Tumor-primed human natural killer cells lyse NK-resistant tumor targets: evidence of a two-stage process in resting NK cell activation

NK cells are defined as those cells that lyse tumor cells without priming. In this study, we show that the preincubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias, and solid tumors even when HLA-matched, allogeneic or autologous. The primed NK cells remained nonresponsive to HLA-C matched and mismatched normal mononuclear cells from multiple donors. CD69, a known NK trigger receptor, was shown to be the predominant trigger on the tumor-primed NK cells because lysis was blocked with the rCD69 protein. The lack of lytic activity against normal hemopoietic cells implied that the ligand for CD69 is tumor restricted, and this was confirmed by experiments using fluorochrome labeled rCD69. It has been recently shown that resting NK cells require prior stimulation with IL-2 before triggering by all known NK-triggering ligands. In this study, we show that a tumor cell can provide the NK priming signal independently of IL-2. These data provide evidence for two NK evasion strategies for tumor cells, namely the prevention of priming (type1 evasion) and failure to trigger (type 2 evasion). Most NK-resistant cell lines are type 1 and fail to prime resting NK cells but are lysed by IL-2-primed NK cells. In contrast, CTV-1 cells prime resting NK cells but fail to trigger (type 2), and coincubation with CTV-1 primes for triggering by type 1 NK-resistant tumor cells. These tumor-activated NK cells lyse a broad spectrum of tumor cells with a degree of specificity never previously reported.     

Co-Expression of the Mosquitocidal Toxins Cyt1Aa and Cry11Aa from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">israelensis</Emphasis> in <Emphasis Type="Italic">Asticcacaulis excentricus</Emphasis>

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.     

Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.     

Weather effects on birds of different size are mediated by long‐term climate and vegetation type in endangered temperate woodlands

Species occurrence is influenced by a range of factors including habitat attributes, climate, weather, and human landscape modification. These drivers are likely to interact, but their effects are frequently quantified independently. Here, we report the results of a 13‐year study of temperate woodland birds in south‐eastern Australia to quantify how different‐sized birds respond to the interacting effects of: (a) short‐term weather (rainfall and temperature in the 12 months preceding our surveys), (b) long‐term climate (average rainfall and maximum and minimum temperatures over the period 1970–2014), and (c) broad structural forms of vegetation (old‐growth woodland, regrowth woodland, and restoration plantings). We uncovered significant interactions between bird body size, vegetation type, climate, and weather. High short‐term rainfall was associated with decreased occurrence of large birds in old‐growth and regrowth woodland, but not in restoration plantings. Conversely, small bird occurrence peaked in wet years, but this effect was most pronounced in locations with a history of high rainfall, and was actually reversed (peak occurrence in dry years) in restoration plantings in dry climates. The occurrence of small birds was depressed—and large birds elevated—in hot years, except in restoration plantings which supported few large birds under these circumstances. Our investigation suggests that different mechanisms may underpin contrasting responses of small and large birds to the interacting effects of climate, weather, and vegetation type. A diversity of vegetation cover is needed across a landscape to promote the occurrence of different‐sized bird species in agriculture‐dominated landscapes, particularly under variable weather conditions. Climate change is predicted to lead to widespread drying of our study region, and restoration plantings—especially currently climatically wet areas—may become critically important for conserving bird species, particularly small‐bodied taxa.     

The contribution of tumor and host tissue factor expression to oncogene-driven gliomagenesis

Glioblastoma multiforme (GBM) is an aggressive form of glial brain tumors, associated with angiogenesis, thrombosis, and upregulation of tissue factor (TF), the key cellular trigger of coagulation and signaling. Since TF is upregulated by oncogenic mutations occurring in different subsets of human brain tumors we investigated whether TF contributes to tumourigenesis driven by oncogenic activation of EGFR (EGFRvIII) and RAS pathways in the brain. Here we show that TF expression correlates with poor prognosis in glioma, but not in GBM. In situ, the TF protein expression is heterogeneously expressed in adult and pediatric gliomas. GBM cells harboring EGFRvIII (U373vIII) grow aggressively as xenografts in SCID mice and their progression is delayed by administration of monoclonal antibodies blocking coagulant (CNTO 859) and signaling (10H10) effects of TF in vivo. Mice in which TF gene is disrupted in the neuroectodermal lineage exhibit delayed progression of spontaneous brain tumors driven by oncogenic N-ras and SV40 large T antigen (SV40LT) expressed under the control of sleeping beauty transposase. Reduced host TF levels in low-TF/SCID hypomorphic mice mitigated growth of glioma subcutaneously but not in the brain. Thus, we suggest that tumor-associated TF may serve as therapeutic target in the context of oncogene-driven disease progression in a subset of glioma.     

Testing evolutionary models of senescence: traditional approaches and future directions

From an evolutionary perspective, the existence of senescence is a paradox. Why has senescence not been more effectively selected against given its associated decreases in Darwinian fitness? Why does senescence exist and how has it evolved? Three major theories offer explanations: (1) the theory of mutation accumulation suggested by PB Medawar; (2) the theory of antagonistic pleiotropy suggested by GC Williams; and (3) the disposable soma theory suggested by TBL Kirkwood. These three theories differ in the underlying causes of aging that they propose but are not mutually exclusive. This paper compares the specific biological predictions of each theory and discusses the methods and results of previous empirical tests. Lifespan is found to be the most frequently used estimate of senescence in evolutionary investigations. This measurement acts as a proxy for an individual’s rate of senescence, but provides no information on an individual’s senescent state or “biological age” throughout life. In the future, use of alternative longitudinal measures of senescence may facilitate investigation of previously neglected aspects of evolutionary models, such as intra- and inter-individual heterogeneity in the process of aging. DNA methylation data are newly proposed to measure biological aging and are suggested to be particularly useful for such investigations.     

A DNA marker assay based on high-resolution melting curve analysis for distinguishing species of the Festuca–Lolium complex

The grass breeding industry is interested in a fast and cheap method of identifying contamination in seeds of Italian and perennial ryegrass (Lolium perenne L. and L. multiflorum Lam., respectively). This study shows that high-resolution melting curve analysis in combination with an unlabelled probe assay is an effective method of detecting single nucleotide polymorphisms (SNPs) in diverse Italian and perennial ryegrass backgrounds. This method proved efficient in differentiating ryegrass species and reducing the effect of additional DNA sequence polymorphisms close to the target SNP on the melting curve profiles. For the identification of contamination in Italian and perennial ryegrass seed production, high-resolution melting curve analysis shows great potential, as it is a single closed-tube PCR reaction with an easy workflow, providing results in <2 h after DNA extraction.