Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

Changes in gray matter induced by learning--revisited

Background

Recently, activation-dependant structural brain plasticity in humans has been demonstrated in adults after three months of training a visio-motor skill. Learning three-ball cascade juggling was associated with a transient and highly selective increase in brain gray matter in the occipito-temporal cortex comprising the motion sensitive area hMT/V5 bilaterally. However, the exact time-scale of usage-dependant structural changes occur is still unknown. A better understanding of the temporal parameters may help to elucidate to what extent this type of cortical plasticity contributes to fast adapting cortical processes that may be relevant to learning.

Principal Findings

Using a 3 Tesla scanner and monitoring whole brain structure we repeated and extended our original study in 20 healthy adult volunteers, focussing on the temporal aspects of the structural changes and investigated whether these changes are performance or exercise dependant. The data confirmed our earlier observation using a mean effects analysis and in addition showed that learning to juggle can alter gray matter in the occipito-temporal cortex as early as after 7 days of training. Neither performance nor exercise alone could explain these changes.

Conclusion

We suggest that the qualitative change (i.e. learning of a new task) is more critical for the brain to change its structure than continued training of an already-learned task.     

A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.     

Residual Cdk1/2 activity after DNA damage promotes senescence

In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/C^Cdh1‐dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.     

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans- homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.     

Predators induce egg retention in prey

To prevent predation on their eggs, prey often avoid patches occupied by predators. As a result, they need to delay oviposition until they reach predator-free patches. Because many species allocate energy to egg production in a continuous fashion, it is not clear what kind of mechanism prey use to delay oviposition. We used females of the phytoseiid mite Neoseiulus cucumeris to study these mechanisms. Females were placed in patches with pollen, a food source they use for egg production, and they were exposed to another phytoseiid mite, Iphiseius degenerans, which is an intraguild predator of N. cucumeris juveniles. We found that the oviposition of N. cucumeris females on patches with the predator was lower than on patches without the predator. Cues left by the intraguild predator were not sufficient to elicit such behaviour. Females of N. cucumeris reduced oviposition when exposed to the predator by retaining the egg inside their body, resulting in a lower developmental rate once these eggs were laid. Hence, females are capable of retaining eggs, but the development of these eggs continues inside the mother’s body. In this way, females gain some time to search for less risky oviposition sites.     

Natural chelators in sea water: Detoxification of Zn2+ by brown algal polyphenols

We have examined growth responses of several species of marine phytoplankton, cultured with and without heavy metal stress, to supplements of polymeric polyphenols from the brown algae Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus L. In the absence of additional heavy metals. supplements of up to 4000 μg. 1 of polyphenols had a small effect on initial growth rates for three of these microalgae and had no effect on maximum cell densities for four species. One very common, heavy metal-sensitive diatom, Skeletonema costatum (Grev.) Cl., showed significantly increasing maximum cell density in the cultures, with increasing addition of polyphenols to the medium. The toxicity of Zn²⁺ (0.5–2.0 mg·1 ¹) to cultures of the diatom Phaeodactylum tricornutum Bohlin was relieved by supplements (100–200 μg·1 ¹) of the brown algal polyphenols. Exudation of these polyphenols from brown seaweeds may contribute to the natural chelating capacity of inshore sea water.     

Molecular and structural basis for redox regulation of beta-actin

An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H₂O₂. It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H₂O₂ acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle β/γ-actin and compare with that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca²⁺. We have determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H₂O₂ in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H₂O₂. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed.