Hr Post-treatment Research Articles

Interactive anticancer effect of nanomicellar curcumin and galbanic acid combination therapy with some common chemotherapeutics in colon carcinoma cells.

In the current investigation, we aimed to study the combined cytotoxicity of curcumin, as a nanomicellar formulation, and galbanic acid (Gal), dissolved in DMSO against the murine C26 and human Caco-2 colon carcinoma cells. Further, curcumin potential for cisplatin and doxorubicin (Dox) co-therapy was studied. The combined cytotoxic effect of these phytochemicals at varying dose ratios were examined using the MTT colorimetric assay. Moreover, the time-dependent toxicity of curcumin, cisplatin, Dox, and pegylated liposomal Dox (Doxil) was determined. The interactive anti-proliferative behavior of these compounds was examined using the CompuSyn software. Nanomicellar curcumin showed considerable cytotoxicity in C26 cells 24 hr post-treatment. Co-treatment of cells with curcumin nanomicelles: Gal had a synergistic effect in C26 (at 10:1 molar ratio), and Caco-2 (at 1:5 molar ratio) cell lines in cell cultures. Nanomicellar curcumin showed strong and mild synergistic inhibitory effects in C26 cells when co-administered with Doxil and cisplatin, respectively. Curcumin nanomicelles and Gal had a synergistic effect in C26 and Caco-2 cell lines. It is speculated that nanomicellar curcumin shows synergistic cancer cell killing if administered 24-hr post-injection of Doxil and cisplatin.

Hydroxychloroquine Sensitivity in Human and Canine Cancer Cell Line Panels Reveals Potential Genetic Signatures of Autophagy‐Dependence and Druggable Targets

BackgroundAutophagy is a lysosomal degradation pathway cells use to survive in stressed environments and has been implicated as a mechanism in cancer chemotherapy resistance. Hydroxychloroquine (HCQ) is an established autophagy inhibitor that is safe and well tolerated in humans. Subsets of cancers have also been found to be dependent on autophagy as a main means of survival even in optimal environmental conditions, and these autophagy‐dependent cancers respond favorably to autophagy inhibition alone or with combination treatments. Since subsets of the same cancer‐type are autophagy‐dependent and others are not, it is critical to understand which tumors will benefit most from autophagy inhibition in the clinic. Analyzing a large set of cell line drug responses can be beneficial in determining which tumor types will benefit from treatment with that drug or drugs with similar mechanisms of action. Further, since human and canine cancers have many commonalities including genetic similarities, comparisons between both species are important for providing new insights to cancer treatments across the oncology field. Multiple cell line data sets exist with publicly available data. The NCI60 is a panel of 60 human lines including breast, colon, lung, melanoma, ovarian, renal, prostate, leukemia, and central nervous system cancers. The Flint Animal Cancer Center (FACC) at Colorado State University has a panel of 36 canine lines with tumor types including osteosarcoma, transitional bladder cell carcinoma, melanoma, leukemia/lymphoma, mast cell, hemangiosarcoma, histiocytic sarcoma, mammary carcinoma, a soft tissue sarcoma, and a thyroid carcinoma.MethodsResponse of the canine FACC cell lines to HCQ was assessed by monitoring growth of fluorescently labeled cells. HCQ data for the human lines was obtained from the NCI60 website. A differential gene expression analysis was completed to determine genes most significantly correlated with HCQ sensitivity.ResultsHCQ Dm values (median dose of drug at which half of the cells are affected) at 96 hr in the canine cell lines ranged widely from less than 2 μM to almost 40 μM. Compared to the human cell lines whose data was collected at 48 hr using a 10 μM dose, canine cell lines appear to be more sensitive overall. However, when directly comparing just the human and canine osteosarcoma cell lines at 96 hr post treatment, they have similar differential HCQ sensitivities between the least and most affected cell lines, indicating that HCQ may have the same affect in other cancer types if the data is analyzed four days following drug dosing instead of just two days like the NCI60 data.ConclusionsThis analysis provides potential to produce a gene signature for tumors that respond favorably to HCQ treatment and may infer which tumors are more autophagy‐dependent and therefore will benefit from autophagy inhibition clinically. It also gives potential druggable gene targets for those tumors that will not benefit from autophagy inhibition (the HCQ‐resistant lines) based on which genes are upregulated in that group of tumors.Support or Funding InformationNCI R01CA190170This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice.

