48h Post-treatment Research Articles

Comparative effect of ivermectin and amitraz on cellular architecture of ovaries, synganglion and Gené's organ of Rhipicephalus microplus (Acari: Ixodidae).

The present study investigated the effect of ivermectin and amitraz on the cellular architecture of vital organs of Rhipicephalus microplus. Adult female ticks were treated with lethal concentrations (LC95) of ivermectin and amitraz, and the ovaries, synganglion, and Gené's organ were processed 48h post treatment. In both the treatment groups, the ultra-thin sections of ovary exhibited deformed oocytes, irregular plasmic membrane and chorion layer, extensive vacuolation in the cytoplasm mainly at periphery of the cell and oocyte-pedicel junction. Marked vacuolations in the cortex and neuropile region with significant structural disorganization of the neural fibers were common alterations observed in the synganglion of ticks exposed to ivermectin and amitraz. The tissue sections of Gené's organ revealed deformed tubular glands with severe loss of cellular limit of secretory epithelium and cytoplasmic vacuolations in the ivermectin treated ticks whereas, the alterations were comparatively less severe in amitraz exposed ticks. The cellular deformities in these vital organs probably impaired reproductive function, nerve signal transmission and metabolic activities and thus affected fecundity and survivability of the treated ticks. The findings suggested that the action of ivermectin and amitraz are not restricted to the nervous system of ticks, but also on other vital organs, ovary and Gené's organ affecting the oviposition. The study provided insights into the development of targeted interventions for tick control strategies.

Understanding the mechanisms of action of atmospheric cold plasma towards the mitigation of the stress induced in molds: The case of Aspergillus chevalieri

Aspergillus chevalieri is a xerophilic/xerotolerant fungi affecting dried food products. In this study the ability of non-thermal cold atmospheric plasma (CAP) at high power density (NOx) to affect biological process inducing the stress responses of A. chevalieri species exposed for 5 min (5’CAP-NOx) and 30 min (30’CAP-NOx) were analysed at 0, 1, 6, 12 and 48-h post treatment (hpt). At 48 hpt with 30’CAP-NOx, 84% of fungal growth reduction was observed. The membrane integrity estimated by confocal investigation after carboxyfluorescein diacetate/propidium iodide staining showed the dead surface mycelium layers exposed to the treatments. Reverse transcription-quantitative real-time PCR revealed an early downregulation, at 0 hpt, followed by upregulation or recovery starting to 1 hpt, of selected key genes involved in fungal stress responses. The cellular response to stress was confirmed by mycelial glutathione accumulation in the early phase after both CAP-NOx treatments, at 0 and 1 hpt, followed by the strong glutathione reduction at 12 and 48 hpt using 30’CAP-NOx treatment. The ability of A. chevalieri to modulate metabolic profile according to treatments was underlined by volatilome investigation, which mainly involved lipid metabolism. This work highlighted the adaptative response mechanisms of A. chevalieri to overcome the CAP-NOx treatment. Industrial relevanceThe application of cold atmospheric plasma (CAP) technology to avoid microbial growth in foods is considered of high interest. Utilization of CAP technology, a nonthermal technique, is encouraged because of its efficiency in maintaining natural aroma and flavor and product shelf-life. Regarding the management of Aspergillus chevalieri, the developments in mechanistic insights indicated that cold plasma affected several targets in fungal cells and was a successful tactic when employed to stop the selection of resistant fungal strains.

CTNI-02. PRECLINICAL DATA AND INITIAL CLINICAL EXPERIENCE IN THE PHASE 1/2A DOSE ESCALATION TRIAL OF RHENIUM-186 NANOLIPOSOME (186RNL) IN LEPTOMENINGEAL METASTASES [LM]: THE RESPECT-LM TRIAL

