A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of approximately 15 months. Treatment is limited by the blood-brain barrier (BBB) which restricts the passage of most drugs to the brain. We previously reported the design and synthesis of a BBB-penetrant macrocyclic cell-penetrating peptide conjugate (M13) covalently linked at the axial position of a Pt(IV) cisplatin prodrug. Here we show the Pt(IV)-M13 conjugate releases active cisplatin upon intracellular reduction and effects potent in vitro GBM cell killing. Pt(IV)-M13 significantly increased platinum uptake in an in vitro BBB spheroid model and intravenous administration of Pt(IV)-M13 in GBM tumor-bearing mice led to higher platinum levels in brain tissue and intratumorally compared with cisplatin. Pt(IV)-M13 administration was tolerated in naïve nude mice at higher dosage regimes than cisplatin and significantly extended survival above controls in a murine GBM xenograft model (median survival 33 days for Pt(IV)-M13 vs 24 days for Pt(IV) prodrug, 22.5 days for cisplatin and 22 days for control). Increased numbers of γH2AX nuclear foci, biomarkers of DNA damage, were observed in tumors of Pt(IV)-M13-treated mice, consistent with elevated platinum levels. The present work provides the first demonstration that systemic injection of a Pt(IV) complex conjugated to a brain-penetrant macrocyclic peptide can lead to increased platinum levels in the brain and extend survival in mouse GBM models, supporting further development of this approach and the utility of brain-penetrating macrocyclic peptide conjugates for delivering non-BBB penetrant drugs to the central nervous system.

Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Get Healthy after Breast Cancer - examining the feasibility, acceptability and outcomes of referring breast cancer survivors to a general population telephone-delivered program targeting physical activity, healthy diet and weight loss.

PURPOSE: This pilot study assessed the feasibility, acceptability and outcomes of referring breast cancer survivors to the 'Get Healthy Service' (GHS), a state health-funded 6-month telephone-delivered lifestyle program. METHODS: Pre-post study with eligible and consenting women following treatment for stages I-III breast cancer referred by nurses in a cancer treatment centre to the GHS. Feasibility was assessed via GHS uptake and completion; acceptability was assessed via patient satisfaction and nurse feedback. Changes in weight, physical activity, diet, quality of life (QoL) and fatigue from baseline to 6 months were examined. RESULTS: Fifty-three women (mean ± SD body mass index, 31.0 ± 5.5 kg/m2; age, 57.3 ± 10.0 years; 14.0 ± 7.1 months post-diagnosis; 43.4% born outside Australia, 49% high school or less education, 32.1% English as a second language) took up the GHS, with 62% completing the program. Almost all (92%) completers had high satisfaction ratings and breast nurses provided positive feedback. Findings from GHS completers (n = 33) show a statistically significant effect from baseline to 6 months for weight loss (mean ± SE; -2.4 ± 0.7 kg; p = 0.002) and total physical activity minutes per week (55 ± 18 min/week; p = 0.006). No significant changes in fruit or vegetable servings per day or takeaways and fast food frequency per week were observed. A significant improvement in mental QoL was observed (3.5 ± 1.6; p = 0.041), but not for physical QoL or fatigue. CONCLUSION: GHS referral appeared feasible, acceptable and effective for a diverse group of women following completion of treatment for breast cancer, yet more remains to be done to fully integrate GHS screening and referral into usual care.

In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

Cell migration in paediatric glioma; characterisation and potential therapeutic targeting.

BACKGROUND: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. METHODS: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. RESULTS: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. CONCLUSIONS: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Effectiveness of lifestyle-based weight loss interventions for adults with type 2 diabetes: a systematic review and meta-analysis.

AIMS: To provide a systematic review and meta-analysis of recent evidence on the effectiveness of lifestyle-based weight loss interventions for adults with type 2 diabetes. METHODS: A search of the literature from January 2003 to July 2013 was conducted (PubMed, Embase, CINAHL and Web of Science). The studies considered eligible were randomized controlled trials evaluating weight loss interventions (diet and physical activity, with or without behavioural strategies) of ≥12 weeks duration, compared with usual care or another comparison intervention. Ten studies were included for review. Some heterogeneity was present in the sample, therefore, random-effects models were used to calculate pooled effects. RESULTS: Intervention duration ranged from 16 weeks to 9 years, with all but one delivered via individual or group face-to-face sessions. From six studies comparing lifestyle intervention with usual care the pooled effect on weight (n = 5795) was -3.33 kg [95% confidence interval (CI) -5.06, -1.60 kg], and on glycated haemoglobin (HbA1c; n = 5784) was -0.29% (95% CI -0.61, 0.03%), with both attenuated in sensitivity analyses. The pooled within-group effect on weight (n = 3063) from all 10 lifestyle intervention groups was -5.33 kg (95% CI -7.33, -3.34 kg), also attenuated in sensitivity analyses. None of the participant or intervention characteristics examined explained the heterogeneity. Only one study assessed whether intervention effects were maintained after the end of the intervention. CONCLUSIONS: Lifestyle-based weight loss intervention trials in type 2 diabetes achieve, on average, modest reductions in weight and HbA1c levels, but results were heavily influenced by one trial. Evidence-based approaches for improving the effectiveness of lifestyle-based interventions in type 2 diabetes are needed, along with future studies reporting on maintenance and cost-effectiveness.

Methylation-specific multiplex ligation-dependent probe amplification identifies promoter methylation events associated with survival in glioblastoma.

DNA methylation plays an important role in cancer biology and methylation events are important prognostic and predictive markers in many tumor types. We have used methylation-specific multiplex ligation-dependent probe amplification to survey the methylation status of MGMT and 25 tumor suppressor genes in 73 glioblastoma cases. The data obtained was correlated with overall survival and response to treatment. The study revealed that methylation of promoter regions in TP73 (seven patients), THBS1 (eight patients) and PYCARD (nine patients) was associated with improved outcome, whereas GATA5 (21 patients) and WT1 (24 patients) promoter methylation were associated with poor outcome. In patients treated with temozolomide and radiation MGMT and PYCARD promoter methylation events remained associated with improved survival whereas GATA5 was associated with a poor outcome. The identification of GATA5 promoter methylation in glioblastoma has not previously been reported. Furthermore, a cumulative methylation score separated patients into survival groups better than any single methylation event. In conclusion, we have identified specific methylation events associated with patient outcome and treatment response in glioblastoma, and these may be of functional and predictive/prognostic significance. This study therefore provides novel candidates and approaches for future prospective validation.

Comparative effectiveness of antinociceptive gene therapies in animal models of diabetic neuropathic pain.

Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67+endomorphin, GAD67+enkephalin or endomorphin+enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.

Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320.

PTEN (Phosphatase and tensin homolog deleted on chromosome 10) expression in stromal fibroblasts suppresses epithelial mammary tumours, but the underlying molecular mechanisms remain unknown. Using proteomic and expression profiling, we show that Pten loss from mammary stromal fibroblasts activates an oncogenic secretome that orchestrates the transcriptional reprogramming of other cell types in the microenvironment. Downregulation of miR-320 and upregulation of one of its direct targets, ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) are critical events in Pten-deleted stromal fibroblasts responsible for inducing this oncogenic secretome, which in turn promotes tumour angiogenesis and tumour-cell invasion. Expression of the Pten-miR-320-Ets2-regulated secretome distinguished human normal breast stroma from tumour stroma and robustly correlated with recurrence in breast cancer patients. This work reveals miR-320 as a critical component of the Pten tumour-suppressor axis that acts in stromal fibroblasts to reprogramme the tumour microenvironment and curtail tumour progression.

MicroRNAs and glioblastoma; the stem cell connection.

Recent data draw close parallels between cancer, including glial brain tumors, and the biology of stem and progenitor cells. At the same time, it has become clear that one of the major roles that microRNAs play is in the regulation of stem cell biology, differentiation, and cell 'identity'. For example, microRNAs have been increasingly implicated in the regulation of neural differentiation. Interestingly, initial studies in the incurable brain tumor glioblastoma multiforme strongly suggest that microRNAs involved in neural development play a role in this disease. This encourages the idea that certain miRs allow continued tumor growth through the suppression of differentiation and the maintenance of the stem cell-like properties of tumor cells. These concepts will be explored in this article.

Plasma acceleration above martian magnetic anomalies.

Auroras are caused by accelerated charged particles precipitating along magnetic field lines into a planetary atmosphere, the auroral brightness being roughly proportional to the precipitating particle energy flux. The Analyzer of Space Plasma and Energetic Atoms experiment on the Mars Express spacecraft has made a detailed study of acceleration processes on the nightside of Mars. We observed accelerated electrons and ions in the deep nightside high-altitude region of Mars that map geographically to interface/cleft regions associated with martian crustal magnetization regions. By integrating electron and ion acceleration energy down to the upper atmosphere, we saw energy fluxes in the range of 1 to 50 milliwatts per square meter per second. These conditions are similar to those producing bright discrete auroras above Earth. Discrete auroras at Mars are therefore expected to be associated with plasma acceleration in diverging magnetic flux tubes above crustal magnetization regions, the auroras being distributed geographically in a complex pattern by the many multipole magnetic field lines extending into space.

Genetic strategies for brain tumor therapy.

Gene therapy is a potentially useful approach in the treatment of human brain tumors, which are notoriously refractory to conventional approaches. Most human clinical trials to date have been unsuccessful in terms of improving patient outcome. Recent improvements in viral vectors, the development of stem cell technology, and increased understanding of the mechanism of action of therapeutic transgenes provide hope that the next generation of gene therapeutics may show increased efficacy in treatment of this devastating disease.

Solar wind-induced atmospheric erosion at Mars: first results from ASPERA-3 on Mars Express.

The Analyzer of Space Plasma and Energetic Atoms (ASPERA) on board the Mars Express spacecraft found that solar wind plasma and accelerated ionospheric ions may be observed all the way down to the Mars Express pericenter of 270 kilometers above the dayside planetary surface. This is very deep in the ionosphere, implying direct exposure of the martian topside atmosphere to solar wind plasma forcing. The low-altitude penetration of solar wind plasma and the energization of ionospheric plasma may be due to solar wind irregularities or perturbations, to magnetic anomalies at Mars, or both.

Conservation implications of the link between biodiversity and ecosystem functioning.

The relationship between biodiversity and individual ecosystem processes is often asymptotic, saturating at relatively low levels, with some species contributing more strongly than others. This has cast doubt on arguments for conservation based on maintenance of the functioning of ecosystems. However, we argue that the link between biodiversity and ecosystem functioning is an important additional argument for conservation for several reasons. (1) Although species differ in importance to ecosystem processes, we do not believe that this argues for preservation of just a few species for two reasons: first, it is nearly impossible to identify all species important to the numerous systems and processes on which humans depend; second, the important species themselves may depend on an unknown number of other species in their communities. (2) Arguments for conservation based on ecosystem functioning are complementary to other utilitarian, ethical and aesthetic justifications. No single reason will convince all people or protect all species, however the combination produces a strong case for conservation of biodiversity. (3) Even if the relationship between biodiversity and ecosystem functioning is asymptotic at local spatial scales and in the short term, effects of biodiversity loss are likely to be important at larger temporal and spatial scales. (4) Initial arguments for the importance of biodiversity for ecosystem functioning were largely based on a precautionary approach (points 1-3). However, we are now moving to a scientific position based on accumulating experimental evidence. The future challenge is the integration of this scientific research with policy.