Nicotinamide adenine dinucleotide (NAD+ ) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD+ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD+ results in impaired mitochondrial and cellular functions. Administration of NAD+ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age-associated physiological decline, and inhibit postischemic NAD+ degradation and cellular death. In this study, we identified a novel link between NAD+ metabolism and mitochondrial dynamics. A single dose (62.5mg/kg) of NMN, administered to male mice, increases hippocampal mitochondria NAD+ pools for up to 24hr posttreatment and drives a sirtuin 3 (SIRT3)-mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species levels via SIRT3-driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 [S616]), with mitochondria. In conclusion, manipulation of mitochondrial NAD+ levels by NMN results in metabolic changes that protect mitochondria against reactive oxygen species and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.

Autophagy induction and PDGFR-\u03b2 knockdown by siRNA-encapsulated nanoparticles reduce chlamydia trachomatis infection

C. trachomatis is the most common sexually transmitted bacterial infection in the world. Although the infection can be easily controlled by the use of antibiotics, several reports of clinical isolates that are resistant to antibiotics have prompted us to search for alternative strategies to manage this disease. In this paper, we developed a nanoparticle formulation (PDGFR-β siRNA-PEI-PLGA-PEG NP) that can simultaneously induce autophagy in human cells and knock down PDGFR-β gene expression, an important surface binding protein for C. trachomatis, as a strategy to reduce vaginal infection of C. trachomatis. PDGFR-β siRNA-PEI-PLGA-PEG NP significantly induced autophagy in human vaginal epithelial cells (VK2/E6E7) 48 hr post treatment by improving autophagic degradation activity without causing inflammation, apoptosis or any decrease in cell viability. Beclin-1, VPS34 (markers for initiation stage of autophagy), UVRAG, TECPR-1 (markers for degradation stage of autophagy) were found to be significantly upregulated after treatment with PDGFR-β siRNA-PEI-PLGA-PEG NP. Furthermore, PDGFR-β siRNA-PEI-PLGA-PEG NP decreased PDGFR-β mRNA expression by 50% and protein expression by 43% in VK2/E6E7 cells 48 hr post treatment. Treatment of cells with PDGFR-β siRNA-PEI-PLGA-PEG NP significantly decreased the intracellular C. trachomatis and extracellular release of C. trachomatis by approximately 65% and 67%, respectively, in vitro through augmenting autophagic degradation pathways and reducing bacterial binding simultaneously.

Identification of a strawberry NPR-like gene involved in negative regulation of the salicylic acid-mediated defense pathway.

Hormonal modulation plays a central role in triggering various resistant responses to biotic and abiotic stresses in plants. In cultivated strawberry (Fragaria x ananassa), the salicylic acid (SA)-dependent defense pathway has been associated with resistance to Colletotrichum spp. and the other pathogens. To better understand the SA-mediated defense mechanisms in strawberry, we analyzed two strawberry cultivars treated with SA for their resistance to anthracnose and gene expression profiles at 6, 12, 24, and 48 hr post-treatment. Strawberry genes related to SA biosynthesis, perception, and signaling were identified from SA-responsive transcriptomes of the two cultivars, and the induction of 17 candidate genes upon SA treatment was confirmed by qRT-PCR. Given the pivotal role of the non-expressor of pathogenesis-related (NPR) family in controlling the SA-mediated defense signaling pathway, we then analyzed NPR orthologous genes in strawberry. From the expression profile, FaNPRL-1 [ortholog of FvNPRL-1 (gene20070 in F. vesca)] was identified as an NPR-like gene significantly induced after SA treatment in both cultivars. With a conserved BTB/POZ domain, ankyrin repeat domain, and nuclear localization signal, FvNPRL-1 was found phylogenetically closer to NPR3/NPR4 than NPR1 in Arabidopsis. Ectopic expression of FvNPRL-1 in the Arabidopsis thaliana wild type suppressed the SA-mediated PR1 expression and the resistance to Pseudomonas syringae pv. tomato DC3000. Transient expression of FvNPRL-1 fused with green fluorescent protein in Arabidopsis protoplasts showed that SA affected nuclear translocation of FvNPRL-1. FvNPRL-1 likely functions similar to Arabidopsis NPR3/NPR4 as a negative regulator of the SA-mediated defense.

Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis invitro and exvivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1hr, but is undetectable by 24hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.

