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Background: Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC. Methods: To reclassify the variants we collected clinical data, initiated tumor and co‐segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies. Results: The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair. Conclusion: By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene‐specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations. [ABSTRACT FROM AUTHOR]

FOXO transcription factors modulate aging-related pathways and influence longevity in multiple species, but the transcriptional targets that mediate these effects remain largely unknown. Here, we identify an evolutionarily conserved FOXO target gene, Oxidative stress-responsive serine-rich protein 1 (OSER1), whose overexpression extends lifespan in silkworms, nematodes, and flies, while its depletion correspondingly shortens lifespan. In flies, overexpression of OSER1 increases resistance to oxidative stress, starvation, and heat shock, while OSER1-depleted flies are more vulnerable to these stressors. In silkworms, hydrogen peroxide both induces and is scavenged by OSER1 in vitro and in vivo. Knockdown of OSER1 in Caenorhabditis elegans leads to increased ROS production and shorter lifespan, mitochondrial fragmentation, decreased ATP production, and altered transcription of mitochondrial genes. Human proteomic analysis suggests that OSER1 plays roles in oxidative stress response, cellular senescence, and reproduction, which is consistent with the data and suggests that OSER1 could play a role in fertility in silkworms and nematodes. Human studies demonstrate that polymorphic variants in OSER1 are associated with human longevity. In summary, OSER1 is an evolutionarily conserved FOXO-regulated protein that improves resistance to oxidative stress, maintains mitochondrial functional integrity, and increases lifespan in multiple species. Additional studies will clarify the role of OSER1 as a critical effector of healthy aging. FOXO transcription factors are known to promote longevity via effects on transcriptional targets. In this study, the authors identify OSER1 as an evolutionarily conserved target of FOXO. OSER1 expression improves resistance to oxidative stress, supports mitochondrial function, and extends lifespan across multiple species. [ABSTRACT FROM AUTHOR]

Unhealthy aging poses a global challenge with profound healthcare and socioeconomic implications. Slowing down the aging process offers a promising approach to reduce the burden of a number of age-related diseases, such as dementia, and promoting healthy longevity in the old population. In response to the challenge of the aging population and with a view to the future, Norway and the United Kingdom are fostering collaborations, supported by a "Money Follows Cooperation agreement" between the 2 nations. The inaugural Norway–UK joint meeting on aging and dementia gathered leading experts on aging and dementia from the 2 nations to share their latest discoveries in related fields. Since aging is an international challenge, and to foster collaborations, we also invited leading scholars from 11 additional countries to join this event. This report provides a summary of the conference, highlighting recent progress on molecular aging mechanisms, genetic risk factors, DNA damage and repair, mitophagy, autophagy, as well as progress on a series of clinical trials (eg, using NAD+ precursors). The meeting facilitated dialogue among policymakers, administrative leaders, researchers, and clinical experts, aiming to promote international research collaborations and to translate findings into clinical applications and interventions to advance healthy aging. [ABSTRACT FROM AUTHOR]

Aging is a risk factor for human health and quality of life. Screening and development of novel supplements and medications to combat aging and delay the incidence of age-related diseases are of great significance. In this study, salidroside (SA), a primary natural small molecule from Rhodiola rosea, was investigated regarding its effects on life and healthspan and the underlying molecular mechanism(s) of anti-aging and antioxidation. Our results showed that SA effectively prolonged lifespan and exhibited anti-aging and antioxidative properties. Computer-assisted methods, label-free interaction analysis, and in vitro assays showed that SA directly bound heat shock protein 90 (HSP90). Furthermore, SA significantly inhibited the ATPase activity of HSP90, affecting the interaction between HSP90 and its interacting proteins and the expression of downstream genes to regulate lifespan and the oxidative stress response. Our findings provided new insights into the pharmacological properties of SA across multiple species and its potential as an anti-aging drug. [ABSTRACT FROM AUTHOR]

Background: DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3′–5′ exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear. Methods: Initially, short-term and long-term survival assays were used to measure the cells' sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used. Results: We show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity. Conclusions: Our findings reveal that MRE11A is a negative regulator of human MMR. [ABSTRACT FROM AUTHOR]

Mitochondria, the versatile organelles crucial for cellular and organismal viability, play a pivotal role in meeting the energy requirements of cells through the respiratory chain located in the inner mitochondrial membrane, concomitant with the generation of reactive oxygen species (ROS). A wealth of evidence derived from contemporary investigations on reproductive longevity strongly indicates that the aberrant elevation of ROS level constitutes a fundamental factor in hastening the aging process of reproductive systems which are responsible for transmission of DNA to future generations. Constant changes in redox status, with a pro-oxidant shift mainly through the mitochondrial generation of ROS, are linked to the modulation of physiological and pathological pathways in gametes and reproductive tissues. Furthermore, the quantity and quality of mitochondria essential to capacitation and fertilization are increasingly associated with reproductive aging. The article aims to provide current understanding of the contributions of ROS derived from mitochondrial respiration to the process of reproductive aging. Moreover, understanding the impact of mitochondrial dysfunction on both female and male fertility is conducive to finding therapeutic strategies to slow, prevent or reverse the process of gamete aging, and thereby increase reproductive longevity. [ABSTRACT FROM AUTHOR]

