Senescence‐associated β‐galactosidase reveals the abundance of senescent CD8+ T cells in aging humans

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second‐generation fluorogenic substrate for β‐galactosidase and multi‐parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence‐associated β‐galactosidase (SA‐βGal) activity with advancing donor age. The greatest age‐associated increases were observed in CD8+ T‐cell populations, in which the fraction of cells with high SA‐βGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA‐βGal activity, but not those with low SA‐βGal activity, were found to exhibit features of telomere dysfunction‐induced senescence and p16‐mediated senescence, were impaired in their ability to proliferate, developed in various T‐cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

Non-erythroid alpha spectrin prevents telomere dysfunction after DNA interstrand cross-link damage

Telomere integrity is critical for telomere function and genomic stability. We previously demonstrated that non-erythroid α-spectrin (αIISp) is present in mammalian cell nuclei where it is important in repair of DNA interstrand cross-links (ICLs) and chromosome stability. We now demonstrate that αIISp is also important for telomere maintenance after ICL damage. It localizes to telomeres in S phase after ICL damage where it has enhanced association with TRF1 and TRF2 and is required for recruitment of the ICL repair protein, XPF, to damage-induced foci at telomeres. In telomerase-positive normal cells depleted of αIISp by siRNA or in Fanconi anemia, complementation group A (FA-A) cells, where αIISp levels are 35–40% of normal, ICL damage results in failure of XPF to localize to telomeres, markedly increased telomere dysfunction-induced foci, followed by catastrophic loss of telomeres. Restoration of αIISp levels to normal in FA-A cells corrects these deficiencies. Our studies demonstrate that αIISp is critical for repair of DNA ICLs at telomeres, likely by facilitating the recruitment of repair proteins similar, but not identical, to its proposed role in repair of DNA ICLs in genomic DNA and that this function in turn is critical for telomere maintenance after DNA ICL damage.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a)

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.     

Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA

The inducible response to H(2)O(2) stress in Bacillus subtilis is under the control of PerR, one of three Fur homologues in this organism. PerR was purified in both an inactive, metal-dependent form and an active, metal-containing form as determined using DNA-binding assays. Active PerR contains both zinc and iron and is designated PerR:Zn,Fe. Added manganous ion competes for binding to the iron site and can restore DNA-binding activity to the metal-dependent form of PerR, presumably generating PerR:Zn,Mn. The DNA-binding activity of PerR:Zn,Fe is eliminated by exposure to H(2)O(2) whereas PerR:Zn,Mn is comparatively resistant. DNA-binding activity can be restored by a thiol-reducing agent, suggesting that redox-active cysteines are involved in peroxide sensing. Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full induction by H(2)O(2). In contrast, in cells grown with iron supplementation, a PerR-repressed gene is completely derepressed by H(2)O(2). These results are consistent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.     

Mutation of cyclin/cdk phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.     

Endorsing Darwin: global biogeography of the epipelagic goose barnacles Lepas spp. (Cirripedia,Lepadomorpha) proves cryptic speciation

It was Darwin that noted the large intraspecific diversity of the goose barnacle Lepas Linnaeus, 1758 and thought about distinct regional varieties. Today, biogeographic compartmentation is known from marine species, but data from globally occurring species remain scarce. We analysed inter‐ and intraspecific divergence within the epipelagic rafter Lepas from tropical and temperate oceans by means of two mitochondrial and one nuclear DNA marker. Besides phylogenetic relations, we resolved biogeography and controlling factors. Inhabiting the Southern Hemisphere, Lepas australis Darwin, 1851 shows separate populations from coastal Chile and from circum‐Antarctic waters, most probably related to temperature differences in the current systems. The cosmopolitan Lepas anatifera Linnaeus, 1758 displays four regional subgroups (coastal Chile, Northeast Pacific/Oregon, the Southern Hemisphere Indopacific, and the Atlantic), and a global group, which might be an ancestral stem group. The differentiation reflects vicariance effects rooted in geological history: the closure of the Neogene Tethys in the Middle East and at the Panama Isthmus, the installation of the cool Benguela Current, differing Pleistocene currents and temperatures, and modern current systems. The extreme ecological generalists Lepas anserifera Linnaeus, 1767 and Lepas pectinata Spengler, 1793 are not differentiated, and might represent true global species. In conclusion, compartmentation of the oceans acts at the species level according to ecospace limits. For Lepas, the multitude of barriers favours allopatric speciation.     

Rugosa und Heterocorallia aus Obervisé-Geröllen der Marbella-Formation (Betische Kordillere,Südspanien)

Limestone pebbles of the post-lower Bashkirian Marbella Formation (Malaguides, Internal Zone of the Betic Cordillera) yielded sixteen species of rugose corals belonging to eleven genera and two species of heterocorals belonging to two genera. The fauna is of uppermost Viséan age (V3b-V3c). Its paleogeographic significance within the western Mediterranean is discussed. Lonsdaleia corbariensis, known up to now only from debris flow deposits of the Mouthoumet massif (Southern France), covers approximately half of the total fauna.Hexaphyllia mirabilis is shown to have juvenile stages with five septa.