A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres^® and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.     

Phosphorylated retinoblastoma protein complexes with pp32 and inhibits pp32-mediated apoptosis

The retinoblastoma gene product (Rb) is a tumor suppressor that affects apoptosis paradoxically. Most sporadic cancers inactivate Rb by preferentially targeting the pathway that regulates Rb phosphorylation, resulting in resistance to apoptosis; this contrasts with Rb inactivation by mutation, which is associated with high rates of apoptosis. How phosphorylated Rb protects cells from apoptosis is not well understood, but there is evidence that Rb may sequester a pro-apoptotic nuclear factor. pp32 (ANP32A) is a pro-apoptotic nuclear phosphoprotein, the expression of which is commonly increased in cancer. We report that hyperphosphorylated Rb interacts with pp32 but not with the closely related proteins pp32r1 and pp32r2. We further demonstrate that pp32-Rb interaction inhibits the apoptotic activity of pp32 and stimulates proliferation. These results suggest a mechanism whereby cancer cells gain both a proliferative and survival advantage when Rb is inactivated by hyperphosphorylation.     

Contrasting scales of oviposition and parasitism in praying mantids

We report on spatial patterns of parasitism of oothecae (egg cases) of praying mantises (Stagmomantis limbata) by torymid wasps, Podagrion spp. Using collections of mapped mantid oothecae from Riparian sites in the Sonoran desert and Grassland sites in the Chiricahua Mountains (both in Arizona, USA), we characterized the spatial distributions of oothecae and parasitism. The likelihood of an egg case suffering some parasitism was higher at Grassland sites, which had high oothecal densities, than at low-density Riparian sites. However, experimental isolation of Grassland oothecae to densities comparable to Riparian sites reduced parasitism rates. At Riparian sites, parasitized oothecae exhibited on average the same extent of parasitism as parasitized oothecae at high densities but with much greater variation. Indeed, large fractions of Riparian oothecae suffered both severe (>50%) and light (<20%) parasitism, whereas most parasitized Grassland oothecae suffered intermediate levels of parasitism. Analysis of first nearest neighbor distances indicated that the parasite load of an ootheca did not depend on its immediate isolation. However, extending the analysis to include subsequent nearest neighbors (using a technique from spatial statistics called the R(K) function), we found that even though oothecae of S. limbata were spatially clustered, some oothecae in a (statistically defined) cluster escaped parasitism when overall oothecal densities were low. This pattern suggests that when oothecae are sparsely distributed, Podagrion wasps exploit only a fraction of the oothecae available locally, even though the oothecae are strongly aggregated relative to their overall density. We suggest this lack of congruency in the scales of oothecal deposition and parasitism at low densities (which is absent when oothecae are at high densities) may be explained in part by behavioral aspects of the parasite's reproduction, including increased host fidelity by relatively sedentary female parasites. Received: June 13, 2000 / Accepted: October 16, 2000     

Plasmodium falciparum gametocyte carriage, emergence, clearance and population sex ratios in anaemic and non-anaemic malarious children

Anaemia in falciparum malaria is associated with an increased risk of gametocyte carriage, but its effects on transmission have not been extensively evaluated in malarious children. Plasmodium falciparum gametocyte carriage, emergence, clearance, population sex ratios (SR) (defined as the proportion of gametocytes that are male), inbreeding rates and temporal changes in SR were evaluated in 840 malarious children. Gametocyte carriage pre-treatment was at a level of 8.1%. Anaemia at enrolment was an independent risk factor for gametocyte carriage post-treatment. The emergence of gametocytes seven days post-treatment was significantly more frequent in anaemic children (7/106 vs. 10/696, p = 0.002). In the initially detected gametocytes, the proportion of children with a male-biased SR (MBSR) (> 0.5) was significantly higher in anaemic children (6/7 vs. 3/10, p = 0.027). Pre-treatment SR and estimated inbreeding rates (proportion of a mother's daughters fertilised by her sons) were similar in anaemic and non-anaemic children. Pre-treatment SR became more female-biased in non-anaemic children following treatment. However, in anaemic children, SR became male-biased. Anaemia was shown to significantly increase gametocyte emergence and may significantly alter the SR of emerging gametocytes. If MBSR is more infective to mosquitoes at low gametocytaemia, then these findings may have significant implications for malaria control efforts in endemic settings where malaria-associated anaemia is common.     

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users.     

Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle

Currently, few factors have been identified that provide the inductive signals necessary to transform the simple otic placode into the complex asymmetric structure of the adult vertebrate inner ear. We provide evidence that Hedgehog signalling from ventral midline structures acts directly on the zebrafish otic vesicle to induce posterior otic identity. We demonstrate that two strong Hedgehog pathway mutants, chameleon (con(tf18b)) and slow muscle omitted (smu(b641)) exhibit a striking partial mirror image duplication of anterior otic structures, concomitant with a loss of posterior otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of the Hedgehog pathway, by injection of sonic hedgehog or dominant-negative protein kinase A RNA, has the reverse effect: ears lose anterior otic structures and show a mirror image duplication of posterior regions. By using double mutants and antisense morpholino analysis, we also show that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in anteroposterior patterning of the zebrafish otic vesicle.     

Characterization,antimicrobial, antioxidant,and anticoagulant activities of silver nanoparticles synthesized from Petiveria alliacea L. leaf extract

Abstract

Phytosynthesis of silver nanoparticles (AgNPs) using leaf extract of Petiveria alliacea (PA) was the focus of this research work. The PA-AgNPs were characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and selected area electron diffraction (SAED) study. Studies were made on the AgNPs for antibacterial, antifungal, anticoagulant, free-radical scavenging, and hydrogen peroxide scavenging activities. The crystalline PA-AgNPs were monodispersed, with a size range of 16.70–33.74?nm and maximum absorption at 410?nm. FTIR analysis displayed prominent peaks at 3430.6, 1711.8, and 1165.9/cm, which showed the existence of phenolic compounds and proteins in the synthesis of AgNPs. PA-AgNPs was active against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus, with 100% inhibition. The PA-AgNPs also displayed good antifungal properties, as the concentrations of 100 and 150?µg/mL had 100% inhibition toward Aspergillus fumigatus and Aspergillus flavus. However, there was 66.67% inhibition of Aspergillus niger. It scavenged both DPPH and H₂O₂ by 70.69 and 89.02%, respectively. PA-AgNPs also prevented the coagulation of human blood. This study, being the first of its kind to use the leaf extract of PA for the synthesis of AgNPs has shown that PA-AgNPs can find biomedical applications.     

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) in Nigerian children 10 years post-adoption of artemisinin-based combination treatments

The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72–76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C₇₂M₇₄N₇₅K₇₆ (42.9%) and mutant C₇₂I₇₄E₇₅T₇₆ (53.8%) were observed. Also, wildtype N₈₆Y₁₈₄ (62.9%) and mutant N₈₆F₁₈₄ (21.1%), Y₈₆Y₁₈₄ (6.4%), and Y₈₆F₁₈₄ (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C₇₂I₇₄E₇₅T₇₆ and C₇₂M₇₄N₇₅K₇₆) and major mdr-1 haplotypes (N₈₆Y₁₈₄, N₈₆F₁₈₄ and Y₈₆Y₁₈₄). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C₇₂I₇₄E₇₅T₇₆ haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.