Higher serum selenium levels have been associated with a reduced risk of several cancers; however, their association with the risk of esophageal adenocarcinoma is unknown. We used a cross-sectional study to investigate the relationship between serum selenium levels and markers of neoplastic progression among persons with Barrett's esophagus.Methods: Medical history, blood, and esophageal tissue specimens were collected from 399 members of a cohort study of Barrett's esophagus patients undergoing endoscopic surveillance. Serum selenium levels were measured by flameless atomic absorption spectrophotometry. DNA content of tissue samples was measured by flow cytometry. Loss of heterozygosity (LOH) at 9p and 17p, chromosomal regions which include the p16 and p53 tumor suppressors, respectively, was detected by automated fluorescent genotyping. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). All statistical tests were two-sided.Results: Persons with serum selenium levels in the upper three quartiles (i.e., >1.5 micro M) were less likely to have high-grade dysplasia (OR = 0.5, 95% CI = 0.3 to 0.9) or aneuploidy (OR = 0.4, 95% CI = 0.2 to 0.8) than those with levels in the lowest quartile. Serum selenium levels in the upper three quartiles were associated with similar reductions in risk of 17p (p53) LOH (OR = 0.5, 95% CI = 0.2 to 0.9) and increased 4N fraction (OR = 0.6, 95% CI = 0.3 to 1.2). By contrast, serum selenium levels were not associated with 9p (p16) LOH (OR = 1.0, 95% CI = 0.5 to 1.7), a marker that appears early in neoplastic progression.Conclusion: Our preliminary results, from a cross-sectional analysis with biologic markers, suggest that higher serum selenium levels may be associated with a reduced risk of esophageal adenocarcinoma among persons with Barrett's esophagus. Because serum selenium was not associated with 9p (p16) LOH, we speculate that selenium may act primarily at later stages of progression toward adenocarcinoma. [ABSTRACT FROM AUTHOR]

Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma that is thought to develop through genomic instability. We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening, thereby increasing the potential of chromosomal ends to fuse, resulting in cycles of chromatin bridge breakage and fusion and chromosomal instability associated with tumor cell progression. Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges—an intermediate in the breakage and fusion of chromatin bridges—in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer. [ABSTRACT FROM AUTHOR]

Presents a study that focused on the use of oligonucleotide-based microarrays to characterize gene expression profiles in each of Barrett's esophagus (BE) tissues. Similarity of BE to each of the normal tissues; Description of the Barrett's esophagus condition; Complication in persons with chronic gastroesophageal reflux disease.

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models. [ABSTRACT FROM AUTHOR]

To determine the role of reactive oxygen species in mammalianlongevity, we generated transgenic mice that overexpress human catalaselocalized to the peroxisome, the nucleus, or mitochondria (MCAT). Medianand maximum life spans were maximally increased (averages of 5 monthsand 5.5 months, respectively) in MCAT animals. Cardiac pathology andcataract development were delayed, oxidative damage was reduced, H[sub2]O[sub 2] production and H[sub 2]O[sub 2]-induced aconitaseinactivation were attenuated, and the development of mitochondrialdeletions was reduced. These results support the free radical theory ofaging and reinforce the importance of mitochondria as a source of theseradicals. [ABSTRACT FROM AUTHOR]

An abstract of the article "Fish As an Alternative Test for LOH in Barrett's Esophagus: Implications for the Mechanism of P53 Loss," by Vaew J. Wongsurawat, Jennifer C. Finley, Patricia C. Galipeau, Carissa A. Sanchez, Carlo Maley, Patricia L. Blount, and colleagues is presented.

Changes in mitochondrial function play a critical role in the basic biology of aging and age-related disease. Mitochondria are typically thought of in the context of ATP production and oxidant production. However, it is clear that the mitochondria sit at a nexus of cell signaling where they affect metabolite, redox, and energy status, which influence many factors that contribute to the biology of aging, including stress responses, proteostasis, epigenetics, and inflammation. This has led to growing interest in identifying mitochondrial targeted interventions to delay or reverse age-related decline in function and promote healthy aging. In this review, we discuss the diverse roles of mitochondria in the cell. We then highlight some of the most promising strategies and compounds to target aging mitochondria in preclinical testing. Finally, we review the strategies and compounds that have advanced to clinical trials to test their ability to improve health in older adults., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The pathology of aging impacts multiple organ systems, including the kidney and skeletal and cardiac muscles. Long-term treatment with the mitochondrial-targeted peptide elamipretide has previously been shown to improve in vivo mitochondrial function in aged mice, which is associated with increased fatigue resistance and treadmill performance, improved cardiovascular diastolic function, and glomerular architecture of the kidney. However, elamipretide is a short tetrameric peptide that is not orally bioavailable, limiting its routes of administration. This study tested whether twice weekly intermittent injections of elamipretide could recapitulate the same functional improvements as continuous long-term infusion. We found that intermittent treatment with elamipretide for 8 months preserved exercise tolerance and left ventricular mass in mice with modest protection of diastolic function and skeletal muscle force production but did not affect kidney function as previously reported using continuous treatment., (© 2023. The Author(s), under exclusive licence to American Aging Association.)

Age-associated diseases are becoming progressively more prevalent, reflecting the increased lifespan of the world's population. However, the fundamental mechanisms of physiologic aging are poorly understood, and in particular, the molecular pathways that mediate cardiac aging and its associated dysfunction are unclear. Here, we focus on certain ion flux abnormalities of the mitochondria that may contribute to cardiac aging and age-related heart failure. Using oxidative phosphorylation, mitochondria pump protons from the matrix to the intermembrane space to generate a proton gradient across the inner membrane. The protons are returned to the matrix by the ATPase complex within the membrane to generate ATP. However, a portion of protons leak back to the matrix and do not drive ATP production, and this event is called proton leak or uncoupling. Accumulating evidence suggests that mitochondrial proton leak is increased in the cardiac myocytes of aged hearts. In this mini-review, we discuss the measurement methods and major sites of mitochondrial proton leak with an emphasis on the adenine nucleotide transporter 1 (ANT1), and explore the possibility of inhibiting augmented mitochondrial proton leak as a therapeutic intervention to mitigate cardiac aging., (© 2023. The Author(s), under exclusive licence to American Aging Association.)

Aims: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD, termed PSC-IBD) have a higher risk of harbouring nonconventional and/or invisible dysplasias, especially in the right/proximal colon, than those with IBD alone. We postulated that DNA content abnormality may be frequently detected in the right/proximal colon in PSC-IBD patients, even in the absence of dysplasia, and that this may predispose to progression to nonconventional and/or invisible dysplasias that are often associated with increased rates of aneuploidy and advanced neoplasia., Methods and Results: DNA flow cytometry was performed on 96 morphologically benign colon biopsies taken throughout the colon from 25 PSC-IBD patients during the surveillance colonoscopy that preceded the next procedure that detected dysplasia. Thirty (31%) of the 96 benign colon biopsies in this dysplasia group demonstrated abnormal DNA content, with a propensity for the right/proximal colon (70%) (P < 0.001). In contrast, only one (1%) of 87 benign colon biopsies from 20 IBD patients without neoplasia (control group) demonstrated DNA content abnormality, and it was from the left colon. For analysis per patient, 48% (12 of 25) of the patients in the dysplasia group had abnormal DNA content compared with 5% (1 of 20) of the control group (P = 0.002). Of the 12 PSC-IBD patients with DNA content abnormality, invisible dysplasia was detected in 10 (83%) patients on follow-up, nine (75%) of whom had nonconventional dysplasia., Conclusion: PSC-IBD patients have an increased risk of developing abnormal DNA content in the right/proximal colon, predating the detection of dysplasia., (© 2023 John Wiley & Sons Ltd.)

Fundic gland polyps (FGPs) develop sporadically (frequently after proton pump inhibitor therapy) or in the setting of a hereditary polyposis syndrome, such as familial adenomatous polyposis (FAP). FAP-related FGPs often demonstrate low-grade dysplasia (LGD) and are frequently associated with APC mutations, even in the absence of dysplasia. Sporadic FGPs with dysplasia are molecularly similar to FAP-related FGPs and demonstrate frequent mutations in APC gene. Despite having similar molecular alterations with colorectal and other adenomatous precursor lesions in the gastrointestinal (GI) tract, FGPs rarely progress to advanced gastric neoplasia (high-grade dysplasia [HGD] or adenocarcinoma), and their role in gastric tumorigenesis remains unclear but likely limited. The clinicopathologic features of 192 patients diagnosed with FGPs, including 86 with FAP-related FGPs (33 with dysplastic FGPs and 53 with nondysplastic FGPs) and 106 with sporadic FGPs (12 with dysplastic FGPs and 94 with nondysplastic FGPs), were analyzed. DNA flow cytometry was performed on 111 FAP-related FGP biopsies, including 32 FGPs with LGD and 79 nondysplastic FGPs, to assess the presence of abnormal DNA content (ie, aneuploidy or elevated 4N fraction). Moreover, 40 sporadic FGP biopsies, including 14 dysplastic (13 LGD and 1 HGD) and 26 nondysplastic FGPs, were examined for DNA content abnormality. Patients with FAP and nondysplastic FGPs were more likely to be younger (mean age, 32 years) and present with multiple FGPs (92%, defined as having ≥2 FGPs) than those with sporadic nondysplastic FGPs (61 years and 65%, respectively; P < .001). They also recorded higher rates of previous or concurrent gastric epithelial dysplasia not occurring in a FGP (8%, P = .016), nongastric GI dysplasia (96%, P < .001), and nongastric GI malignancy (17%, P = .001) compared with those with sporadic nondysplastic FGPs (0%, 52%, and 2%, respectively). The sporadic group was more frequently associated with proton pump inhibitor therapy (78%, P < .001), gastric intestinal metaplasia (24%, P = .004), and a family history of gastric cancer (10%, P = .027) than the FAP group (19%, 6%, and 0%, respectively). Almost all FAP-related FGPs had a polypoid endoscopic appearance (98% vs 84% for sporadic FGPs; P = .009). The mean size of the largest FAP-related FGPs (0.5 cm) was similar to that of sporadic FGPs (0.7 cm) (P = .069). None of the 147 patients with FAP-related or sporadic nondysplastic FGPs were associated with subsequent detection of advanced gastric neoplasia within a mean follow-up time of 54 months (range, <1 to 277 months). However, 2 (4%) of the 45 patients with FAP-related or sporadic dysplastic FGPs developed advanced gastric neoplasia within a mean follow-up time of 59 months (range, <1 to 236 months). One (3%) of the 33 patients with FAP and dysplastic FGPs developed signet ring cell adenocarcinoma, whereas 1 (8%) of the 12 patients with sporadic dysplastic FGPs developed HGD (P = .445). However, none of the FAP-related and sporadic FGP biopsies, regardless of the presence or absence of dysplasia, demonstrated DNA content abnormality. In conclusion, FGPs lack large-scale chromosomal changes that are characteristic of the typical adenoma-carcinoma sequence involved in the development of other GI malignancies. Progression to advanced gastric neoplasia is rare in FGPs, which may be partly explained by the apparent lack of the chromosomal instability phenotype in these lesions., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software. The computational pipeline of TurnoveR performs critical steps to determine protein turnover rates, including isotopologue demultiplexing, precursor-pool correction, statistical analysis, and generation of data reports and visualizations. This workflow is compatible with many mass spectrometric platforms and recapitulates turnover rates and differential changes in turnover rates between treatment groups calculated in previous studies. We expect that the addition of TurnoveR to the widely used Skyline proteomics software will facilitate wider utilization of protein turnover analysis in highly relevant biological models, including aging, neurodegeneration, and skeletal muscle atrophy.

The age-related decline in skeletal muscle mass and function is known as sarcopenia. Sarcopenia progresses based on complex processes involving protein dynamics, cell signaling, oxidative stress, and repair. We have previously found that 8-week treatment with elamipretide improves skeletal muscle function, reverses redox stress, and restores protein S-glutathionylation changes in aged female mice. This study tested whether 8-week treatment with elamipretide also affects global phosphorylation in skeletal muscle consistent with functional improvements and S-glutathionylation. Using female 6-7-month-old mice and 28-29-month-old mice, we found that phosphorylation changes did not relate to S-glutathionylation modifications, but that treatment with elamipretide did partially reverse age-related changes in protein phosphorylation in mouse skeletal muscle., (© 2022. The Author(s).)