Abstract INTRODUCTION Leptomeningeal metastases (LM) is a clinical complication that occurs when cancer cells invade the leptomeninges and cerebrospinal fluid of patients with malignant tumors. Once diagnosed, limited treatment options exist, and survival is poor. Rhenium-186 Nanoliposome (186RNL) is a liposomal encapsulated beta emitter with a short path length of 1.8 mm, thereby allowing high specific activity brachytherapy with limited exposure to surrounding tissues. METHODS Preclinical efficacy was assessed using C6-Luc glioma cells in Wistar rats or MDA-MB-231 in Athymic Nude Rats via intraventricular injection. At 15 days post inoculation the animals were treated with 0.69 mCi of 186RNL. Absorbed doses were assessed with gamma camera imaging at 0h, 24h, and 48h post-treatment. Tumor growth was assessed by luciferase bioluminescence. A multicenter phase 1/2a dose (3 + 3 design) escalation trial was then initiated with 186RNL in LM patients to determine the maximum feasible dose, overall response rate (ORR) and overall survival distribution. RESULTS A significant difference in survival between the control and treatment groups (n = 8 each) was observed in the C6/Wistar model at 2 weeks post treatment, with 50% survival in the control group and 100% survival in the treatment group (p = 0.0087). Similar efficacy was observed in the MDA-MB-236/Athymic model. Thus far two patients have received 186RNL with 6.6 mCi in 5ml via Ommaya reservoir. Post treatment imaging shows full CSF distribution by 4 hours and persistence of activity through 7 days following administration. The dose was well-tolerated with no related AEs through day 56 following treatment. CSF cell count by BioCept analysis was reduced by > 90% with durability through day 28. CONCLUSION Intraventricular delivered 186RNL is promising for treatment of LM, with very early evidence of efficacy in patients. Enrollment is ongoing.

Non-invasive neuromodulation in the acute treatment of migraine: a systematic review and meta-analysis of randomized controlled trials.

A systematic review and meta-analysis was performed to determine the efficacy of non-invasive neuromodulation modalities for the treatment of acute migraine. Although pharmacological treatments are the gold standard for the management of acute migraine, some patients may require non-pharmacological treatment options. Non-invasive neuromodulation may provide an alternative, and techniques include transcranial magnetic stimulation (TMS), non-invasive vagal nerve stimulation (nVNS), non-painful remote electrical stimulation (NRES), and external trigeminal nerve stimulation (e-TNS). This systematic review and meta-analysis was performed following PRISMA guidelines. We searched PUBMED, EMBASE, ClinicalTrials.gov, Cochrane Center Register of Controlled Trials, and LILACS databases. We included randomized controlled clinical trials studying patients with migraine treated with any form of non-invasive neuromodulation. Primary outcome was pain freedom within 2h post-treatment. Secondary outcomes were pain relief within 2-h post-treatment and sustained pain freedom and sustained pain relief 48h post-treatment. Pooled analysis demonstrated a significant effect of non-invasive neuromodulation on pain-free rates within 2h (RR, 1.66; 95% CI, 1.35 to 2.05; P < 0.00001) and pain relief rates within 2h (RR, 1.52; 95% CI, 1.13 to 2.05; P = 0.005) post-treatment. Non-invasive neuromodulation had no significant effect on sustained pain freedom at 48h (RR, 1.56; 95% CI, 0.68 to 3.59; P = 0.29) or sustained pain relief at 48h (RR, 1.47; 95% CI, 0.57 to 3.77; P = 0.43) after administration. Neuromodulation has demonstrated some efficacy in acute migraine management and may be considered in the treatment paradigm of acute migraine in patients with contraindications to pharmacological therapies.

LB733 Effect of vitamin C on gene transcription in ex-vivo skin model

Ascorbic acid (Vitamin C) is a potent antioxidant and is also suggested to affect genome activity via modifiable epigenomic processes as an enhancer of demethylases, consequently spatially and temporally altering gene expression. Utilizing ex-vivo skin models and standard RNA sequencing (NOVASEQTM/HISEQ), changes in gene expression within 24 h and 48h posttreatment were observed. Single, topical application of 10% buffered ascorbic acid (AA) altered skin’s transcriptional profile at both 24 and 48 hours post-treatment, with a total of 229 and 280 affected gene transcripts, respectively.

