Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Host-selection behaviour by genetically identical aphids with different plant preferences

The behaviour of summer and autumn winged forms of the black bean aphid, Aphis fabae Scopoli (Homoptera: Aphididae), was compared on two plants utilized at different stages of the insect’s life cycle. Adult autumn migrants (gynoparae) are monophagous, colonizing spindle (Euonymus europaeus), whereas polyphagous summer winged aphids (alate virginoparae) are associated with a variety of herbaceous plants, including broad bean (Vicia faba). When aphids from a single clone were given access to a spindle leaf and a bean seedling in choice tests, many virginoparae settled and larviposited on both plant species over 24 h. By contrast, gynoparae showed a clear preference for spindle, with 93.5% of settled adults and 98.3% of larvae on this plant species. Close‐up video monitoring showed that gynoparae discriminated beans from spindle within a 5‐min period, whereas virginoparae behaved similarly on both plant species. For gynoparae, the major behavioural difference on the two plants appeared after a brief (epidermal) stylet penetration, with many insects taking flight within a few seconds of stylet withdrawal from bean. Factors detected during stylet insertion by gynoparae must therefore inhibit take‐off on spindle. Electrical recording experiments showed that aphids often punctured a cell membrane during brief probes on both plant species, and intracellular stylet activities always included a waveform associated with ingestion. When gynoparae puncture spindle cells their behaviour is probably modified by intracellular metabolites detected via gustation of ingested epidermal cell sap. These cues may inhibit the take‐off reflex which otherwise follows probing.     

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.     

CHROMOSOME NUMBERS IN CHIHUAHUAN DESERT CACTACEAE. TRANS-PECOS TEXAS

Chromosome numbers are presented for 100 collections of Cactaceae from the Trans-Pecos region of Texas. A total of 65 taxa representing 52 species and 12 genera were counted, including first reports for 45 taxa and new ploidy levels for four taxa. Notable among those listed are counts for Opuntia schottii var. grahamii (n = 11, ca. 22), O. stanlyi (n = 22), O. arenaria (n = 11), O. phaeacantha var. spinosibacca (n = 22), O. lindheimeri var. lindheimeri (n = 11), O. strigil (n = 11), Echinocereus enneacanthus var. cf. dubius (n = ca. 22), E. pectinatus var. neomexicanus (n = 22), and Thelocactus bicolor var. bicolor (n = 22). Endomitosis was found to be present in O. phaeacantha var. spinosibacca, Mammillaria pottsii, and Neolloydia intertexta var. dasyacantha. Meiotic irregularities were noted in some species belonging to the genera Opuntia and Echinocereus. Phytogeographic considerations are inferred from the chromosomal data for O. polyacantha, O. lindheimeri, and O. ficusindica. A base number of x = 11 in Cactaceae is supported.     

Silver Impregnation of Degenerating Axons; Comparisons of Postoperative Intervals, Fixatives and Staining Methods

By introducing variations in procedures and comparing density and patterns of degeneration exhibited, we found that: (1) in the rat, cat, and monkey, the optimum survival time was about 1 wk; this period yielding the maximum amount of argyrophilic reaction and the most consistent staining among several different parts of the brain. Survival times of up to 3 wk did not seriously impair the interpretation of argyrophilial patterns; (2) some qualitative changes in the nuclear patterns of degeneration suggested that the use of a range of survival times may provide useful data; (3) staining differences and similarities in different neural systems of known connections were about the same in the 3 species of animals; (4) formalin, buffered formalin, and a 4%:1% paraformaldehyde-glutaraldehyde mixture were satisfactory perfusion solutions; however, staining was less intense when the Nauta-Gygax method followed the last fixative mixture; (5) The Fink-Heimer I method yielded more consistent results than did the Fink-Heimer II or the Nauta-Gygax methods.