Several modifiable risk factors for neurodegeneration and dementia have been identified, although individuals vary in their vulnerability despite a similar risk of exposure. This difference in vulnerability could be explained at least in part by the variability in DNA repair mechanisms' efficiency between individuals. Therefore, the aim of this study was to test associations between documented, prevalent genetic variation (single nucleotide polymorphism, SNP) in DNA repair genes, cognitive function, and brain structure. Community-living participants (n = 488,159; 56.54 years (8.09); 54.2% female) taking part in the UK Biobank study and for whom cognitive and genetic measures were available were included. SNPs in base excision repair (BER) genes of the bifunctional DNA glycosylases OGG1 (rs1052133, rs104893751), NEIL1 (rs7402844, rs5745906), NEIL2 (rs6601606), NEIL3 (rs10013040, rs13112390, rs13112358, rs1395479), MUTYH (rs34612342, rs200165598), NTHL1 (rs150766139, rs2516739) were considered. Cognitive measures included fluid intelligence, the symbol–digit matching task, visual matching, and trail-making. Hierarchical regression and latent class analyses were used to test the associations between SNPs and cognitive measures. Associations between SNPs and brain measures were also tested in a subset of 39,060 participants. Statistically significant associations with cognition were detected for 12 out of the 13 SNPs analyzed. The strongest effects amounted to a 1–6% difference in cognitive function detected for NEIL1 (rs7402844), NEIL2 (rs6601606), and NTHL1 (rs2516739). Associations varied by age and sex, with stronger effects detected in middle-aged women. Weaker associations with brain measures were also detected. Variability in some BER genes is associated with cognitive function and brain structure and may explain variability in the risk for neurodegeneration and dementia. [ABSTRACT FROM AUTHOR]

Current research on human aging has largely been guided by the milestone paper "hallmarks of aging," which were first proposed in the seminal 2013 paper by Lopez‐Otin et al. Most studies have focused on one aging hallmark at a time, asking whether the underlying molecular perturbations are sufficient to drive the aging process and its associated phenotypes. More recently, researchers have begun to investigate whether aging phenotypes are driven by concurrent perturbations in molecular pathways linked to not one but to multiple hallmarks of aging and whether they present different patterns in organs and systems over time. Indeed, preliminary results suggest that more complex interactions between aging hallmarks must be considered and addressed, if we are to develop interventions that successfully promote healthy aging and/or delay aging‐associated dysfunction and diseases. Here, we summarize some of the latest work and views on the interplay between hallmarks of aging, with a specific focus on mitochondrial dysfunction. Indeed, this represents a significant example of the complex crosstalk between hallmarks of aging and of the effects that an intervention targeted to a specific hallmark may have on the others. A better knowledge of these interconnections, of their cause‐effect relationships, of their spatial and temporal sequence, will be very beneficial for the whole aging research field and for the identification of effective interventions in promoting healthy old age. [ABSTRACT FROM AUTHOR]

Background: The growing prevalence of Alzheimer's disease (AD) is becoming a global health challenge without effective treatments. Defective mitochondrial function and mitophagy have recently been suggested as etiological factors in AD, in association with abnormalities in components of the autophagic machinery like lysosomes and phagosomes. Several large transcriptomic studies have been performed on different brain regions from AD and healthy patients, and their data represent a vast source of important information that can be utilized to understand this condition. However, large integration analyses of these publicly available data, such as AD RNA-Seq data, are still missing. In addition, large-scale focused analysis onmitophagy, which seems to be relevant for the aetiology of the disease, has not yet been performed. Methods: In this study, publicly available raw RNA-Seq data generated from healthy control and sporadic AD post-mortemhuman samples of the brain frontal lobe were collected and integrated. Sex-specific differential expression analysis was performed on the combined data set after batch effect correction. From the resulting set of differentially expressed genes, candidate mitophagy-related genes were identified based on their known functional roles in mitophagy, the lysosome, or the phagosome, followed by Protein-Protein Interaction (PPI) and microRNA-mRNA network analysis. The expression changes of candidate genes were further validated in human skin fibroblast and induced pluripotent stem cells (iPSCs)-derived cortical neurons from AD patients and matching healthy controls. Results: From a large dataset (AD: 589; control: 246) based on three different datasets (i.e., ROSMAP, MSBB, & GSE110731), we identified 299 candidate mitophagy-related differentially expressed genes (DEG) in sporadic AD patients (male: 195, female: 188). Among these, the AAA ATPase VCP, the GTPase ARF1, the autophagic vesicle forming protein GABARAPL1 and the cytoskeleton protein actin beta ACTB were selected based on network degrees and existing literature. Changes in their expression were further validated in AD-relevant human in vitro models, which confirmed their down-regulation in AD conditions. Conclusion: Through the joint analysis of multiple publicly available data sets, we identify four differentially expressed key mitophagy-related genes potentially relevant for the pathogenesis of sporadic AD. Changes in expression of these four genes were validated using two AD-relevant human in vitro models, primary human fibroblasts and iPSC-derived neurons. Our results provide foundation for further investigation of these genes as potential biomarkers or disease-modifying pharmacological targets. [ABSTRACT FROM AUTHOR]