LMD-14. Preclinical safety and activity of intraventricular Rhenium-186 Nanoliposome (186RNL) for leptomeningeal metastases

IntroductionLeptomeningeal metastases (LM) is a clinical complication that occurs when cancer cells invade the leptomeninges and cerebrospinal fluid of patients with malignant tumors. Once diagnosed, limited treatment options exist, and survival is poor. Rhenium-186 Nanoliposome (186RNL) is a liposomal encapsulated beta emitter with a short path length of 1.8 mm, thereby allowing high specific activity brachytherapy with limited exposure to surrounding tissues.MethodsTo establish the maximum tolerated dose (MTD) of 186RNL by intraventricular (IT) injection, eight cohorts of Wistar rats (n=3 each) were injected IT with increasing activity of 186RNL at doses of 0 (control), 0.480, 0.800, 1.000, 1.150, and 1.340 mCi. Toxicity was assessed by daily food and water intake, daily weights, and observing for neurological deficits. To assess efficacy, C6-Luc glioma cells were injected IT and 15 days post inoculation the animals were treated with 0.69 mCi of 186RNL. Absorbed doses were assessed with gamma camera imaging at 0h, 24h, and 48h post-treatment. Tumor growth was assessed by luciferase bioluminescence.ResultsNo evidence of adverse 186RNL-related effects was observed in rats through 3 months following administration of up to 1.34 mCi with an absorbed dose of up to 1075 Gy. Hence, the MTD exceeded the doses evaluated in this study. A significant difference in survival between the control and treatment groups (n=8 each) was observed at 2 weeks post treatment, with 50% survival in the control group and 100% survival in the treatment group (p=0.0087). The only significant treatment-related histologic finding among treated rats was slight focal thickening of the leptomeninges, suggesting a mild reactive hypertrophy.ConclusionIntraventricular delivery of 186RNL is well tolerated and improves animal survival at 2 weeks in a rat model of LM.

Cephalic Neuronal Vesicle Formation is Developmentally Dependent and Modified by Methylmercury and sti-1 in Caenorhabditis elegans.

Methylmercury (MeHg) is a potent neurotoxicant. The mechanisms underlying MeHg-induced neurotoxicity are not fully understood. Several studies have shown that protein chaperones are involved in MeHg toxicity. The protein co-chaperone, stress inducible protein 1 (STI-1), has important functions in protein quality control of the chaperone pathway. In the current study, dopaminergic (DAergic) cephalic (CEP) neuronal morphology was evaluated in the Caenorhabditis elegans (C. elegans) sti-1 knockout strain. In the control OH7193 strain (dat-1::mCherry + ttx-3::mCherry), we characterized the morphology of CEP neurons by checking the presence of attached vesicles and unattached vesicles to the CEP dendrites. We showed that the attached vesicles were only present in adult stage worms; whereas they were absent in the younger L3 stage worms. In the sti-1 knockout strain, MeHg treatment significantly altered the structures of CEP dendrites with discontinuation of mCherry fluorescence and shrinkage of CEP soma, as compared to the control. 12 h post treatment on MeHg-free OP50-seeded plates, the discontinuation of mCherry fluorescence of CEP dendrites in worms treated with 0.05 or 0.5µM MeHg returned to levels statistically indistinguishable from control, while in worms treated with 5µM MeHg a higher percentage of discontinuation of mCherry fluorescence persisted. Despite this strong effect by 5µM MeHg, CEP attached vesicles were increased upon 0.05 or 0.5µM MeHg treatment, yet unaffected by 5µM MeHg. The CEP attached vesicles of sti-1 knockout strain were significantly increased shortly after MeHg treatment, but were unaffected 48h post treatment. In addition, there was a significant interactive effect of MeHg and sti-1 on the number of attached vesicles. Knock down sti-1 via RNAi did not alter the number of CEP attached vesicles. Taking together, our data suggests that the increased occurrence of attached vesicles in adult stage worms could initiate a substantial loss of membrane components of CEP dendrites following release of vesicles, leading to the discontinuation of mCherry fluorescence, and the formation of CEP attached vesicles could be regulated by sti-1 to remove cellular debris for detoxification.