Aging and poor diet are independent risk factors for heart disease, but the impact of high-sucrose (HS) consumption in the aging heart is understudied. Aging leads to impairments in mitochondrial function that result in muscle dysfunction (e.g., cardiac remodeling and sarcopenia). We tested whether HS diet (60%kcal sucrose) would accelerate muscle dysfunction in 24-month-old male CB6F1 mice. By week 1 on HS diet, mice developed significant cardiac hypertrophy compared to age-matched chow-fed controls. The increased weight of the heart persisted throughout the 4-week treatment, while body weight and strength declined more rapidly than controls. We then tested whether HS diet could worsen cardiac dysfunction in old mice and if the mitochondrial-targeted drug, elamipretide (ELAM), could prevent the diet-induced effect. Old and young mice were treated with either ELAM or saline as a control for 2 weeks, and provided with HS diet or chow on the last week. As demonstrated in the previous experiment, old mice had age-related cardiac hypertrophy that worsened after one week on HS and was prevented by ELAM treatment, while the HS diet had no detectable effect on hypertrophy in the young mice. As expected, mitochondrial respiration and reactive oxygen species (ROS) production were altered by age, but were not significantly affected by HS diet or ELAM. Our findings highlight the vulnerability of the aged heart to HS diet that can be prevented by systemic targeting of the mitochondria with ELAM.

We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs elamipretide (SS-31) and nicotinamide mononucleotide (NMN) to reverse aging-associated changes in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are altered with age, while also inducing additional changes. Age-related increases in protein acetylation were predominantly in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling. We further assessed how these age-related changes associated with diastolic function (Ea/Aa) and systolic function (fractional shortening under higher workload) measurements from echocardiography. These results identify a subset of protein abundance and acetylation changes in muscle, mitochondrial, and structural proteins that appear to be essential in regulating diastolic function in old hearts., (© 2022. The Author(s), under exclusive licence to American Aging Association.)

The isolation of intact single adult cardiomyocytes from model animals, mouse and rat, is an essential tool for cardiac molecular and cellular research. While several methods are reported for adult mouse cardiomyocyte isolation, the viability and yield of the isolated cells have been variable. Here, we describe step-by-step protocols for high viability and yield cardiomyocyte isolation from mouse and rat, based on the use of a stable pressure Langendorff perfusion system. After the animal is euthanized or terminally anesthetized, the heart is removed from the chest and subject to Langendorff perfusion. Then, the heart is digested by perfusion with collagenase and hyaluronidase. After thorough digestion, the cardiomyocytes are dispersed and gradually recovered, the extracellular Ca 2+ concentration adjusted, and cells are then ready for use. This protocol will facilitate research that requires isolated adult mouse and rat cardiomyocytes., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