Background: Testicular germ cell tumours (TGCTs) have a high sensitivity to chemotherapy and a high cure rate, although with serious adverse effects. In the search for tumour suppressive drugs, the RANKL inhibitor Denosumab, used to treat osteoporosis, came up as a candidate since RANKL signalling was recently identified in the testis.Methods: Expression of RANKL, RANK and OPG, and the effects of RANKL inhibition were investigated in human TGCTs, TGCT-derived cell-lines, and TGCT-xenograft models. Serum RANKL was measured in TGCT-patients.Results: RANKL, RANK, and OPG were expressed in germ cell neoplasia in situ (GCNIS), TGCTs, and TGCT-derived cell lines. RANKL-inhibition reduced proliferation of seminoma-derived TCam-2 cells, but had no effect on embryonal carcinoma-derived NTera2 cells. Pretreatment with Denosumab did not augment the effect of cisplatin in vitro. However, inhibition of RANKL in vivo reduced tumour growth exclusively in the TCam-2-xenograft model and Denosumab-treatment decreased proliferation in human GCNIS cultures. In TGCT-patients serum RANKL had no prognostic value.Conclusions: This study shows that the RANKL signalling system is expressed in GCNIS and seminoma where RANKL inhibition suppresses tumour growth in vitro and in vivo. Future studies are needed to determine whether RANKL is important for the malignant transformation or transition from GCNIS to invasive tumours. [ABSTRACT FROM AUTHOR]

T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a highly prevalent degenerative disease and a leading cause of vision loss worldwide. Evidence for an inflammatory component in the development of AMD exists, yet the exact mechanisms remain unclear. Bisretinoid N-retinylidene-N-retinylethanolamine (A2E) in retinal pigmental epithelial (RPE) cells, and in extracellular deposits constitutes a hallmark of AMD, but its role in the pathology of AMD is elusive. Here, we tested the hypothesis that A2E is responsible for the heightened inflammatory activity in AMD. To this end, we measured ex vivo mRNA expression of the cytokines TNF-α, IL-6, and IL-10 in whole blood samples after stimulation with A2E in a clinical sample of 27 patients with neovascular AMD and 24 patients with geographic atrophy secondary to AMD. Patients' spouses (n = 30) were included as non-affected controls. After stimulation with A2E, no statistical differences were found in the median expression level of TNF-α, IL-6, IL-10 between the control group, and the neovascular AMD and the geographic atrophy group. Our findings do not support evidence for the hypothesis, that A2E per se contributes to heightened inflammatory activity in AMD. [ABSTRACT FROM AUTHOR]

Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N -glycosylation gene N -acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition. MGAT5 knockout (KO) in HEK293 cells induces metabolic changes resulting in increased intracellular UDP-GlcNAc, increased glycolysis, enhanced spare respiratory capacity and higher citrate flux from the mitochondria. MGAT5 KO cells express constitutively high MICA, mainly regulated on the transcriptional level through opening of the chromatin at the MICA promoter. MICA expression in MGAT5 KO cells is dependent on citrate turnover and histone acetylation. Blocking citrate flux inhibits MICA expression in numerous cancer cell lines, and we propose that this is a central metabolic regulation of MICA and immune surveillance. [ABSTRACT FROM AUTHOR]