Low Intensity Focused Ultrasound Modulation of Vincristine Induced Neuropathy

Previously, we showed internal low intensity focused ultrasound (liFUS) improves nociceptive thresholds in rats with vincristine-induced neuropathy (VIN) for 48-h post-treatment. Here, we perform more rigorous behavioral testing with the internal device and introduce external liFUS treatment. Behavioral testing confirmed VIN (Von Frey fibers, VFF; hot plate, HPT; locomotion, OFT). This was followed by internal or external liFUS treatment (2.5 W or 8 W, for 3 min, respectively) to the left L5 dorsal root ganglia (DRG). A thermocouple placed at the DRG documented temperature changes during treatment, to confirm the modulatory nature of our treatment. Behavioral testing was performed pre-liFUS, and for five consecutive days post-liFUS. Groups included: (1) VIN/liFUS, (2) saline/liFUS, (3) VIN/sham liFUS, and (4) saline/sham liFUS. Significant improvements in mechanical (VFF) and thermal (HPT) nociceptive thresholds were seen in the VIN/liFUS group following both internal and external treatment. Hematoxylin and Eosin, and Fluorojade staining showed no histological damage to the DRG. Internal liFUS treatment produced a mean temperature rise of 3.21 ± 0.30 °C, whereas external liFUS resulted in a mean temperature rise of 1.78 °C ± 0.21 °C. We demonstrate that, in a VIN rat model, external liFUS treatment of the L5 DRG significantly reduces nociceptive sensitivity thresholds without causing tissue damage.

Chemical profile, characterization and acaricidal activity of essential oils of three plant species and their nanoemulsions against Tyrophagus putrescentiae, a stored-food mite.

Essential oils of Ocimum basilicum (L.), Achillea fragrantissima (Forssk.) and Achillea santolina (L.) were obtained by hydrodistillation and analyzed using gas chromatography (GC) and GC/mass spectrometry (MS). Oil-in-water nanoemulsions (10% active ingredient) were prepared through a high-energy (ultrasonication) emulsification process. Nanoemulsions were characterized by viscosity, pH, thermodynamic stability, droplet size, polydispersity index (PDI) and scanning electron microscopy (SEM) measurements. The plant oils and their nanoemulsions showed considerable acaricidal activity against the mold mite, Tyrophagus putrescentiae (Schrank) (Sarcoptiformes: Acaridae). In a contact toxicity bioassay and 48h post treatment, O. basilicum oil was the most toxic, followed by A. fragrantissima and A. santolina, where LC50 values were 8.4, 14.1 and 21.8µl/cm2, respectively. LC50 for benzyl benzoate, a standard acaricide was 9.8µl/cm2. Upon fumigation, responses also varied according to the test oil. Based on the 48-h LC50 values, the same manner of activity was also observed, where O. basilicum was the most toxic followed by A. fragrantissima and A. santolina. When prepared as nanoemulsions (particle size from 78.5 to 104.6) and tested as fumigants, toxicity of the oils was increased drastically with LC50 values of 2.2, 4.7, and 9.6µl/l air for O. basilicum, A. fragrantissima and A. santolina, respectively. The oils showed a moderate to strong residual acaricidal activity, where O. basilicum oil was the most effective. The results suggest that appropriate nanoemulsions containing the tested oils can be developed to control T. putrescentiae after the required toxicological assessments.

Effect of Platelet-rich Plasma on Implant Bone Defects in Rabbits Through the FAK/PI3K/AKT Signaling Pathway.

Platelet-rich plasma (PRP) has been shown to be a beneficial growth factor for bone tissue healing and is used in implantology. The aim of this study was to investigate the effects of PRP on bone defects in rabbits. Twenty rabbits were used to establish the implant bone defect model in this study. An intrabony defect (5mm × 5mm × 3mm) was created in alveolar bone in the lower jar of each rabbit. The wound was treated with PRP. The expression of platelet-derived growth factor BB (PDGFBB) was assessed by enzyme-linked immunosorbent assay (ELISA). Focal adhesion kinase (FAK) and related phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) levels were measured by Western blot. The results show that PRP could significantly improve the bone healing process when compared with control, and 10% PRP could markedly increase fibroblast proliferation 48-h post treatment. PDGFBB was higher in the PRP group than that in the control group. PRP treatment also could elevate the phosphorylation of FAK and PI3K/AKT, although the inhibitor of PDGFR could reverse this trend. These results suggest that PRP treatment improves the bone healing process through the FAK/PI3K/AKT pathway.