Microbubble-assisted MRI-guided focused ultrasound for hyperthermia at reduced power levels

Purpose: Ultrasound contrast agent microbubbles were combined with magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) as a means to achieve mild hyperthermia at reduced power levels.Methods: MRgFUS hyperthermia (42°C for 20 min) was evaluated in rabbit thigh muscle or Vx2 tumors using infusions of microbubbles (Definity, 20 µL/kg) or saline (sham) administered over 5 min. The impact of treatments on drug uptake was assessed with liposomal doxorubicin (Caelyx, 2.5 mg/kg). Applied power levels before and after the injection of microbubbles or saline were compared, and drug uptake was evaluated with fluorometry of tissues harvested 24 hr post-treatment.Results: MRgFUS hyperthermia in muscle and tumors resulted in accurate temperature control (mean =42.0°C, root mean square error (RMSE) = 0.3°C). The power dropped significantly following the injection of microbubbles in muscle and tumors compared to exposures without microbubbles (–21.9% ± 12.5% vs –5.9% ± 7.8%, p = .009 in muscle; –33.8% ± 9.9% vs –3.0% ± 7.2%, p < .001 in tumors). Cavitation monitoring indicated emission of subharmonic, ultraharmonic, and elevated levels of fourth to sixth harmonic frequencies following microbubble injection. The drug delivery was elevated significantly in muscle with the use of microbubble-assisted relative to conventional heating (0.5 ± 0.5 ng/mg vs 0.20 ± 0.04 ng/mg, p = .05), whereas in tumors similar levels were found (11 ± 3 ng/mg vs 16 ± 4 ng/mg, p = .13).Conclusions: The finding that microbubbles reduce the applied power requirements for hyperthermia has considerable clinical implications. The elevated levels of drug found in muscle but not tumor tissue suggest a complex interplay between the heating effects of microbubbles with those of enhanced permeabilization and possible vascular damage.

Acute effects of 3,4-methylenedioxymethamphetamine (MDMA) and R(-) MDMA on actigraphy-based daytime activity and sleep parameters in rhesus monkeys.

3,4-Methylenedioxymethamphetamine (MDMA) affects monoaminergic pathways that play a critical role in sleep-wake cycles. Dopaminergic mechanisms are thought to mediate the sleep-disrupting effects of stimulant drugs. However, the mechanisms underlying the effects of MDMA on sleep-wake cycles and the effects of R(-) MDMA, a stereoisomer that lacks dopaminergic activity, on sleep remain unknown. The aim of the present study was to investigate the effects of racemic MDMA and R(-) MDMA on daytime activity and sleep-like parameters evaluated with actigraphy in adult rhesus macaques (Macaca mulatta, n = 6). Actiwatch monitors were attached to the monkeys' collars and actigraphy recording was conducted during baseline conditions and after the administration of acute intramuscular injections of saline (vehicle), racemic MDMA (0.3, 1.0, or 1.7 mg/kg), or R(-) MDMA (0.3, 1.0, or 1.7 mg/kg) at 9 or 16 h (3 h before "lights off"). Morning treatments had no effects on sleep-like parameters. Racemic MDMA decreased general daytime activity during the first hour after injection and increased daytime activity at 3 hr posttreatment. Although afternoon administration of racemic MDMA increased sleep latency, it improved other sleep parameters, decreasing wake time after sleep onset (WASO) and increasing sleep efficiency for subjects with low baseline sleep efficiency. Afternoon treatment with R(-) MDMA improved sleep measures, increasing sleep efficiency and decreasing sleep latency and WASO, while having no effects on daytime activity. The stimulant and sleep-disrupting effects of racemic MDMA are likely mediated by dopaminergic and noradrenergic mechanisms, while serotonergic pathways appear to be involved in the sleep-promoting effects of MDMA. (PsycINFO Database Record

Proteomic Changes in Chick Brain Proteome Post Treatment with Lathyrus Sativus Neurotoxin, \u03b2-N-Oxalyl-L-\u03b1,\u03b2-Diaminopropionic Acid (L-ODAP): A Better Insight to Transient Neurolathyrism