Infection of aquatic insects with trematode metacercariae carrying Ehrlichia risticii, the cause of Potomac horse fever.

We provide evidence of Ehrlichia risticii Holland, the agent of Potomac horse fever, in trematode stages found in aquatic insects collected from a pasture stream in northern California, using nested polymerase chain reaction (PCR) amplification and sequence analyses of the 16S rRNA, 51 kDa major antigen and groEL heat shock protein genes. E. risticii was detected in metacercariae found in the immatures and adults of the following insects: caddisflies (Trichoptera), mayflies (Ephemeroptera), damselflies (Odonata, Zygoptera), dragonflies (Odonata, Anisoptera), and stoneflies (Plecoptera). The prevalence of E. risticii was 31.9% (n = 454 individuals) in aquatic insects (13 of 17 species were positive). Prevalence within orders was as follows: 43.5% (n = 207) in caddisflies, 15.2% (n = 92) in mayflies, 13.9% (n = 115) in damselflies, 10.0% (n = 10) in dragonflies, and 80.0% (n = 30) in stoneflies. This study demonstrates a broad intermediate host range for trematodes that act as vector for E. risticii. Insects are likely to play an important role in the epidemiology of this disease.

Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report.

Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.

Effects of sustained-release methoprene and a combined formulation of liquid methoprene and Bacillus thuringiensis israelensis on insects in salt marshes.

Aquatic insects are an important component of the food web in salt marshes, therefore it is necessary to test whether pesticides used to control mosquitoes in salt marshes are safe for nontarget insects. We tested the nontarget effects of a combined formulation (duplex) of Bacillus thuringiensis israelensis (B.t.i.) and liquid methoprene (an insect development regulator) or sustained-release methoprene pellets (Altosid(R) pellets) by applying these materials to replicated salt marsh ponds at maximum label rates. Untreated ponds served as controls. We measured effects of the pesticides by rearing immature mosquitoes (Aedes dorsalis) and water boatmen (Trichocorixa reticulata) in predator-exclusion cages and by monitoring uncaged populations of invertebrates using replicated sweep-net samples. Both pesticides killed caged mosquitoes, and the activity of the Altosid(R) pellets continued through 99 days. There were no detectable effects of either pesticide on the survival or maturation of T. reticulata, or on abundances of uncaged invertebrates. The long-term activity of the pellets could help minimize mosquito abatement activity in salt marshes where there are breeding birds or endangered species. However, other studies suggest that this advantage needs to be balanced against the risks that sustained-release formulations could lead to development of resistance in mosquitoes or that initially undetected nontarget effects could build over time.

Field efficacy and nontarget effects of the mosquito larvicides temephos, methoprene, and Bacillus thuringiensis var. israelensis in Florida mangrove swamps.

We compared the efficacy and nontarget effects of temephos, Bacillus thuringiensis var. israelensis (B.t.i.), and methoprene applied by helicopter to control mosquito larvae in mangrove swamps on Sanibel Island, FL, in May 1997. Three sites per treatment and 3 untreated sites were used. Temephos (Abate) was applied at 37 ml/ha (43% active ingredient [AI]), B.t.i. granules (Vectobac G) were applied at 5.606 kg/ha (200 International Toxic Units/mg), and methoprene (Altosid ALL) was applied at 213 ml/ha (5% AI). Efficacy was quantified by monitoring the survival of caged and uncaged larval Aedes taeniorhynchus. We quantified mortality of sentinel nontarget amphipods (Talitridae) at all sites, monitored the effect of temephos on flying arthropods using light traps, and collected dead insects in tarps suspended under mangroves in areas treated with either temephos or methoprene. Each pesticide showed good overall efficacy but occasional failures occurred. No detectable mortality of amphipods or flying insects attributable to pesticides was found. The inconsistent field efficacies of the pesticides indicate a need for reinspection of treated sites in this habitat.