MECHANISM OF HOUSE FLY RESISTANCE: I- Toxicity of Cypermethrin, Imidacoloprid and Spinosad to Larvae and Adults of the Laboratory Strain

The house fly, Musca domestica L., is a vector for more than100 human and animal diseases and has the ability to developeresistance to different insecticides. This study evaluated toxicity ofCypermethrin, Imidacloprid and Spinosad, in addition to, their mixturesunder laboratory conditions against larvae and adults of M. domesticafor laboratory strain. The LC50s of the three tested insecticides were579.30, 415.46 and 16.32ppm, respectively, against larvae 48h posttreatment. The toxic effect of these insecticides against M. domesticaadults was assessed 24h post treatment. The LC50s of Cypermethrin,Imidacloprid and Spinosad were 208.75, 238.3 and 86.05 ppm,respectively, against the laboratory strain. This study also showedefficacy of the binary mixtures of these insecticides, where the mixtureof Cypermethrin+Imidacloprid and Cypermethrin+Spinosad showedpotentiation at ratios 1:1, 1:2 and 2:1. On other hand, the mixture ofImidaclporid+Spinosad showed an additive effect at all mixing ratios.

Modulating Survivin as a Radioresistant Factor, Caspase-3, and Apoptosis by Omega-3 Docosahexaenoic Acid Sensitizes Mutant-p53 Colorectal Cancer Cells to γ-Irradiation.

Mutant-p53 colorectal cancer (CRC) cells are often resistant to radiotherapy. Loss of suppressor activity of wt-p53 on survivin is responsible for the enhanced expression of survivin as a radioresistant factor in tumor cells. Yet, no survivin-modulating drug has been approved for clinical application in CRC. Thus, the search for safe compounds that modulate survivin expression and induce apoptosis irrespective of p53 status may potentiate the efficacy of radiotherapy in mutant-p53 CRC cells. Omega-3 docosahexaenoic acid (DHA) induces apoptosis in malignant cells without cytotoxicity in normal cells. However, little is known whether in vitro concentrations of DHA equal to the human plasma levels are able to modulate expression of survivin and sensitize mutant-p53 CRC cells to γ-irradiation. Radioresistant mutant-p53 HT-29 cells were pretreated with 50- and 100-μM DHA for 48-h before 2-, 4-, 6-, 8-, and 10-Gy of γ-irradiation. Thereafter, proliferation rates were measured after 6 d. HT-29 cells were also pretreated with 50- and 100-μM DHA for 4-h before 2- and 10-Gy of γ-irradiation after which, cell number, survivin expression, caspase-3 activation, apoptosis, and ED50 (γ-irradiation dose causing 50% growth inhibition) were evaluated. Pretreatment of HT-29 cells with 50- and 100-μM DHA for 48-h followed by 2- to 10-Gy of γ-irradiation induced a dose-dependent additive decrease in cell proliferation rate and ED50 values were decreased by 88%, 44%, 41%, and 27% for 500-, 1500-, 2500-, and 5000 cells per well pretreated with 100-μM DHA respectively. Pretreatment of 5 × 105 HT-29 cells per well with 100-μM DHA for 4-h followed by 2- or 10-Gy of irradiation resulted in 53% and 86% decreases in cell numbers, 2- and 5.1-fold activation in caspase-3 followed by 66% and 60% decreases in survivin mRNA levels respectively. DHA in combination with radiation increased total apoptotic rate 48-h post-treatment. DHA decreases survivin expression and induces caspase-3 activation irrespective of p53 status. Significant decreases in ED50 values at concentrations of DHA equal to human plasma levels, suggesting that DHA could be used as an attractive radiosensitizer agent in CRC patients with mutant-p53.