Neurolathyrism is a neurodegenerative disorder characterized by spastic paraplegia resulting from the excessive consumption of Lathyrus sativus (Grass pea). β-N-Oxalyl-L-α,β-diaminopropionic acid (L-ODAP) is the primary neurotoxic component in this pea. The present study attempted to evaluate the proteome-wide alterations in chick brain 2 hr and 4 hr post L-ODAP treatment. Proteomic analysis of chick brain homogenates revealed several proteins involved in cytoskeletal structure, signaling, cellular metabolism, free radical scavenging, oxidative stress and neurodegenerative disorders were initially up-regulated at 2 hr and later recovered to normal levels by 4 hr. Since L-ODAP mediated neurotoxicity is mainly by excitotoxicity and oxidative stress related dysfunctions, this study further evaluated the role of L-ODAP in apoptosis in vitro using human neuroblastoma cell line, IMR-32. The in vitro studies carried out at 200 μM L-ODAP for 4 hr indicate minimal intracellular ROS generation and alteration of mitochondrial membrane potential though not leading to apoptotic cell death. L-ODAP at low concentrations can be explored as a stimulator of various reactive oxygen species (ROS) mediated cell signaling pathways not detrimental to cells. Insights from our study may provide a platform to explore the beneficial side of LODAP at lower concentrations. This study is of significance especially in view of the Government of India lifting the ban on cultivation of low toxin Lathyrus varieties and consumption of this lentil.

Pathogenicity of Coagulase Negative Staphylococcus chicken isolates to 10 Days Old Broiler Chickens

One hundred and sixty, 1-day old broiler chicks were grouped into 4 equal groups, at the 10th day birds of groups 1- 3 were s.c inoculated with 0.5 ml containing y 1.5x 108 of S.xylosis, S. sciuri and S.lentus; respectively and group 4 was noninfected control. Clinical signs in infected groups started at 2-3dpi as general signs. Signs disappear in Ciprofloxacillin treated subgroups 24 hr post treatment and lasted to the 7th day in non treated. Average body weight gain in S.xylosis infected non treated was the highest (813.90 gm), followed by S. scuiri (778.50gm ) and 773.75 in S. lentus infected treated . FCR was the highest in control (1.69 treated and 1.74 non treated) followed by S. scuiri infected (1.81 non treated and 1.82 treated) and the lowest 1.94 was in S.lentus infected non treated. S. sciuri was reisolated from intestine and spleen (5th dpi) and from intestine (7 and 10 dpi). While S.lentuswas reisolated from intestine , liver and spleen (3rd dpi) ; from intestine and spleen (5th dpi) and intestine (10th dpi). Histopathological lesion was recorded in infected group as hemorrhages with sinusoidal dilation, focal areas of vacuolar degeneration , fatty degeneration and shrinkage of hepatocytes in liver, necrotic changes of lymphocytes and vacuolion of corpuscle in spleen. Leucocytic infiltration , degeneration and necrosis of epithelium surface and intraepithelial as well as submucosal leucoytic infiltration were seen in intestine. In conclusion the injected organisms induce mild subclinical disease with recording of histopathological lesions in liver, spleen and intestine. This area needs more investigation to explore pathogenicity of CoNS in chickens.

TNF-α blockade suppresses pericystic inflammation following anthelmintic treatment in porcine neurocysticercosis.

