No abstract

The DNA-methylating drug temozolomide, which induces cell death through apoptosis, is used for the treatment of malignant glioma. Here, we investigate the mechanisms underlying the ability of temozolomide to induce senescence in glioblastoma cells. Temozolomide-induced senescence was triggered by the specific DNA lesion O 6methylguanine (O 6 MeG) and characterized by arrest of cells in the G 2 -M phase. Inhibitor experiments revealed that temozolomide-induced senescence was initiated by damage recognition through the MRN complex, activation of the ATR/CHK1 axis of the DNA damage response pathway, and mediated by degradation of CDC25c. Temozolomideinduced senescence required functional p53 and was dependent on sustained p21 induction. p53-deficient cells, not expressing p21, failed to induce senescence, but were still able to induce a G 2 -M arrest. p14 and p16, targets of p53, were silenced in our cell system and did not seem to play a role in temozolomide-induced senescence. In addition to p21, the NF-kB pathway was required for senescence, which was accompanied by induction of the senescence-associated secretory phenotype. Upon temozolomide exposure, we found a strong repression of the mismatch repair proteins MSH2, MSH6, and EXO1 as well as the homologous recombination protein RAD51, which was downregulated by disruption of the E2F1/DP1 complex. Repression of these repair factors was not observed in G 2 -M arrested p53deficient cells and, therefore, it seems to represent a specific trait of temozolomide-induced senescence.Significance: These findings reveal a mechanism by which the anticancer drug temozolomide induces senescence and downregulation of DNA repair pathways in glioma cells.

Environmental DNA (eDNA) assays for single‐ and multi‐species detection show promise for providing standardized assessment methods for diverse taxa, but techniques for evaluating multiple taxonomically divergent assemblages are in their infancy. We evaluated whether microfluidic multiplex metabarcoding on the Fluidigm Access Array™ platform and high‐throughput sequencing could identify diverse stream and riparian assemblages from 48 taxon‐general and taxon‐specific metabarcode primers. eDNA screening was paired with electrofishing along a stream continuum to evaluate congruence between methods. A fish hatchery located midway along the stream continuum provided a dispersal barrier, and a point source for non‐native White Sturgeon (Acipencer transmontanus). Microfluidic metabarcoding had 87% accuracy with respect to electrofishing and detected all 13 species electrofishing observed. Taxon‐specific barcoding primers were more successful than taxon‐general universal metabarcoding primers at classifying sequences to species. Both types of markers detected a transition from downstream sites dominated by multiple fish species, to upstream sites dominated by a single species; however, we failed to detect a complementary transition in amphibian occupancy. White Sturgeon was only detected at the hatchery outflow, indicating eDNA transport was not detectable ~2.4 km from its source. Overall, we identified 878 predicted taxa. Most sequences (50.1%) derived from fish (Actinopteri, Petromyzontidae), oomycetes (21.3%), arthropoda (classes Insecta, Decapoda; 16.5%), and apicomplexan parasites (3.8%). Taxa accounting for ~1% or less of sequences included freshwater red algae, diatoms, amphibians, and beaver. Our work shows that microfluidic metabarcoding can survey multiple phyla per assay, providing fine discrimination required to resolve closely related species, and enable data‐driven prioritization for multiple forest health objectives.

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Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.