Extensive preclinical investigation of polymersomal formulation of doxorubicin versus Doxil-mimic formulation

Due to the severe cardiotoxicity of doxorubicin, its usage is limited. This shortcoming could be overcome by modifying pharmacokinetics of the drugs via preparation of various nanoplatforms.Doxil, a well-known FDA-approved nanoplatform of doxorubicin as antineoplastic agent, is frequently used in clinics in order to reduce cardiotoxicity of doxorubicin. Since Doxil shows some shortcomings in clinics including hand and food syndrome and very slow release pattern thus, there is a demand for the development and preparation of new doxorubicin nanoformulation with fewer side effects.The new formulation of the doxorubicin, synthesized previously by our group was extensively examined in the current study. This new formulation is doxorubicin encapsulated in PEG-PLGA polymersomes (PolyDOX). The main aim of the study was to compare the distribution and treatment efficacy of a new doxorubicin-polymersomal formulation (PolyDOX) with regular liposomal formulation (Doxil-mimic) in murine colon adenocarcinoma model. Additionally, the pathological, hematological changes, pharmacodynamics, biodistribution, tolerated dose and survival rate in vivo were evaluated and compared.Murine colon cancer model was induced by subcutaneous inoculation of BALB/c mice with C26 cells. Afterwards, either Doxil-mimic or PolyDOX was administered intravenously.The obtained results from biodistribution study showed a remarkable difference in the distribution of drugs in murine organs. In this regard, Doxil-mimic exhibited prolonged (48h) presence within liver tissues while PolyDOX preferentially accumulate in tumor and the presence in liver 48h post-treatment was significantly lower than that of Doxil-mimic.Obtained results demonstrated comparable final length of life for mice receiving either Doxil-mimic or PolyDOX formulations whereas tolerated dose of mice receiving Doxil-mimic was remarkably higher than those receiving PolyDOX.Therapeutic efficacy of formulation in term of tumor growth rate after one injection of formulations (5mg/kg, 10mg/kg or 15mg/kg) demonstrated better efficacy at lower dose for PolyDOX.Analysis of Kaplan Meier curve was in favor of both formulations in their treatment–dose. Pathological and hematological surveys of mice treated with both formulations did not show considerable difference except for a small atrophy in liver observed after successive administration of Doxil-mimic.It could be concluded that PolyDOX can potentially limit off-site effects of Doxil due to its biodegradability and sustained release properties while it exhibited favorable safety profile comparable to Doxil.

Lupeol evokes anticancer effects in oral squamous cell carcinoma by inhibiting oncogenic EGFR pathway

Epidermal growth factor receptor (EGFR) pathway is overexpressed in head and neck cancer (HNC). Lupeol, a natural triterpene (phytosterol found in fruits, vegetables, etc.), has been reported to be effective against multiple cancer indications. Here we investigate the antitumor effects of Lupeol and underlying mechanism in oral cancer. Lupeol-induced antitumor response was evaluated in two oral squamous cell carcinoma (OSCC) cell lines (UPCI:SCC131 and UPCI:SCC084) by viability (MTT), proliferation, and colony formation assays. Lupeol-mediated induction of apoptosis was examined by caspase 3/7 assay and flow cytometry. Effect of Lupeol on EGFR in the presence or absence of EGF was delineated by Western blot. The mRNA stability assay was performed to check the role of Lupeol on COX-2 mRNA regulation. Lupeol inhibited proliferation of OSCC cells in vitro by inducing apoptosis 48h post treatment. Ligand-induced phosphorylation of EGFR and subsequent activation of its downstream molecules such as protein kinase B (PKB or AKT), I kappa B (IκB), and nuclear factor kappa B (NF-κB) was also found to be, in part, suppressed. Interestingly, Lupeol suppressed expression of COX-2 at mRNA and protein level in a time-dependent manner. Primary explants from oral squamous cell carcinoma tissues further confirmed significant inhibition of proliferation (Ki67) in Lupeol-treated explants as compared to untreated control at 48h. Together these data suggest that Lupeol may act as a potent inhibitor of the EGFR signaling in OSCC and therefore imply its role in triggering antitumor efficacy.