A ketogenic diet (KD; high-fat, low-carbohydrate) can benefit refractory epilepsy, but underlying mechanisms are unknown. We used mice inducibly expressing a mutated form of the mitochondrial DNA repair enzyme UNG1 (mutUNG1) to cause progressive mitochondrial dysfunction selectively in forebrain neurons. We examined the levels of mRNAs and proteins crucial for mitochondrial biogenesis and dynamics. We show that hippocampal pyramidal neurons in mutUNG1 mice, as well as cultured rat hippocampal neurons and human fibroblasts with H2O2 induced oxidative stress, improve markers of mitochondrial biogenesis, dynamics and function when fed on a KD, and when exposed to the ketone body β-hydroxybutyrate, respectively, by upregulating PGC1α, SIRT3 and UCP2, and (in cultured cells) increasing the oxygen consumption rate (OCR) and the NAD+/NADH ratio. The mitochondrial level of UCP2 was significantly higher in the perikarya and axon terminals of hippocampus CA1 pyramidal neurons in KD treated mutUNG1 mice compared with mutUNG1 mice fed a standard diet. The β-hydroxybutyrate receptor GPR109a (HCAR2), but not the structurally closely related lactate receptor GPR81 (HCAR1), was upregulated in mutUNG1 mice on a KD, suggesting a selective influence of KD on ketone body receptor mechanisms. We conclude that progressive mitochondrial dysfunction in mutUNG1 expressing mice causes oxidative stress, and that exposure of animals to KD, or of cells to ketone body in vitro, elicits compensatory mechanisms acting to augment mitochondrial mass and bioenergetics via the PGC1α-SIRT3-UCP2 axis (The compensatory processes are overwhelmed in the mutUNG1 mice by all the newly formed mitochondria being dysfunctional). [ABSTRACT FROM AUTHOR]

Exonuclease 1 (EXO1) is a multifunctional 5′ → 3′ exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the humanEXO1gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification. [ABSTRACT FROM AUTHOR]

Aim: LIFESTAT is an interdisciplinary project that leverages approaches and knowledge from medicine, the humanities and the social sciences to analyze the impact of statin use on health, lifestyle and well-being in cohorts of Danish citizens. The impetus for the study is the fact that 10% of the population in the Scandinavian countries are treated with statins in order to maintain good health and to avoid cardiovascular disease by counteracting high blood levels of cholesterol. The potential benefit of treatment with statins should be considered in light of evidence that statin use has prevalent and unintended side effects (e.g. myalgia, and glucose and exercise intolerance). Methods: The LIFESTAT project combines invasive human experiments, biomedical analyses, nationwide surveys, epidemiological studies, qualitative interviews, media content analyses, and ethnographic participant observations. The study investigates the biological consequences of statin treatment; determines the mechanism(s) by which statin use causes muscle and mitochondrial dysfunction; and analyzes achievement of treatment goals, people’s perception of disease risk, media influence on people’s risk and health perception, and the way people manage to live with the risk (personally, socially and technologically). Conclusions: The originality and success of LIFESTAT depend on and derive from its interdisciplinary approach, in which the disciplines converge into thorough and holistic study and describe the impact of statin use on the everyday life of statin users. This has the potential for much greater benefit than any one of the disciplines alone. Integrating traditional disciplines provides novel perspectives on potential current and future social, medical and personal benefits of statin use. [ABSTRACT FROM AUTHOR]

Summary The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5′ end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4’s helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4’s unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR. [ABSTRACT FROM AUTHOR]

Cardiac mitochondrial dysfunction has been implicated in heart failure of diverse etiologies. Generalized mitochondrial disease also leads to cardiomyopathy with various clinical manifestations. Impaired mitochondrial homeostasis may over time, such as in the aging heart, lead to cardiac dysfunction. Mitochondrial DNA (mtDNA), close to the electron transport chain and unprotected by histones, may be a primary pathogenetic site, but this is not known. Here, we test the hypothesis that cumulative damage of cardiomyocyte mtDNA leads to cardiomyopathy and heart failure. Transgenic mice with Tet-on inducible, cardiomyocyte-specific expression of a mutant uracil-DNA glycosylase 1 (mutUNG1) were generated. The mutUNG1 is known to remove thymine in addition to uracil from the mitochondrial genome, generating apyrimidinic sites, which obstruct mtDNA function. Following induction of mutUNG1 in cardiac myocytes by administering doxycycline, the mice developed hypertrophic cardiomyopathy, leading to congestive heart failure and premature death after ~2 mo. The heart showed reduced mtDNA replication, severely diminished mtDNA transcription, and suppressed mitochondrial respiration with increased Pgc-1α, mitochondrial mass, and antioxidative defense enzymes, and finally failing mitochondrial fission/fusion dynamics and deteriorating myocardial contractility as the mechanism of heart failure. The approach provides a model with induced cardiac-restricted mtDNA damage for investigation of mtDNA-based heart disease. [ABSTRACT FROM AUTHOR]

Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5' flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1 in strand displacement. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance. [ABSTRACT FROM AUTHOR]

