Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8 + T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8⁺ T cells responses in the elderly has come back into focus since the COVID‐19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8⁺ T cells in aging. We focused on the different subpopulations and time‐resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8⁺ T cells from elderly mice are much faster than those of CD8⁺ T cells from adult mice. This is true not only in the total CD8⁺ T cell population but also for their effector (T_EM) and central memory (T_CM) T cell subpopulations. TIRF experiments reveal that CD8⁺ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8⁺ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell‐intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.     

Can size distributions of European lake fish communities be predicted by trophic positions of their fish species?

An organism''s body size plays an important role in ecological interactions such as predator–prey relationships. As predators are typically larger than their prey, this often leads to a strong positive relationship between body size and trophic position in aquatic ecosystems. The distribution of body sizes in a community can thus be an indicator of the strengths of predator–prey interactions. The aim of this study was to gain more insight into the relationship between fish body size distribution and trophic position in a wide range of European lakes. We used quantile regression to examine the relationship between fish species'' trophic position and their log‐transformed maximum body mass for 48 fish species found in 235 European lakes. Subsequently, we examined whether the slopes of the continuous community size distributions, estimated by maximum likelihood, were predicted by trophic position, predator–prey mass ratio (PPMR), or abundance (number per unit effort) of fish communities in these lakes. We found a positive linear relationship between species'' maximum body mass and average trophic position in fishes only for the 75% quantile, contrasting our expectation that species'' trophic position systematically increases with maximum body mass for fish species in European lakes. Consequently, the size spectrum slope was not related to the average community trophic position, but there were negative effects of community PPMR and total fish abundance on the size spectrum slope. We conclude that predator–prey interactions likely do not contribute strongly to shaping community size distributions in these lakes.     

The PLEKHA7–PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell–cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7–PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.     

Fluorescence and Absorption Spectra of Biological Dyes

Data include charts and tables of absorbance and fluorescence of 4 coupling agents for immunity reactions, 34 typical fluorescent dyes and reagents, and 12 dyes or reagents having no fluorescence in solution but which may be fluorescent in the dry state. Since this report consists largely of reference data, the original must be consulted for details. Reprint source: The Perkin-Elmer Corporation, Norwalk, Connecticut, U. S. A.     

Species-specific primers for predation studies of the pollen beetle, Meligethes aeneus (Coleoptera, Nitidulidae)

Species‐specific primers were developed for the pollen beetle (a pest in oilseed rape) for studies of predation by natural insect predators. Two forward and three reverse primers were designed within the mitochondrial COI gene and used in combination to amplify fragments in the size range of 163–290 bp. Remains of pollen beetle DNA were consistently detected in Pardosa spiders up to 24 h after ingestion but dropped drastically at 48 h. These primers will facilitate studies on biological control of this oilseed rape pest. Detection time was not correlated with fragment length as might be expected as the DNA gradually degrades into progressively shorter fragments over time.     

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.     

Bacterial Factors Associated with Lethal Outcome of Enteropathogenic Escherichia coli Infection: Genomic Case-Control Studies

Background

Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome.

Methods

We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality.

Results

Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains.

Conclusions

Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.     

HER-2/neu-mediated down-regulation of biglycan associated with altered growth properties

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-β(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ～5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.     

Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure

A glasshouse experiment was undertaken to provide baseline data on the variation between conventional maize (Zea mays L.) varieties and genetically modified maize plants expressing the insecticidal Bacillus thuringiensis protein (Bt, Cry1Ab). The objective was to determine whether the variation in soil parameters under a range of conventional maize cultivars exceeded the differences between Bt and non-Bt maize cultivars. Variations in plant growth parameters (shoot and root biomass, percentage carbon, percentage nitrogen), Bt protein concentration in shoots, roots and soil, soil nematode abundance and soil microbial community structure were determined. Eight paired varieties (i.e. varieties genetically modified to express Bt protein and their near-isogenic control varieties) were investigated, together with a Bt variety for which no near-isogenic control was available (NX3622, a combined transformant expressing both Bt and herbicide tolerance) and a conventional barley (Hordeum vulgare L.) variety which was included as a positive control. The only plant parameter which showed a difference between Bt varieties and near-isogenic counterparts was the shoot carbon to nitrogen ratio; this was observed for only two of the eight varieties, and so was not attributable to the Bt trait. There were no detectable differences in the concentration of Bt protein in plant or soil with any of the Bt-expressing varieties. There were significant differences in the abundance of soil nematodes, but this was not related to the Bt trait. Differences in previously published soil nematode studies under Bt maize were smaller than these varietal effects. Soil microbial community structure, as determined by phospholipid fatty acid (PLFA) analysis, was strongly affected by plant growth stage but not by the Bt trait. The experimental addition of purified Cry1Ab protein to soil confirmed that, at ecologically relevant concentrations, there were no measurable effects on microbial community structure.