Abstract B22: Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine

Abstract Erufosine is a third generation alkylphosphocholine, which has been shown to inhibit the proliferation of oral squamous cell carcinoma (OSCC) cells. It simultaneously induces apoptosis and autophagy by modulating the Akt–mTOR signaling pathway and is known to down regulate the expression of cell cycle related proteins, but the exact mechanism of action of this membrane seeking compound is still unknown. For investigating the mechanism of action in a systematic way, we studied the effects of erufosine in a panel of OSCC cell lines (Cal-27, FaDu and HN-5). The concentration effecting the 50% inhibition in cell proliferation (IC50) in response to erufosine was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using concentrations ranging from 0.6µM – 100µM at various time points (24h, 48h and 72h) after exposure. Based on these results, FaDu cells were exposed to erufosine at concentrations effecting IC25, IC50 and IC75, and then the mRNA of respectively treated cells was isolated and subjected to microarray analysis by Illumina Chip array. Selected genes with highly modulated mRNA expression were further investigated by quantitative RT-PCR and Western Blot. IC50 values in response to erufosine, following exposure for 24h, 48h and 72h exposure ranged from 20µM to 9.7µM for Cal-27 -, 27µM to 14µM for FaDu - , and 43µM to 37µM for HN-5 cells. Analysis of microarray data by the Ingenuity Pathways Analysis (IPA; Ingenuity® Systems, www.ingenuity.com) software revealed that 725 genes were modulated at 16h, averaged for all concentrations, with 47 genes being modulated more than 3 fold. The respective 24h gene expression analysis revealed that 1215 genes were significantly modulated, with 77 genes deregulated over 3 fold, whereas a total of 1305 genes were altered at the end of 48h, with 93 genes being deregulated over 3 fold. In a first approach, we concentrated on genes related to the c-MYC signaling pathway. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) was found to be upregulated 9.2, 7.9 and 9.9 fold, averaged for all concentrations, at 16h, 24h and 48h, respectively. The Max dimerization protein 1 (MDX1) was upregulated 3.7 fold and 3.3 fold over the untreated control at 24h and 48h post treatment. The combined modulation of the two mRNA species hints at the involvement of c-MYC as a target of erufosine treatment. These results were further validated by qRT-PCR, which also confirmed the modulation of these genes across the OSCC cell panel. In line with this, the Western blots also showed a clear decrease in c-MYC protein levels and a corresponding increase in p21 in a concentration- and time-dependent manner. In summary, the known effect of erufosine on cell cycle related proteins as well as the increased mRNA (p21, MDX-1) and protein (p21) expression reported here is in accordance with the decreased expression of c-MYC at protein level. These findings support the hypothesis that c-MYC is an indirect target of erufosine in OSCC cells. Citation Format: Shariq S. Ansari, Martin R. Berger. Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B22.

O12. Analysis of the molecular and cellular mechanisms regulated by magnesium sulphate in an in vitro model of the human placenta

Introduction Pre-eclampsia is a multi-systemic pregnancy disorder and characterised as a hypertensive condition which consists of symptoms such as high blood pressure and proteinuria in women in their second trimester (around 20weeks gestation). It globally affects 2–8% of all pregnancies and remains the leading pregnancy disorder. Magnesium sulphate (MgSO 4 ) is commonly administered for the prevention and treatment of life-threatening seizures in women with preeclampsia and fetal neuroprotection before anticipated preterm birth. Despite this the mechanisms of action of MgSO 4 are poorly understood, partly due to a lack of good cellular models. We have developed an in vitro placental model to investigate the effects of MgSO 4 on (1) cytotrophoblast differentiation (syncytialisation) and (2) on preventing placental inflammation. Objectives To investigate the effects of MgSO 4 on (1) cytotrophoblast differentiation (syncytialisation) and (2) on preventing placental inflammation. Methods Cytotrophoblast viability was examined at 24h and 48h post treatment with Lipopolysaccharide (LPS) (10–1000ng/ml) and MgSO 4 (100nM–1mM) by MTT assay. Pyknotic nuclei morphology was identified by immunofluorescence. Syncytial formation was assessed by β -HCG production via ELISA methodology and assessed by desmoplakin architectural staining of cells treated with MgSO 4 (1mM), LPS (200ng) and Forksolin (25 μ M) for 72h. Quantitative real-time PCR was used to evaluated ACVR2A mRNA and TNF- α mRNA expression. Results MgSO 4 has a dose dependent effect on cytotrophoblast viability. BeWo cells treated with 200ng/ml LPS had a statistically significant effect on cell viability (30% viable cells) after 48h. 1mM MgSO 4 pre-treatment of cells resulted in an increase in viability to 57% compared to controls. Similarly, 1mM MgSO 4 pre-treatment reduced development of pyknotic nuclei compared to controls. MgSO 4 reduced the mRNA expression of ACVR2A and TNF- α . Finally MgSO 4 may have the potential to alter cytotrophoblast differentiation. Conclusion MgSO 4 provides protection against LPS-induced inflammation by modulating syncytialisation and reducing pro-inflammatory cytokine expression in vitro .