Nonampullary duodenal adenomas (NADAs) develop sporadically or in the setting of a hereditary syndrome such as familial adenomatous polyposis (FAP). Although they are thought to progress into duodenal adenocarcinomas via an adenoma to carcinoma sequence similar to colorectal cancer, limited data suggested that they may be biologically dissimilar to colorectal adenomas. The clinicopathologic features of 71 patients diagnosed with NADAs (37 FAP and 34 sporadic) were analyzed. From the 71 patients, 89 NADA biopsies (42 FAP and 47 sporadic) were evaluated by DNA flow cytometry. Eighty-two samples showed low-grade dysplasia, and 7 demonstrated high-grade dysplasia (HGD). Twenty-one low-grade adenomas of the ileal pouch (n=19) and jejunum (n=2) from 15 FAP patients who underwent total proctocolectomy were also analyzed by DNA flow cytometry. The FAP patients were more likely to be younger (mean: 28 y) and have multifocal disease (92%) than the sporadic patients (66 y and 24%, respectively) (P<0.001). Most NADAs presented as polypoid lesions (87%) in the duodenal bulb and/or second portion of the duodenum (94%). Sporadic NADAs (mean: 2.4 cm) were significantly larger than FAP-related NADAs (1.3 cm) (P=0.005). Three (4%) patients (2 sporadic and 1 FAP) had high-grade NADAs at the first endoscopy, while the remaining 68 (96%) patients had low-grade dysplasia. Two additional sporadic and 1 FAP patients developed HGD on follow-up. Although the overall detection rate of advanced neoplasia (either HGD or adenocarcinoma) was similar between the FAP (n=5; 14%) and sporadic groups (n=4; 12%) (P=1.000), 3 FAP patients (all with Spigelman stage III to IV) developed adenocarcinoma in the duodenum (n=2) or in the ileal pouch (n=1) within a mean follow-up time of 76 months, while no adenocarcinoma was found in the sporadic group. Of the 37 FAP patients, 29 (78%) had a history of total proctocolectomy, and 15 (52%) developed low-grade adenomas in the ileal pouch with (n=2) or without (n=13) jejunal involvement (vs. 0% in the sporadic patients, P<0.001). All 15 patients had ≥Spigelman stage II. Aneuploidy was detected in only 1 (1%) sporadic NADA with HGD, whereas the remaining 109 duodenal, ileal pouch, and jejunal adenomas showed normal DNA content. The overall 3-, 9-, and 15-year detection rates of adenocarcinoma (in the duodenum and ileal pouch) in all NADA patients were 1.4%, 7.2%, and 18.8%, respectively. Three-, 9-, and 15-year detection rates of adenocarcinoma in the FAP patients were 2.7%, 9.7%, and 22.6%, respectively, while these rates remained at 0% in the sporadic patients. In conclusion, FAP-related NADAs have distinct clinicopathologic features compared with their sporadic counterpart. However, the vast majority of both FAP-related and sporadic NADAs (99%) lack the DNA content abnormality that is characteristic of the typical adenoma-carcinoma sequence involved in other gastrointestinal carcinogenesis. Although adenocarcinoma is more likely to develop in FAP patients with a high adenoma burden, probably due to the higher likelihood that some advanced lesions are missed endoscopically, FAP-related and sporadic NADAs may have a comparable risk of developing advanced neoplasia on a per-adenoma basis., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Department of Pathology, University of California at San Francisco. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light., Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay., Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable., Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.

Most gastric cancers (GCs) are thought to develop via gastric intestinal metaplasia (GIM)-dysplasia-carcinoma pathway. Patients with extensive and/or incomplete GIM have been reported to have a higher risk of GC. GIM can also display dysplasia-like cytoarchitectural atypia limited to the bases of gastric pits without surface involvement. However, only a small proportion of GIM patients will develop gastric neoplasia, and it remains questionable if GIM is a direct precursor. A cohort of 82 GC patients with GIM who underwent gastrectomy were analyzed. DNA flow cytometry was performed on 109 GIM samples (including 88 predominantly complete GIM and 21 predominantly incomplete GIM subclassified based on morphology) obtained from adjacent mucosa of the 82 GCs. Only 2 (2%) of the 109 GIM samples demonstrated aneuploidy, both from 2 minority patients (Asian and Hispanic) with limited and complete GIM and no cytoarchitectural atypia. The remaining 107 GIM samples showed mild to focally moderate basal gland (metaplastic) atypia limited to the bases of gastric pits, but they all demonstrated normal DNA content regardless of anatomic location, histologic GIM subtype, or varying degrees of basal gland atypia. In conclusion, the vast majority of the GIM samples (98%) lack the aneuploidy that is characteristic of gastric dysplasia or cancer. This indicates that aneuploidy usually occurs after the development of gastric dysplasia rather than at the stage of GIM. The finding also suggests that the presence of GIM alone may not be sufficient to suggest an increased risk for GC and that the inclusion of other high-risk features (ie, extensive GIM, dysplasia, racial minorities, and/or family history of GC in a first-degree relative) and/or aneuploidy ought to play a role in the selection of GIM patients who may warrant endoscopic surveillance. Finally, GIM with mild to focally moderate basal gland atypia is likely to represent metaplastic atypia in most cases., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Department of Pathology, University of California at San Francisco. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide., (© 2021. American Aging Association.)