BackgroundNeurocysticercosis (NCC) is an infection of the brain with the larval cyst of the tapeworm, Taenia solium. Cysticidal treatment induces parasite killing resulting in a post inflammatory response and seizures, which generally requires corticosteroid treatment to control inflammation. The nature of this response and how to best control it is unclear. We investigated the anti-inflammatory effects of pretreatment with etanercept (ETN), an anti-tumor necrosis factor agent, or dexamethasone (DEX), a high potency corticosteroid, on the post treatment inflammatory response in naturally infected pigs with neurocysticercosis after a single dose of the cysticidal drug praziquantel (PZQ).Methodology/Principal findingsWe followed the methods from a previously developed treatment model of NCC in naturally infected swine. The four study groups of infected pigs included 3 groups treated with PZQ on day 0: PZQ-treated alone (100 mg/kg PO; n = 9), pretreated with dexamethasone (DEX, 0.2 mg/kg IM administered on days -1, +1 and +3; n = 6), and pretreated with etanercept (ETN, 25 mg IM per animal on days -7 and 0; n = 6). The fourth group remained untreated (n = 3). As measured by quantitative RT-PCR, ETN pretreatment depressed transcription of a wide range of proinflammatory, regulatory and matrix protease encoding genes at 120 hr post PZQ treatment in capsules of cysts that demonstrated extravasated Evans Blue (EB) (a measure of blood brain barrier dysfunction) compared to animals not receiving ETN. Transcription was significantly depressed for the proinflammatory genes tumor necrosis factor (TNF)-α, and interferon (IFN)-γ; the inflammation regulating genes cytotoxic T-lymphocyte-associated protein (CTLA)4, interleukin (IL)-13 and transforming growth factor (TGF)-β; the tissue remodeling genes matrix metalloprotease (MMP)1 and 9, tissue inhibitors of metalloproteases (TIMP)1 and 2, and the genes regulating endothelial function vascular endothelial growth factor (VEGF)1, angiopoietin (Ang)1, Ang 2, and platelet endothelial cell adhesion molecule (PECAM)-1. In contrast, transcription was only modestly decreased in the DEX pretreated pigs compared to PZQ alone, and only for TNF-α, IL-6, IFN-γ, TGF-β and Ang1. IL-10 was not affected by either ETN or DEX pretreatments. The degree of inflammation, assessed by semi-quantitative inflammatory scores, was modestly decreased in both ETN and DEX pretreated animals compared to PZQ treated pigs whereas cyst damage scores were moderately decreased only in cysts from DEX pretreated pigs. However, the proportion of cysts with EB extravasation was not significantly changed in ETN and DEX pretreated groups.Conclusions/SignificanceOverall, TNF-α blockade using ETN treatment modulated expression of a large variety of genes that play a role in induction and control of inflammation and structural changes. In contrast the number of inflammatory cells was only moderately decreased suggesting weaker effects on cell migration into the inflammatory capsules surrounding cysts than on release of modulatory molecules. Taken together, these data suggest that TNF-α blockade may provide a viable strategy to manage post-treatment pericystic inflammation that follows antiparasitic therapy for neurocysticercosis.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

PKM2 activation sensitizes cancer cells to growth inhibition by 2-deoxy-D-glucose.

Cancer metabolism has emerged as an increasingly attractive target for interfering with tumor growth. Small molecule activators of pyruvate kinase isozyme M2 (PKM2) suppress tumor formation but have an unknown effect on established tumors. We demonstrate that TEPP-46, a PKM2 activator, results in increased glucose consumption, providing the rationale for combining PKM2 activators with the toxic glucose analog, 2-deoxy-D-glucose (2-DG). Combination treatment resulted in reduced viability of a range of cell lines in standard cell culture conditions at concentrations of drugs that had no effect when used alone. This effect was replicated in vivo on established subcutaneous tumors. We further demonstrated the ability to detect acute metabolic differences in combination treatment using hyperpolarized magnetic resonance spectroscopy (MRS). Combination treated tumors displayed a higher pyruvate to lactate 13C-label exchange 2 hr post-treatment. This ability to assess the effect of drugs non-invasively may accelerate the implementation and clinical translation of drugs that target cancer metabolism.

Pharmaco‐parasitological evaluation of the ricobendazole plus levamisole nematodicidal combination in cattle

The goals of the current study were to evaluate the potential pharmacokinetic (PK) interactions and the clinical efficacy occurring after the subcutaneous (s.c.) administration of ricobendazole (RBZ) and levamisole (LEV) given both separately and co-administered to calves naturally infected with susceptible gastrointestinal nematodes. The clinical efficacy was shown in two seasons, winter and spring, with predominance of different nematode populations. Groups of 15 calves were treated with RBZ alone, LEV alone and RBZ+LEV combination, and an untreated group was kept as a Control. RBZ and LEV plasma concentrations were quantified by HPLC. The clinical efficacy was determined by the faecal egg count reduction test. RBZ and LEV have similar plasma persistence, being detected in plasma over 24hr post-treatment. No PK interactions were observed after the combined treatment, with similar PK parameters (p>.05) obtained for the single-drug and the combination-based strategy. In winter, the observed clinical efficacies were 96%, 99% and 100% for groups treated with RBZ, LEV and RBZ+LEV, respectively; however, in spring, the efficacies were 95%, 93% and 96% for the same groups. Remarkably, the combination was the only treatment that achieved 100% clinical efficacy against both Haemonchus spp and Ostertagia spp in winter; but the increased presence of Ostertagia spp. in spring (28% in untreated group) determined a tendency to reduced efficacies compared to winter time (only 10% of Ostertagia spp. in untreated group), even for the combined treatment. Overall, in a scenario where the nematode population is susceptible, the RBZ+LEV treatment may be a valid combination in cattle to delay the development of resistance, especially in winter when this combination achieved 100% of efficacy. Thus, selection of anthelmintic resistance will never occur. In fact, this is one of the greatest challenges for the whole cattle production system: to be one step ahead of anthelmintic resistance.