Chronic inflammation due to bacterial overgrowth of the stomach predisposes to the development of gastric cancer and is also associated with high levels of reactive oxygen species (ROS). In recent years increasing attention has been drawn to microRNAs (miRNAs) due to their role in the pathogenesis of many human diseases including gastric cancer. Here we studied the impact of infection by the gram-positive bacteria Enterococcus faecalis (E. faecalis) on global miRNA expression as well as the effect of ROS on selected miRNAs. Human gastric adenocarcinoma cell line MKN74 was infected with living E. faecalis for 24 h or for 5 days or with E. faecalis lysate for 5 days. The miRNA expression was examined by microarray analysis using Affymetrix GeneChip miRNA Arrays. To test the effect of ROS, MKN74 cells were treated with 100 mM tert-Butyl hydroperoxide (TBHP). Following 5 days of E. faecalis infection we found 91 differentially expressed miRNAs in response to living bacteria and 2 miRNAs responded to E. faecalis lysate. We verified the down-regulation of the miR-17-92 and miR-106-363 clusters and of other miRNAs involved in the oxidative stress-response by qRT-PCR. We conclude that only infection by living E. faecalis bacteria caused a significant global response in miRNA expression in the MKN74 cell culture. E. faecalis infection as well as ROS stimulation down-regulated the expression of the miR-17-92 cluster. We believe that these changes could reflect a general response of gastric epithelial cells to bacterial infections. [ABSTRACT FROM AUTHOR]

In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare. [ABSTRACT FROM AUTHOR]

Background: Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. Methods: To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Results: Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Conclusions: Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-κB inflammatory response as well as impaired DNA damage response and cell cycle control gene expression. Transcript profiling: Array Express accession number E-MEXP-3496. [ABSTRACT FROM AUTHOR]

Germline mutations in the human DNA mismatch repair (MMR) genes MSH2 and MLH1 are associated with the inherited cancer disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer or HNPCC. A proportion of MSH2 and MLH1 mutations found in suspected LS patients give rise to single amino acid substitutions. The functional consequences in regard to pathogenicity of many of these variants are unclear. We have examined the functionality of a panel of MLH1 missense mutations found in LS families, by testing the variant proteins in functional assays, addressing subcellular localization, and protein-protein interaction with the dimer partner PMS2 and the MMR-associated exonuclease 1. We show that a significant proportion of examined variant proteins have functional defects in either subcellular localization or protein-protein interactions, which is suspected to lead to the cancer phenotype observed in patients. Moreover, the obtained results correlate well with reported MMR activity and with in silico analysis for a majority of the variants. Hum Mutat 33:1647-1655, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as 'variants of uncertain significance' (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. We describe different approaches for the functional analysis of MMR gene VUS and propose development of a validated diagnostic framework. Furthermore, we call attention to common misconceptions about functional assays and endorse development of an integrated approach comprising validated assays for diagnosis of VUS in patients suspected of LS. Hum Mutat 33:1617-1625, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress–related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung epithelium and immune cells. Macrophages have important immune defence functions by engulfing insoluble foreign materials, including particles, although they might also promote or enhance inflammation. We investigated the effect of co-culturing type II lung epithelial A549 cells with macrophages upon treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine-DNA glycosylase or oxoguanine DNA glycosylase (hOGG1) sensitive sites, in mono-cultures of A549 or THP-1a and co-cultures of A549 and THP-1a cells. The strongest genotoxic effects were observed in A549 mono-cultures and SRM2975 was more potent than SRM1650b. The ROS production only increased in cells exposed to SRM2975, with strongest concentration-dependent effect in the THP-1a mono-cultures. The basal respiration level in THP-1a cells increased on exposure to SRM1650b and SRM2975 without indication of mitochondrial dysfunction. This is consistent with activation of the cells and there was no direct relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells. [ABSTRACT FROM PUBLISHER]

Lynch syndrome (LS) is an autosomal dominant disorder that predisposes to colon, endometrial, and other cancers. LS is caused by a heterozygous germline mutation in one of the DNA mismatch repair (MMR) genes. A significant proportion of all mutations found in suspected LS patients comprises single amino acid alterations. The pathogenicity of these variants of uncertain significance (VUS) is difficult to assess, precluding diagnosis of carriers and their relatives. Here we present a rapid cell-free assay to investigate MMR activity of MSH2 or MSH6 VUS. We used this assay to analyze a series of MSH2 and MSH6 VUS, selected from the Leiden Open Variation Database. Whereas a significant fraction of the MSH2 VUS has lost MMR activity, suggesting pathogenicity, the large majority of the MSH6 VUS appears MMR proficient. We anticipate that this assay will be an important tool in the development of a comprehensive and widely applicable diagnostic procedure for LS-associated VUS. Hum Mutat 33:488-494, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Several enzymes of the metabolic pathways responsible for metabolism of cytosolic ribonucleotides and deoxyribonucleotides are located in mitochondria. Studies described in this paper suggest dysfunction of the mitochondria to affect these metabolic pathways and limit the available levels of cytosolic ribonucleotides and deoxyribonucleotides, which in turn can result in aberrant RNA and DNA synthesis. Mitochondrial dysfunction has been linked to genomic instability, and it is possible that the limiting effect of mitochondrial dysfunction on the levels of nucleotides and resulting aberrant RNA and DNA synthesis in part can be responsible for this link. This paper summarizes the parts of the metabolic pathways responsible for nucleotide metabolism that can be affected by mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Somatic defects in the mismatch repair system constitute an important pathway in colorectal carcinogenesis. We have examined the expression of mismatch repair proteins in sporadic stage IV colorectal tumors and their derived metastases. Sporadic tumors were further examined for differences in expression between the tumor transition zone and the invasive front. Expression of hMSH2, hMLH1, and hPMS2 was screened immunohistochemically in 92 stage IV tumors and derived liver metastases. In cases with loss of mismatch repair protein expression, lymph node metastases were also examined. Clinicopathological parameters and Ki-67 staining indexes were evaluated and compared. Four tumors displayed a complete loss of hMLH1/hPMS2 expression at the transition zone; however, three of these expressed both proteins at the invasive front and in liver and lymph node metastases. A further four were predominantly hMLH1/hPMS2 negative at the transition zone, but with distinct subclones of hMLH1/hPMS2-expressing cells at the transition zone. All of these tumors expressed hMLH1/hPMS2 at the invasive front and in liver metastases, with three also expressing hMLH/hPMS2 in lymph node metastases. No significant difference in the proliferative index was observed for the hMLH1/hPMS2-compromised group. In stage IV tumors re-expression of hMLH1/hPMS2 occurred, leading to different patterns of expression within the primary tumor and between tumor and metastases. This may have functional importance for the chemosensitivity of metastases compared to the primary tumor. [ABSTRACT FROM AUTHOR]

DNA mismatch repair (MMR) is a highly conserved pathway that corrects both base–base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood. Interestingly, CNOT6 is one of four deadenylase subunits in the conserved CCR4-NOT complex and it targets poly(A) tails of mRNAs for degradation. CNOT6 is overexpressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and androgen-independent prostate cancer cells, which suggests that an altered expression of CNOT6 may play a role in tumorigenesis. Here, we report that a depletion of CNOT6 sensitizes human U2OS cells to N-methyl-N′nitro-N-nitrosoguanidine (MNNG) and leads to enhanced apoptosis. We also demonstrate that the depletion of CNOT6 upregulates MMR and decreases the mutation frequency in MMR-proficient cells. Furthermore, the depletion of CNOT6 increases the stability of mRNA transcripts from MMR genes, leading to the increased expression of MMR proteins. Our work provides insight into a novel CNOT6-dependent mechanism for regulating MMR. [ABSTRACT FROM AUTHOR]

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with Kd values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes. [ABSTRACT FROM AUTHOR]

Inherited predisposition to disease is often linked to reduced activity of a disease associated gene product. Thus, quantitation of the influence of inherited variants on gene function can potentially be used to predict the disease relevance of these variants. While many disease genes have been extensively characterized at the functional level, few assays based on functional properties of the encoded proteins have been established for the purpose of predicting the contribution of rare inherited variants to disease. Much of the difficulty in establishing predictive functional assays stems from the technical complexity of the assays. However, perhaps the most challenging aspect of functional assay development for clinical testing purposes is the absolute requirement for validation of the sensitivity and specificity of the assays and the determination of positive predictive values (PPVs) and negative predictive values (NPVs) of the assays relative to a 'gold standard' measure of disease predisposition. In this commentary, we provide examples of some of the functional assays under development for several cancer predisposition genes ( BRCA1, BRCA2, CDKN2A, and mismatch repair [MMR] genes MLH1, MSH2, MSH6, and PMS2) and present a detailed review of the issues associated with functional assay development. We conclude that validation is paramount for all assays that will be used for clinical interpretation of inherited variants of any gene, but note that in certain circumstances information derived from incompletely validated assays may be valuable for classification of variants for clinical purposes when used to supplement data derived from other sources. Hum Mutat 29(11), 1314-1326, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Most biotransformation studies to date have concentrated on single-substance exposures. The biotransformation ability of organisms exposed to a single substance, however, may differ from that of organisms coexposed to binary or complex mixtures. The marine polychaete Capitella sp. I was exposed to either 31 μg/g dry weight sediment of benzo[a]pyrene (BaP) in a single-substance exposure or to a mixture of 31 and 28 μg/g dry weight sediment of BaP and fluoranthene (Flu), respectively (coexposure). Biotransformation capability and DNA damage were compared between treatments. Worms exposed to BaP alone accumulated total BaP equivalents to a significantly higher degree than coexposed worms (final body burden: 117.14 ± 3.52 μg BaP equivalents/g dry wt tissue and 69.78 ± 8.58 μg BaP equivalents/g dry wt tissue, respectively), probably because of a higher degree of BaP biotransformation in the latter. In addition, a larger percentage of the accumulated BaP equivalents was in the form of parent compound in worms exposed to BaP alone compared to coexposed worms on day 10 (87 and 56%, respectively). In coexposed worms, Flu was both accumulated and biotransformed to a considerably greater degree than BaP (final Flu body burden: 181.85 ± 18.46 μg Flu equivalents/g dry wt tissue, 24% as parent compound). Neither treatment resulted in significant effects on growth or DNA damage. The present study shows that the extent to which bioaccumulation factors estimated from single-substance exposures have relevance for the field situation will depend on the degree to which accumulation is influenced by the presence of other contaminants. These results therefore are relevant for assessing risks from contaminated sediments.fluor [ABSTRACT FROM AUTHOR]

Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome is caused by DNA variations in the DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and PMS2. Many of the mutations identified result in premature termination of translation and thus in loss-of-function of the encoded mutated protein. These DNA variations are thought to be pathogenic mutations. However, some patients carry other DNA mutations, referred to as unclassified variants (UVs), which do not lead to such a premature termination of translation; it is not known whether these contribute to the disease phenotype or merely represent rare polymorphisms. This is a major problem which has direct clinical consequences. Several criteria can be used to classify these UVs, such as: whether they segregate with the disease within pedigrees, are absent in control individuals, show a change of amino acid polarity or size, provoke an amino acid change in a domain that is evolutionary conserved and/or shared between proteins belonging to the same protein family, or show altered function in an in vitro assay. In this review we discuss the various functional assays reported for the HNPCC-associated MMR proteins and the outcomes of these tests on UVs identified in patients diagnosed with or suspected of having HNPCC. We conclude that a large proportion of MMR UVs are likely to be pathogenic, suggesting that missense variants of MMR proteins do indeed play a role in HNPCC. Hum Mutat 28(11), 1047-1054, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is a hallmark of cancer cells. However, genetic response to mitochondrial dysfunction during carcinogenesis is unknown. To elucidate genetic response to mitochondrial dysfunction we used Saccharomyces cerevisiae as a model system. We analyzed genome-wide expression of nuclear genes involved in signal transduction and transcriptional regulation in a wild-type yeast and a yeast strain lacking the mitochondrial genome (rho0). Our analysis revealed that the gene encoding cAMP-dependent protein kinase subunit 3 (PKA3) was upregulated. However, the gene encoding cAMP-dependent protein kinase subunit 2 (PKA2) and the VTC1, PTK2, TFS1, CMK1, and CMK2 genes, involved in signal transduction, were downregulated. Among the known transcriptional factors, OPI1, MIG2, INO2, and ROX1 belonged to the upregulated genes, whereas MSN4, MBR1, ZMS1, ZAP1, TFC3, GAT1, ADR1, CAT8, and YAP4 including RFA1 were downregulated. RFA1 regulates DNA repair genes at the transcriptional level. RFA is also involved directly in DNA recombination, DNA replication, and DNA base excision repair. Downregulation of RFA1 in rho0 cells is consistent with our finding that mitochondrial dysfunction leads to instability of the nuclear genome. Together, our data suggest that gene(s) involved in mitochondria-to-nucleus communication play a role in mutagenesis and may be implicated in carcinogenesis. [ABSTRACT FROM AUTHOR]

Human exonuclease 1 (hEXO1) has been implicated in DNA mismatch repair (MMR), replication, and recombination, but the nature of its interaction with these cellular processes is still ambiguous. We show that hEXO1 colocalizes with proliferating cell nuclear antigen (PCNA) at DNA replication sites and that the C-terminal region of hEXO1 is sufficient for this localization. We also show that both hMLH1-hPMS2 (MutLa) and hMLH1-hEXO1 complexes are formed in a reaction mixture containing all three proteins. Moreover, hEXO1 5' double-stranded exonuclease activity on a homoduplex substrate but not on a substrate containing a G/T mismatch was inhibited by complex formation with hMSH2-hMSH6 (MutSa) or MutLa. Taken together, the results support a model in which hEXO1 plays a role in events at the replication sites as well as a functional role in the MMR and/or recombination processes.Oncogene (2004) 23, 1457-1468. doi:10.1038/sj.onc.1207265 Published online 15 December 2003 [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons, such as fluoranthene (Flu), are of environmental concern because of their persistence, toxicity, and mutagenic properties. In this study we examined the genotoxicity of Flu to Capitella sp. I using the comet assay. We assessed patterns of DNA damage as a function of uptake route and as a function of exposure and depuration time and related levels of DNA damage to published information on Flu-metabolite formation. Exposure to approximately 30 µg Flu/g dry-weight sediment or 50 µg Flu/L seawater resulted in significant DNA damage. The degree of DNA damage was time dependent during both exposure and depuration, and although exposure route had no effect on the maximum degree of DNA damage occurring, it did influence the time course of damage. Levels of damage declined despite continued exposure to Flu, providing evidence for the induction of one or more DNA repair mechanisms. Comparison with Flu-metabolite profiles suggests that DNA damage is associated with the production of aqueous metabolites. The transitory nature of the DNA damage and repair process may contribute important insights into the mechanisms of toxicant effects at the molecular level but limits the usefulness of such endpoints as biomarkers of exposure or effect in ecotoxicological studies. [ABSTRACT FROM AUTHOR]

