Cytotoxicity of Exogenous Acetoacetate in Lithium Salt Form Is Mediated by Lithium and Not Acetoacetate.

BACKGROUND/AIM: The ketogenic diet has recently gained interest as potential adjuvant therapy for cancer. Many researchers have endeavored to support this claim in vitro. One common model utilizes treatment with exogenous acetoacetate in lithium salt form (LiAcAc). We aimed to determine whether the effects of treatment with LiAcAc on cell viability, as reported in the literature, accurately reflect the influence of acetoacetate. MATERIALS AND METHODS: Breast cancer and normal cell lines were treated with acetoacetate, in lithium and sodium salt forms, and cell viability was assessed. RESULTS: The effect of LiAcAc on cells was mediated by Li ions. Our results showed that the cytotoxic effects of LiAcAc treatment were significantly similar to those caused by LiCl, and also treatment with NaAcAc did not cause any significant cytotoxic effect. CONCLUSION: Treatment of cells with LiAcAc is not a convincing in vitro model for studying ketogenic diet. These findings are highly important for interpreting previously published results, and for designing new experiments to study the ketogenic diet in vitro.

Immune checkpoint protein VSIG4 as a biomarker of aging in murine adipose tissue.

Adipose tissue is recognized as a major source of systemic inflammation with age, driving age-related tissue dysfunction and pathogenesis. Macrophages (Mφ) are central to these changes yet adipose tissue Mφ (ATMs) from aged mice remain poorly characterized. To identify biomarkers underlying changes in aged adipose tissue, we performed an unbiased RNA-seq analysis of ATMs from young (8-week-old) and healthy aged (80-week-old) mice. One of the genes identified, V-set immunoglobulin-domain-containing 4 (VSIG4/CRIg), encodes a Mφ-associated complement receptor and B7 family-related immune checkpoint protein. Here, we demonstrate that Vsig4 expression is highly upregulated with age in perigonadal white adipose tissue (gWAT) in two mouse strains (inbred C57BL/6J and outbred NIH Swiss) independent of gender. The accumulation of VSIG4 was mainly attributed to a fourfold increase in the proportion of VSIG4+ ATMs (13%-52%). In a longitudinal study, VSIG4 expression in gWAT showed a strong correlation with age within a cohort of male and female mice and correlated strongly with physiological frailty index (PFI, a multi-parameter assessment of health) in male mice. Our results indicate that VSIG4 is a novel biomarker of aged murine ATMs. VSIG4 expression was also found to be elevated in other aging tissues (e.g., thymus) and was strongly induced in tumor-adjacent stroma in cases of spontaneous and xenograft lung cancer models. VSIG4 expression was recently associated with cancer and several inflammatory diseases with diagnostic and prognostic potential in both mice and humans. Further investigation is required to determine whether VSIG4-positive Mφ contribute to immunosenescence and/or systemic age-related deficits.

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation.

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.

Monocytes of Different Subsets in Complexes with Platelets in Patients with Myocardial Infarction.

Acute myocardial infarction (AMI) is associated with activation of various cells, including platelets that form monocyte-platelet complexes (MPCs). Here, we analysed MPC in vivo and in vitro and investigated the abilities of different monocyte subclasses to form MPC, the characteristics of the cells involved in MPC formation and MPC changes in AMI. We identified MPC by co-staining for platelet antigen CD41a and monocyte antigens CD14 and CD16. Platelet activation was evaluated from expression of phosphatidylserine as revealed by annexin V. Our results confirm published data and provide new information regarding the patterns of MPC in AMI patients. We found that the patterns of platelet aggregation with monocytes were different in AMI patients and controls: (1) in AMI patients, MPC formed by intermediate monocytes carry more platelets whereas in healthy controls more platelets aggregated with classical monocytes; (2) the numbers of MPC in AMI patients, being already higher than in controls, were further increased if these patients suffered various in-hospital complications; (3) on the basis of the CD41a fluorescence of the antibody-stained MPC, some of the aggregates seem to consist of monocytes and platelet-derived extracellular vesicles (EVs); (4) aggregation of monocytes with platelet EV occurred in in vitro experiments; and (5) these experiments demonstrated that monocytes from AMI patients aggregate with both platelets and platelet EVs more efficiently than do monocytes from controls. MPC in AMI patients may play an important role in this pathology.

Stability analysis of a model gene network links aging, stress resistance, and negligible senescence.

Several animal species are considered to exhibit what is called negligible senescence, i.e. they do not show signs of functional decline or any increase of mortality with age. Recent studies in naked mole rat and long-lived sea urchins showed that these species do not alter their gene-expression profiles with age as much as other organisms do. This is consistent with exceptional endurance of naked mole rat tissues to various genotoxic stresses. We conjectured, therefore, that the lifelong transcriptional stability of an organism may be a key determinant of longevity. We analyzed the stability of a simple genetic-network model and found that under most common circumstances, such a gene network is inherently unstable. Over a time it undergoes an exponential accumulation of gene-regulation deviations leading to death. However, should the repair systems be sufficiently effective, the gene network can stabilize so that gene damage remains constrained along with mortality of the organism. We investigate the relationship between stress-resistance and aging and suggest that the unstable regime may provide a mathematical basis for the Gompertz "law" of aging in many species. At the same time, this model accounts for the apparently age-independent mortality observed in some exceptionally long-lived animals.

Mining gene expression data for pollutants (dioxin, toluene, formaldehyde) and low dose of gamma-irradiation.

General and specific effects of molecular genetic responses to adverse environmental factors are not well understood. This study examines genome-wide gene expression profiles of Drosophila melanogaster in response to ionizing radiation, formaldehyde, toluene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. We performed RNA-seq analysis on 25,415 transcripts to measure the change in gene expression in males and females separately. An analysis of the genes unique to each treatment yielded a list of genes as a gene expression signature. In the case of radiation exposure, both sexes exhibited a reproducible increase in their expression of the transcription factors sugarbabe and tramtrack. The influence of dioxin up-regulated metabolic genes, such as anachronism, CG16727, and several genes with unknown function. Toluene activated a gene involved in the response to the toxins, Cyp12d1-p; the transcription factor Fer3's gene; the metabolic genes CG2065, CG30427, and CG34447; and the genes Spn28Da and Spn3, which are responsible for reproduction and immunity. All significantly differentially expressed genes, including those shared among the stressors, can be divided into gene groups using Gene Ontology Biological Process identifiers. These gene groups are related to defense response, biological regulation, the cell cycle, metabolic process, and circadian rhythms. KEGG molecular pathway analysis revealed alteration of the Notch signaling pathway, TGF-beta signaling pathway, proteasome, basal transcription factors, nucleotide excision repair, Jak-STAT signaling pathway, circadian rhythm, Hippo signaling pathway, mTOR signaling pathway, ribosome, mismatch repair, RNA polymerase, mRNA surveillance pathway, Hedgehog signaling pathway, and DNA replication genes. Females and, to a lesser extent, males actively metabolize xenobiotics by the action of cytochrome P450 when under the influence of dioxin and toluene. Finally, in this work we obtained gene expression signatures pollutants (dioxin, toluene), low dose of gamma-irradiation and common molecular pathways for different kind of stressors.

Selective anticancer agents suppress aging in Drosophila.

Mutations of the PI3K, TOR, iNOS, and NF-κB genes increase lifespan of model organisms and reduce the risk of some aging-associated diseases. We studied the effects of inhibitors of PI3K (wortmannin), TOR (rapamycin), iNOS (1400W), NF-κB (pyrrolidin dithiocarbamate and QNZ), and the combined effects of inhibitors: PI3K (wortmannin) and TOR (rapamycin), NF-κB (pyrrolidin dithiocarbamates) and PI3K (wortmannin), NF-κB (pyrrolidine dithiocarbamates) and TOR (rapamycin) on Drosophila melanogaster lifespan and quality of life (locomotor activity and fertility). Our data demonstrate that pharmacological inhibition of PI3K, TOR, NF-κB, and iNOS increases lifespan of Drosophila without decreasing quality of life. The greatest lifespan expanding effect was achieved by a combination of rapamycin (5 µM) and wortmannin (5 µM) (by 23.4%). The bioinformatic analysis (KEGG, REACTOME.PATH, DOLite, and GO.BP) showed the greatest aging-suppressor activity of rapamycin, consistent with experimental data.