Abstract 5210: Differential cytotoxic effects of Piperlongumine analogues and their radiotherapeutic response in breast cancer

Abstract Background: Breast cancer is the most common cancer in women in the United Kingdom. The glutathione (GSH) system is a major redox buffering system involved in regulating radioresponse of cancer cells. Overexpression has been shown to cause multidrug and radiation resistance. Its modulation may increase radiosensitivity and improve radiotherapy efficacy. Piperlongumine (PL) can reportedly inhibit GSH system function and reduce proliferation of a variety cancer cells in vitro. The current study investigates novel PL analogues to determine efficacy in triple-negative (TNBC) and luminal breast cancer cell lines, as both single agents and in combination with ionising radiation. Methods: Two novel PL analogues (LH91 and LH92) were used along with the parental drug, PL. Cell proliferation assays were conducted in MDA-MB-231 (TNBC) and T47D (luminal) lines at various drug concentrations and times. At 48hr post treatment, cells were also assessed for clonogenic survival. Clonogenic radiosensitisation was assessed by treating with PL agents or L-buthionine-S, R-sulfoximine (BSO, a well characterised GSH radiosensitiser) for 48hr then irradiation with 0-8Gy X-rays. ROS levels were evaluated using H2DCFDA flow cytometry following IC50 drug treatment alone or with subsequent exposure to 1mM H2O2 for 1hr. Results: PL, LH91 and LH92 inhibited proliferation and decreased clonogenic survival in both lines. From proliferation assays, MDA-MB-231 cells were more sensitive to PL at 48hr (P=0.047) and 72hr (P=0.01) with an IC50 dose of approx. 4μM versus approx. 12μM in T47D’s. There was a significant difference between IC50 doses of LH91 in MDA-MB-231 (5μM) and T47D (12μM) cell lines at 72hr (P=0.006), however this was not observed at 48hr treatment (P=0.178). In contrast, there was no differential sensitivity of LH92 in cell proliferation between the two cell lines at 48 and 72hr. Interestingly, in clonogenic assays, MDA-MB-231’s were more sensitive to LH92 (P=0.01) with an IC50 of 4μM versus 11μM in T47D’s. There were no significant differences in clonogenic survival between each line after treatment with PL or LH91. No radiosensitisation was observed with PL or PL analogues in MDA-MBA-231 however BSO, as a drug comparator, enhanced radiation response with a sensitizer enhancement ratio (SER) of 1.13 at 1% iso-survival. Radiation and drug combinations in T47D cells are ongoing. There were no changes in ROS levels with the drug alone in MDA-MB-231; however, with subsequent exposure of H2O2, there was a 1.7-fold increase in ROS level with LH91 at 48hr (P=0.002). Conclusion: The TNBC cell line is more sensitive to the cytotoxic effect of PL agents (PL and LH91 in cell proliferation and LH92 in clonogenic assays) in comparison to the luminal cell line; however no altered radiosensitisation was observed. The mechanism of such differential sensitivity is yet to be determined. Note: This abstract was not presented at the meeting. Citation Format: Nurul Akmaryanti Abdullah, John Moses, Li Chen Han, Sarah Storr, Aula Ammar, Stewart G. Martin. Differential cytotoxic effects of Piperlongumine analogues and their radiotherapeutic response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2017-5210

Changes in Oligodendroglial and Microglial Cell Populations in the Embryonic Rat Spinal Cord Following Maternal Immune Activation

Epidemiological studies suggest that maternal immune activation (MIA) may contribute to the development of neurological disorders such as cerebral palsy, schizophrenia, autism, epilepsy and potentially, neurodegenerative diseases. These studies, and those in animal models, have suggested that the effects of MIA on the developing nervous system, and thus the longer term consequences for the offspring, may be spatially and temporally regulated. However, much work remains in order to elucidate fully the CNS regions, cell populations and timing of gestational vulnerability.The in vivo effects of MIA on cellular development in the spinal cord (SC) at embryonic day (E)12, 14 and 16 were investigated in this study. Dams received a single intraperitoneal injection of 50μg/kg lipopolysaccharide (LPS), 100 μg/kg LPS, or saline. Embryos were sacrificed either 5 hours post‐injection or, following their delivery, two weeks postnatally(P). Embryos and pups were fixed and frozen prior to cryosectioning at 15μm. Rostral, intermediate and caudal SC sections were immunofluorescently stained with Olig‐2 (an oligodendrocyte marker), Iba‐1 (a microglial marker), nestin (a radial glial marker) and reelin antisera.Olig2‐positive cells were counted in white matter (WM) and grey matter (GM) regions of the SC. Olig2‐positive cell number in E16 embryos decreased 5 hours following treatment with 100μg/kg LPS but not with treatment with 50μg/kg LPS. At E14 Olig2‐positive cells showed no change at 5 hours in response to 50μg/kg LPS treatment.Iba‐1+ cells decreased in GM and WM of the E16 SC 5Hr post treatment with 100μg/kg LPS.Reelin fluorescence intensity is decreased in the GM of the E16 SC 5 hours after 100μg/kg LPS treatment. Reelin fluorescence intensity is unaltered in E16 spinal cord in response to 50μg/kg LPS. Reelin appears to co‐localise with a specific population of neurons at E16 and P14.In conclusion, MIA appears to have an acute effect on the development of key cell populations at specific time points. Ongoing work is extending these analyses and will seek to identify how such perturbations affect ongoing SC development into the early postnatal period.Support or Funding InformationAnatomical SocietyGraduate Entry Medical School University of Limerick

Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens.

Natural or synthetic Toll-like receptor (TLR) ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide) polymer nanoparticles (PLGA NPs) was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line) were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4) induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN)-γ and IFN-β until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens) by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1β until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens.

Abstract 1997: Inhibition of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia

Abstract Splicing factor SF3B1 is frequently mutated in about 10-15% of CLL patients. SF3B1 mutations are predominately associated with faster disease progression and poor overall survival, and mutations are typically enriched in approximately 20% relapsed and chemorefractory CLL patients. Therefore, there is a growing interest in using SF3B1 as a therapeutic target for CLL. In this study, we investigated the effectiveness of Sudemycin D6 (SD6), a small molecule SF3B1 inhibitor, in reducing cell viability and promoting apoptosis in CLL cells. We then isolated RNA from both the SD6-treated and control cells at 2, 6, and 24hr post-treatment and performed RNA sequencing analysis to investigate the global impact. We demonstrated that SD6 effectively inhibits cell growth and induces apoptosis in CLL cells at a nanomolar concentration in both a time and dosage dependent manner. Furthermore, global analysis of the RNA sequencing data revealed that treatment with SD6 causes an aberrant splicing signature with significant increase in global intron retention. The increase in global intron retention peaked at 6hr then significantly diminished at 24hr post-treatment. Ontology analysis of the intron-retaining transcripts using the Ingenuity Pathway Analysis (IPA) software package suggested that B-cell receptor (BCR) signaling, protein ubiquitination, and PI3K signaling were among the top canonical pathways affected by SD6 treatment. Using RT-PCR analysis, we confirmed intron retention events in several components of the BCR pathway, such as BTK, BLNK, and PIK3CD, and an exon-skipping event in the exons encoding the kinase domain of Bruton's Tyrosine Kinase. The increase in intron retention significantly correlated with a decrease in overall mRNA and protein expression levels of the affected downstream BCR pathway proteins like AKT, PI3Kδ, and PLCγ2. Furthermore, SD6 treatment induced a time-dependent exon-skipping event in the apoptotic regulator MCL1, resulting in alternative splicing of the long, anti-apoptotic isoform of MCL1 into the short, pro-apoptotic isoform. SD6 treatment also caused downregulation of anti-apoptotic gene TRAF1 at both the mRNA and protein level, which could contribute to SD6-induced apoptosis. Cumulatively, these results strongly suggest that SF3B1 is a promising target for therapeutic treatment in CLL. Our study also provides solid rationale for future clinical development of spliceosome inhibitors and combination therapies based on spliceosome inhibitors. Citation Format: Qimei Han, Austin Young Shull, Fang Shi, Chandraiah Lagisetti, Thomas Webb, Justin Choi, Huidong Shi. Inhibition of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1997.

The Effect Of Exercise 24-hours Before Chemotherapy On Cardiac Function And Symptoms In Breast Cancer

Rodent studies have demonstrated that a single submaximal exercise bout can attenuate markers of cardiac damage caused by adriamycin chemotherapy injection 24 hr later. However, it is unclear whether this finding extends to humans. PURPOSE: To determine the effects of exercise 24 hr prior to the first chemotherapy treatment on cardiac function in breast cancer patients (BCP). METHODS: Patients were randomized to either: 1) no vigorous exercise for 72 hr prior to first treatment (n = 8), or 2) no vigorous exercise for 48 hrs, then 30 min of vigorous exercise (70% age-predicted heart rate reserve) 24 hr prior to first treatment (n = 9). Chemotherapy treatment included 60 mg/m2 adriamycin and 600 mg/m2 cyclophosphamide. Two-dimensional echocardiography and blood pressure were assessed pre- and 24-48 hr post-treatment. Key cardiac measures included speckle tracking-derived global longitudinal strain (GLS) and standard volumetric measures (e.g., end-diastolic volume (EDV)). Rotterdam Symptom Checklists were administered prior to the first and second treatments. Standardized physical and psychological distress scores were tabulated. Analysis included mixed methods ANOVA with p≤0.05. RESULTS: There were no significant interactions or group differences for GLS, EDV, systolic blood pressure, or resting heart rate. There were significant (p≤0.05) main effects for time (pre vs. post treatment) for GLS (-19.3 ± 1.5 to -21.2 ± 1.8 %), systolic blood pressure (107 ± 14 to 102 ± 14 mmHg) and a trend for EDV (80 ± 13 to 88 ± 16 mL, p=0.06). There was no significant change in heart rate (70 ± 10 to 68 ± 11 bpm). Physical distress increased post-treatment (14 ± 8 to 26 ± 11, p<0.01), while there was a trend toward a decrease in psychological distress (26 ± 13 to 18 ± 17, p=0.07). CONCLUSIONS: Within this initial small sample of BCP, there does not seem to be an effect of aerobic exercise 24 hr prior to receipt of the first adriamyacin chemotherapy treatment on the cardiac function parameters or symptoms measured. The acute effect of chemotherapy is an increase in GLS; and the trend in EDV may be suggestive of an increase in blood volume, but this requires further investigation. Receipt of the first treatment significantly increases physical symptoms, but may decrease the psychological distress associated with anticipation of the first treatment.

Proteomic analysis of Pteropus alecto kidney cells in response to the viral mimic, Poly I:C.

BackgroundBats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses. While many of these viruses cause severe and often fatal disease in humans, bats are able to coexist with these viruses without clinical signs of disease. The mechanism conferring this antiviral response is not fully understood. Here, we investigated the differential protein expression of immortalised Pteropus alecto kidney cells (PaKiT03) following transfection with the viral mimic, Poly I:C. Two complementary proteomic approaches, difference gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ) were used to quantify changes in protein expression following Poly I:C stimulation at 4, 8 and 20 hr post treatment (hpt).ResultsThe expression of ISG54 gene, a known responder to virus infection and Poly I:C treatment, was significantly induced in transfected cells compared with mock-transfected cells. Through iTRAQ analysis we show that Poly I:C up-regulates key glycolytic enzymes at 4 hpt within PaKiT03 cells. In contrast, at 20 hpt PaKiT03 cells down-regulated ribosomal subunit proteins. The analysis with DIGE of Poly I:C transfected PaKiT03 cells showed over 215 individual spots differentially regulated, however only 25 spots could be unambiguously identified by LC-MS/MS. Immunoblotting confirmed the up-regulation of Eno1 and Tpi1 in PaKiT03 cells following Poly I:C transfection. A comparison with human cells (HEK293T and HeLa) and one additional bat cell line (PaLuT02), demonstrated that glycolytic pathways are also induced in these cell types, but at different intensities.ConclusionThe two techniques, DIGE and iTRAQ identified largely overlapping sets of differentially expressed proteins, however DIGE unambiguously identified significantly less proteins than iTRAQ. Poly I:C induced a rapid metabolic shift towards glycolysis within the PaKiT03 cells at 4 hpt, presumably as a consequence of increased energy requirements. On the other hand ribosomal subunit proteins were seen as down-regulated by iTRAQ, these proteins may be the limiting factors in the translational machinery available for virus replication. This study provides new insight into the antiviral response of bat cells, highlighting the importance of energy metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0081-6) contains supplementary material, which is available to authorized users.