An In Vitro Characterization of Functional Interactions Between Purified Telomere Repeat Binding Factors 1 and 2 and Rad51 Recombinase

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and non-telomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat-binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90 nucleotide telomeric and non-telomeric substrates into telomeric and non-telomeric plasmid templates. We report that pre-incubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation when pre-incubated with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation, and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated non-telomeric D-loop formation, possibly explaining how TRF2 promotes non-telomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.Doctor of Philosoph

Short communication: NKG2C+ NK cells contribute to increases in CD16+CD56- cells in HIV type 1+ individuals with high plasma viral load.

Chronic HIV-1 infection results in the expansion of both NKG2C+ and CD16+CD56- human natural killer cells. NKG2C+ cells proliferate in response to human cytomegalovirus (HCMV) and expansion of the dysfunctional CD56-CD16+ natural killer (NK) cells is associated with HIV-1 viremia. Here we report an association between increased proportions of CD56-CD16+ NK cells in viremic HIV-1+ individuals and an increased contribution of NKG2C+ cells to this subset. These data, in addition to anti-HCMV IgG serology, indicate a potential contribution of both HCMV and HIV-1 to NK cell dysfunction in HIV-1-infected individuals

High Density Faraday Cup Array or Other Open Trench Structures and Method of Manufacture Thereof

A detector array and method for making the detector array. The detector array includes a substrate including a plurality of trenches formed therein, and a plurality of collectors electrically isolated from each other, formed on the walls of the trenches, and configured to collect charged particles incident on respective ones of the collectors and to output from the collectors signals indicative of charged particle collection. In the detector array, adjacent ones of the plurality of trenches are disposed in a staggered configuration relative to one another. The method forms in a substrate a plurality of trenches across a surface of the substrate such that adjacent ones of the trenches are in a staggered sequence relative to one another, forms in the plurality of trenches a plurality of collectors, and connects a plurality of electrodes respectively to the collectors

Faraday Cup Array Integrated with a Readout IC and Method for Manufacture Thereof

A detector array and method for making the detector array. The array includes a substrate including a plurality of trenches formed therein, and includes a plurality of collectors electrically isolated from each other, formed on the walls of the trenches, and configured to collect charge particles incident on respective ones of the collectors and to output from said collectors signals indicative of charged particle collection. The array includes a plurality of readout circuits disposed on a side of the substrate opposite openings to the collectors. The readout circuits are configured to read charge collection signals from respective ones of the plurality of collectors

TRF1 and TRF2 Differentially Modulate Rad51-Mediated Telomeric and Nontelomeric Displacement Loop Formation in Vitro

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication

Reproductive success and pollination of the Tuncurry Midge Orchid (Genoplesium littorale) (Orchidaceae) by Chloropid Flies

The Midge Orchids (Genoplesium R.Br.) (Orchidaceae) are thought to attract pollinators by nectar reward. All verified records of Genoplesium pollinators are small flies of the families Chloropidae and Milichiidae, suggesting pollinator specificity. We investigated pollination of the Critically Endangered Tuncurry Midge Orchid, Genoplesium littorale D.L.Jones. In common with other Genoplesium species, G. littorale is pollinated exclusively by chloropid flies. Although there is specificity at the pollinator family level, G. littorale is oligophilous, being pollinated by five putative chloropid species in two genera, Conioscinella and Cadrema. Most visitors were female with females greatly predominating among flies bearing pollinaria. Examination of flowers on ten inflorescences showed G. littorale is outcrossing with high pollen vector activity; pollinaria had been removed from 71% of post anthesis flowers. A set of criteria for distinguishing outcrossed, autogamous and apomictic flowers based on observations of pollinaria removal, pollination of stigmas and fruit set on individual flowers ruled out the occurrence of autogamy and apomixis in G. littorale. Fruit set on inflorescences averaged 44% percent prior to seed dispersal and varied significantly among sub-populations. Nectar is produced in the groove of the labellum callus, although flowers emitted no odour detectable by humans. Detailed examination of 29 flowers revealed no chloropid eggs, indicating the pollination syndrome is not brood site mimicry. The absence of strong dung or carrion-like odours also makes sapromyophily unlikely. The Genoplesium pollination syndrome is nectar reward, but may also represent an example of ‘kleptomyiophily’, recently described in Aristolochia rotunda. Herbivory reduced reproductive capacity by half overall, varied significantly among sub-populations and may be a significant threatening process for G. littorale. Strategies to reduce herbivory in this critically endangered species should be investigated