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC–hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC–hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1–hEXO1 complex formation and hMutLα heterodimer assembly essential for MMR activity. Oncogene (2001) 20, 3590–3595. [ABSTRACT FROM AUTHOR]

Aging is the consequence of a lifelong accumulation of stochastic damage to tissues and cellular components. Advancing age closely associates with elevated markers of innate immunity and low-grade chronic inflammation, probably reflecting steady increasing incidents of cellular and tissue damage over the life course. The DNA sensing cGAS-STING signaling pathway is activated by misplaced cytosolic self-DNA, which then initiates the innate immune responses. Here, we hypothesize that the stochastic release of various forms of DNA from the nucleus and mitochondria, e.g., because of DNA damage, altered nucleus integrity, and mitochondrial damage, can result in chronic activation of inflammatory responses that characterize the aging process. This cytosolic self-DNA-innate immunity axis may perturb tissue homeostasis and function that characterizes human aging and age-associated pathology. Proper techniques and experimental models are available to investigate this axis to develop therapeutic interventions. [ABSTRACT FROM AUTHOR]

The 20th century saw an unprecedented increase in average human lifespan as well as a rapid decline in human fertility in many countries of the world. The accompanying worldwide change in demographics of human populations is linked to unanticipated and unprecedented economic, cultural, medical, social, public health and public policy challenges, whose full implications on a societal level are only just beginning to be fully appreciated. Some of these implications are discussed in this commentary, an outcome of Cultures of Health and Ageing, a conference co-sponsored by the University of Copenhagen (UCPH) and the Center for Healthy Ageing at UCPH, which took place on 20–21 June 2014 in Copenhagen, Denmark. Questions discussed here include the following: what is driving age-structural change in human populations? how can we create ‘age-friendly’ societies and promote ‘ageing-in-community’? what tools will effectively promote social engagement and prevent social detachment among older individuals? is there a risk that further extension of human lifespan would be a greater burden to the individual and to society than is warranted by the potential benefit of longer life? [ABSTRACT FROM AUTHOR]

The 20th century saw an unprecedented increase in average human lifespan as well as a rapid decline in human fertility in many countries of the world. The accompanying worldwide change in demographics of human populations is linked to unanticipated and unprecedented economic, cultural, medical, social, public health and public policy challenges, whose full implications on a societal level are only just beginning to be fully appreciated. Some of these implications are discussed in this commentary, an outcome of Cultures of Health and Ageing, a conference co-sponsored by the University of Copenhagen (UCPH) and the Center for Healthy Ageing at UCPH, which took place on 20–21 June 2014 in Copenhagen, Denmark. Questions discussed here include the following: what is driving age-structural change in human populations? how can we create ‘age-friendly’ societies and promote ‘ageing-in-community’? what tools will effectively promote social engagement and prevent social detachment among older individuals? is there a risk that further extension of human lifespan would be a greater burden to the individual and to society than is warranted by the potential benefit of longer life? [ABSTRACT FROM PUBLISHER]

DNA mismatch repair defects in certain cell types confer resistance to the cytotoxic effects of alkylating agents, suggesting that a normally functioning DNA mismatch repair pathway can actually mediate alkylation-induced cell death. In eukaryotic cells this phenomenon is only observed in cells lacking adequate DNA methyltransferase for the repair of O6-methylguanine (O6MeG) DNA lesions. It has been proposed that O6MeG may act as a substrate for DNA mismatch repair when paired with cytosine and when mispaired with thymine and that repeated futile DNA mismatch repair at O6MeG DNA lesions is cytotoxic. Here we show that the Escherichia coli MutS DNA mismatch repair binding protein does indeed bind specifically to O6MeG DNA lesions. In contrast, MutS does not bind DNA containing another O-alkylated base, namely O4-methylthymine, or another kind of modified guanine, namely 8-oxoguanine. These results provide direct biochemical evidence for the involvement of DNA mismatch repair in specifically processing O6MeG DNA lesions. [ABSTRACT FROM PUBLISHER]

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical σ70 promoter with 18bp spacing between the -10 and -35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feed-back and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of β-galactosidase activity at all growth rates tested, in contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-A confirming the different modes of regulation. The cde-4::minlTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene. [ABSTRACT FROM AUTHOR]