In vitro and in planta fungicide properties of ozonated water against the esca-associated fungus Phaeoacremonium aleophilum

Ozone could be used as a sanitary agent in the food and agricultural industries. The present study investigates whether ozonated water could be used to control conidia dispersal of the esca-associated fungus Phaeoacremonium aleophilum. The fungicide properties of ozonated water was firstly assessed in vitro by exposing spores to several concentrations of ozonated water (2.2, 4.5, or 13.5g/m3 of ozone dissolved into water) and observing the germination rate on plate. Secondarily an in planta approach was conducted on grapevine cuttings of cv. Cabernet Sauvignon clone 15. P. aleophilum conidia were inoculated in pruning wounds, which then received ozonated (4.5g/m3) or sterile water. P. aleophilum DNA was quantified by a quantitative polymerase chain reaction (qPCR) 4 and 9 weeks post-inoculation. The effect of ozonated water on plant-defense gene expression was monitored by reverse-transcriptase qPCR (RT-qPCR) 48h post treatment. The results indicate that ozonated water totally suppresses spore germination in vitro. In addition, at 9 weeks post-inoculation, fungal development was significantly reduced by 50% in planta. RT-qPCR analysis shows that ozonated water did not induce plant-defense-related genes 48h post treatment. The fungicide properties of ozonated water and the absence of gene induction in planta make however ozonated water a promising candidate for limiting grapevine infection by P. aleophilum in nurseries.

Activity of Andrographolide and Its Derivatives on HPV16 Pseudovirus Infection and Viral Oncogene Expression in Cervical Carcinoma Cells

Andrographolide (Androg) has been reported to contain antiviral and antitumor activities, but the effects of Androg on human papillomavirus (HPV) infection and cervical cancer have not been elucidated. This study investigated the effects of Androg and its derivatives, namely, 14-deoxy-11,12-didehydroandrographolide (14-DDA) and 3,19-isopropylidene andrographolide (IPAD), on HPV16 pseudovirus (HPV16PsV) infectivity, HPV16 E6 oncogene expression and cervical cancer cell apoptosis. The result demonstrated that all compounds inhibited HPV16PsV infection and that 14-DDA showed the highest potency. Only Androg suppressed long control region (LCR) transcription activity of HPV16 in transiently transfected C33A cells and significantly inhibited E6 oncogene expression in SiHa cells in a dose-dependent manner. A twofold subcytotoxic concentration of IPAD exhibited an inhibitory effect on E6 oncogene expression at 48-h posttreatment. Interestingly, p53 protein was restored in a downstream process and was detected earlier by IPAD treatment than by Androg treatment. This result corresponded to the level of cell apoptosis and cell cycle arrest at the G2/M phase. E6 oncogene expression was also suppressed in CaSki cells treated with Androg and IPAD leading to cell apoptosis. These findings imply that Androg and its derivatives have different activities and may be effective agents for HPV prevention and cervical cancer treatment.

Insecticidal alkanes from Bauhinia scandens var. horsfieldii against Plutella xylostella L. (Lepidoptera: Plutellidae)

The insecticidal activity of various extracts prepared from the dried stem of Bauhinia scandens var. horsfieldii was determined against Plutella xytostella second instars via topical application. The best activity was observed in case of dichloromethane extracts with an LD50 of 2.76 and 2.15μg/larva after 24 and 48h, respectively. Further processing of this extract provided a mixture of long chain alkanes as active material exhibiting a LD50 of 2.81μg/larva, 48h post-treatment. Further screening of this mixture showed that heptacosane and hexacosane were the major compounds and were toxic to P. xylostella larvae with respective LD50 of 1.58 and 2.58μg/larva after 48h of treatment. This suggested that both compounds have additive effect in the alkane mixture. Combined evaluation of heptacosane and hexacosane proved that they were additive in activity when the two compounds were mixed in a natural ratio in which they occur in the alkane mixture. The impact of these compounds on detoxification enzymes was determined to show the possibility of resistance development against these compounds.

Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke.