Significant Reduction In Viability Research Articles

Label-Free Monitoring of Uptake and Toxicity of Endoprosthetic Wear Particles in Human Cell Cultures.

The evaluation of the biological effects of endoprosthetic wear particles on cells in vitro relies on a variety of test assays. However, most of these methods are susceptible to particle-induced interferences; therefore, label-free testing approaches emerge as more reliable alternatives. In this study, impedance-based real-time monitoring of cellular viability and metabolic activity were performed following exposure to metallic and ceramic wear particles. Moreover, label-free imaging of particle-exposed cells was done by high-resolution darkfield microscopy (HR-ODM) and field emission scanning electron microscopy (FESEM). The isolated human fibroblasts were exposed to CoCr28Mo6 and alumina matrix composite (AMC) ceramic particles. HR-ODM and FESEM revealed ingested particles. For impedance measurements, cells were seeded on gold-plated microelectrodes. Cellular behavior was monitored over a period of 48 h. CoCr28Mo6 and AMC particle exposure affected cell viability in a concentration-dependent manner, i.e., 0.01 mg/mL particle solutions led to small changes in cell viability, while 0.05 mg/mL resulted in a significant reduction of viability. The effects were more pronounced after exposure to CoCr28Mo6 particles. The results were in line with light and darkfield microcopy observations indicating that the chosen methods are valuable tools to assess cytotoxicity and cellular behavior following exposure to endoprosthetic wear particles.

Ganglioside Hp-s1 Analogue Inhibits Amyloidogenic Toxicity in Alzheimer's Disease Model Cells.

Alzheimer's disease (AD) is characterized by extracellular deposition of amyloid plaques, which are predominantly composed of amyloid-β (Aβ) peptide derived from amyloid precursor protein (APP) cleavage. APP interacts with tropomyosin receptor kinase A, a neurotrophic receptor associated with gangliosides and mediating neuronal survival and differentiation through the extracellular signal-regulated protein kinase (ERK) pathway. The ganglioside Hp-s1's analogue Hp-s1A exerts neuritogenic activity; however, its effect on AD pathology remains unknown. To test the hypothesis that Hp-s1A is a potential candidate to treat AD, we established the AD-modeled cell line by expressing human Swedish and Indiana APP gene (APP-Swe/Ind) in N2a mouse neuroblastoma cells. The cells were treated with Hp-s1A or monosialoganglioside GM1 for comparison. The AD model cells expressing APP-Swe/Ind exhibited a significant reduction in viability, as well as neurite outgrowth rate, in comparison to the control cells expressing APP-695. APP C-terminal fragment-β (CTFβ) and Aβ42 were increased in the AD cell lysates and the culture media, respectively. With the treatment of either Hp-s1A or GM1 at 1 μM, the AD model cells showed a significant increase in viability; however, only Hp-s1A reduced CTFβ levels in these cells. Further analysis of the culture media revealed that Hp-s1A also reduced Aβ42 production from AD model cells. The phosphorylation of ERK was elevated and the neurite outgrowth rate was restored with Hp-s1A treatment. In conclusion, the ganglioside analogue Hp-s1A inhibited amyloidogenic processing of APP and promoted neurotrophic activity and survival of AD model cells. Hp-s1A has great potential in AD therapeutic development.

Targeting ninjurin 2 by miR-764 regulates hydrogen peroxide (H2O2)-induced neuronal cell death

Ninjurin 2 (NINJ2) is a novel adhesion molecule expressed in neurons and glial cells. The current study determined if specific microRNA (miRNA) can regulate NINJ2 expression in human neuronal cells. Sequence analysis of NINJ2 mRNA 3′-untranslated region (3′-UTR) revealed that microRNA-764 (miR-764) putatively targets NINJ2. In SH-SY5Y/SK-N-BE neuronal cells and primary human neurons, lentivirus-mediated overexpression of miR-764 conferred significant repression on NINJ2 3′-UTR luciferase activity and downregulation of NINJ2 mRNA/protein. Conversely, transfection of the miR-764 inhibitor increased NINJ2 mRNA and protein expression in the neuronal cells. Function studies show that NINJ2 downregulation by miR-764 induced significant viability reduction and apoptosis in the neuronal cells. Further, CRISPR/Cas9-mediated NINJ2 knockout mimicked and abolished miR-764-induced actions in SH-SY5Y cells. Conversely, lentivirus-mediated NINJ2 overexpression or transfection of miR-764 inhibitor protected neuronal cells from hydrogen peroxide (H2O2)-induced cell death and apoptosis. Collectively, these results show that NINJ2 is a pro-survival factor in human neuronal cells. miR-764 regulates NINJ2 expression and neuron functions.

Analysis of Damage on the Streptococcus mutans Immersed in Ozonated Water: Preliminary Study for Application as Mouth Rinse

ABSTRACTOzonated water has been demonstrated to induce significant results in terms of the elimination of microorganisms. The present study assessed the damage to Streptococcus mutans after exposure to ozonated water; the ozone generator was adjusted to provide an outlet concentration of 60 mg/L, the samples were submitted to different ozonation times 1, 2, 4, 6, and 10 mi. Scanning electron microscopy and atomic force images were obtained to identify damage to the bacteria, followed by reactive oxygen species (ROS) evaluation and microbial viability. The results showed a significant reduction in viability and the images evidenced the generation of gaps on the microbial wall and surface layer alterations. Ozone can induce significant damage to S. mutans, thus suggesting that the use of ozonated water to prevent carious lesion formation is extremely promising.

Abstract 2330: CaV1.3 calcium channel expression in prostate cancer cells during androgen-deprivation conditions

Abstract Background Prostate cancer (PCa) remains one of the most frequently diagnosed cancers in males in the western world. Despite efforts to reduce tumour burden through treatments including androgen-deprivation therapy (ADT), patients often relapse 2 years post-therapy due to emergence of castration-resistant disease. The importance of ion channels including calcium channels in cancer biology is increasingly recognized. CACNA1D encodes the calcium channel, CaV1.3, which is overexpressed in PCa biopsies. In addition to canonical ion transport, the c-terminal region of CaV1.3 may modulate gene expression. The purpose of this study was to elucidate the expression of CaV1.3 during PCa progression and explore the effects of calcium channel blockers. Methods A cell line panel comprising normal prostate epithelium (RWPE-1), androgen-sensitive PCa (LNCaP, VCaP) and castration-resistant PCa (C4-2B, DU145 and PC3) were used. LNCaP were cultured in charcoal-stripped serum-containing media to mimic ADT or with the anti-androgen enzalutamide (10μM) for 4-14 days. qPCR, Western blotting and immunofluorescence imaging were performed. Cell viability assays were performed in the absence/presence of ADT, enzalutamide and the CaV1.3 blocker nifedipine (1μM). Apoptosis was measured with flow cytometry in cells stained with Annexin V/Propidium Iodide (AV/PI). Results CACNA1D mRNA had higher expression in LNCaP, VCaP and C4-2B vs. RWPE-1 (p≤0.05; N=3). Western blotting/densitometry showed higher protein expression of CaV1.3 in LNCaP, VCaP and C4-2B relative to RWPE-1 (P≤0.05; N=3). Immunofluorescence indicated cytoplasmic and perinuclear localisation of CaV1.3 in the majority of cell lines and diffuse cytoplasmic staining in VCaP (N=2). ADT and enzalutamide-treated LNCaP displayed time-dependent increased CACNA1D expression over 14 days however, CaV1.3 protein expression peaked at 4 days (N=3). Cell viability assays of LNCaP treated with ADT or enzalutamide (4 days) showed significant reduction in viability when nifedipine was combined with the treatment (N=3; P≤0.05). Similarly, increased % of apoptotic LNCaP cells were observed when nifedipine was added to treated LNCaP vs. ADT or enzalutamide alone (N=2). Conclusion CaV1.3 expression is differentially expressed in cells lines modelling PCa progression and enhanced during ADT. Blockade of CaV1.3 in combination with ADT treatments further reduced cell viability and increased the apoptotic population. Citation Format: Niamh McKerr, Conor Breen, Kirtiman Srivastava, Ian G. Mills, Karen D. McCloskey. CaV1.3 calcium channel expression in prostate cancer cells during androgen-deprivation conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2330.

Protective effects of honokiol against oxidative stress-induced apoptotic signaling in mouse podocytes treated with H2O2.

Honokiol (HNK), an important bioactive compound purified from Magnolia officinalis Cortex, has been demonstrated to have manifold beneficial anti-oxidative, anti-inflammatory, anti-bacterial and antitumor pharmacological effects. In the present study, the association of HNK in the signaling mechanism associated with hydrogen peroxide (H2O2)-induced apoptosis of cultured mouse podocytes was investigated. HNK did not cause significant changes in podocyte viability when its concentration remained below 20 µM. MTS assay and flow cytometry confirmed that H2O2 significantly enhanced the rates of apoptosis while produce significant reduction in viability of podocytes. Following 24 h of pre-treatment with different concentrations of HNK, the viability of adherent podocytes increased and apoptosis significantly decreased in a dose-dependent manner below 20 µM. Reverse transcription-polymerase chain reaction and western blot results indicated that HNK significantly decreased the expression of mRNA and cleaved protein of caspase-3 and caspase-9 in podocytes pre-treated with H2O2. Furthermore, phosphorylation of the signaling molecules protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) 1/2 appeared to increase following HNK treatment. In conclusion, HNK largely eliminated the role of promoting podocyte apoptosis in an oxidative stress environment, which was a protective factor on podocytes cultured with H2O2. The anti-oxidative stress mechanisms of HNK are partly due to suppressing the expression of caspase-3 and caspase-9 and upregulating phosphorylated-Akt and -Erk 1/2.

Metabolic changes associated with metformin potentiates Bcl-2 inhibitor, Venetoclax, and CDK9 inhibitor, BAY1143572 and reduces viability of lymphoma cells.

Metformin exerts direct anti-tumor effects by activating AMP-activated protein kinase (AMPK), a major sensor of cellular metabolism in cancer cells. This, in turn, inhibits pro-survival mTOR signaling. Metformin has also been shown to disrupt complex 1 of the mitochondrial electron transport chain. Here, we explored the lymphoma specific anti-tumor effects of metformin using Daudi (Burkitt), SUDHL-4 (germinal center diffuse large B-cell lymphoma; GC DLBCL), Jeko-1 (Mantle-cell lymphoma; MCL) and KPUM-UH1 (double hit DLBCL) cell lines. We demonstrated that metformin as a single agent, especially at high concentrations produced significant reductions in viability and proliferation only in Daudi and SUDHL-4 cell lines with associated alterations in mitochondrial oxidative and glycolytic metabolism. As bcl-2 proteins, cyclin dependent kinases (CDK) and phosphoinositol-3- kinase (PI3K) also influence mitochondrial physiology and metabolism with clear relevance to the pathogenesis of lymphoma, we investigated the potentiating effects of metformin when combined with novel agents Venetoclax (bcl-2 inhibitor), BAY-1143572 (CDK9 inhibitor) and Idelalisib (p110δ- PI3K inhibitor). Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM of metformin resulted in 1.4 fold and 8.8 fold decreases, respectively, in IC-50 values of Venetoclax. By contrast, 3-fold and 10 fold reduction in IC-50 values of BAY-1143572 in Daudi and Jeko-1 cells respectively was seen in the presence of 10 mM of metformin. No change in IC-50 value for Idelalisib was observed across cell lines. These data suggest that although metformin is not a potent single agent, targeting cancer metabolism with similar but more effective drugs in novel combination with either bcl-2 or CDK9 inhibitors warrants further exploration.

Abstract P1-09-03: Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors

Abstract Background Estrogen-receptor positive (ER+) breast cancer (BC) accounts for over 75% of diagnosed cases. Despite treatment, a large proportion relapse with de novo or acquired endocrine resistant disease, making it one of the greatest challenges for BC research. Using a kinome siRNA library screen, we identified MPS1 that is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to endocrine therapy and palbociclib. Until now, the target population for MPS1 inhibitors has focused on triple negative BC. Our unexpected finding shows, potential efficacy of MPS1 inhibitors in ER+ BC resistant to endocrine therapies and palbociclib, a prominent contemporary first-line combination for advanced disease. Methods ER+ BC cell lines (MCF7, SUM44, ZR75.1, HCC1428 and T47D) adapted to estrogen independent growth (LTED) and sequential resistance to palbociclib (991R) were subjected to a siRNA screen targeting 709 kinases. Z-scores were used to identify the most robust candidates. Cell viability upon MPS1 inhibition with CCT289346 (MPS1i) was assessed 2D and 3D. The class effect was confirmed with other compounds targeting MPS1. Impact of MPS1i on ER co-localisation and ER-transactivation was assessed using confocal microscopy and reporter assays, respectively. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. BrdU incorporation and cell cycle were assessed by FACS. PARP cleavage was used to measure apoptosis. Global gene expression analysis of MPS1 was carried out in two independent neoadjuvant studies of aromatase inhibitor (AI) treated patients. Results Kinome knockdown identified targets associated with the G2/M checkpoint as strongly implicated in the LTED phenotype. In particular, MPS1 was the top common hit in LTED and 991R cell lines. Increase in MPS1 was evident in MCF7-LTED at both the transcript and protein level. Notably, the MPS1 inhibitor CCT289346 caused a significant reduction in viability of the majority of LTED and 991R cell lines tested. Table. Sensitivity of ER+ BC cell lines to CCT289346Cell LineModelIC50 nMMCF7Wt90 LTEDESR1wt80 LTEDESR1Y537C50 TAMR55 ICIR50 LTEDICIR60 991R40 LTED991R45 LTED991R-ICIR25T47DWt110 LTED110 991R50 LTED991R60SUM44LTED40 Upon inhibition of MPS1, cells demonstrated shorter time in mitosis, aberration of cell cycle and amplified mitotic errors, resulting in increased apoptosis. To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoadjuvant AI therapy. In the anastrozole cohort, on-treatment gene expression of MPS1 (p<0.0001) was significantly associated with poor response to anastrozole based on a 2-week residual Ki67 score <10%. In the letrozole cohort, increased on-treatment expression of MPS1 (p=0.0118) was associated with poor response based of tumor shrinkage ≥50%. Conclusion This novel finding shows MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC models resistant to endocrine therapy and palbociclib providing a new therapeutic strategy. Citation Format: Nikitorowicz-Buniak J, Pancholi S, Simigdala N, Ribas R, Linardopoulos S, Dowsett M, Johnston SR, Martin L-A. Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-03.

In Vitro Studies of the Antimicrobial and Free-Radical Scavenging Potentials of Silver Nanoparticles Biosynthesized From the Extract of Desmostachya bipinnata

The aim of this study was to perform green synthesis of silver nanoparticles (AgNPs) from the leaf extract of Desmostachya bipinnata (Dharba), a medicinally important herb which is widely used across India. Synthesized AgNPs were analyzed by UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDAX). The results have confirmed that green synthesis of AgNPs leads to the fabrication of sphere-shaped particles with a diameter of 53 nm. Furthermore, these AgNPs were subjected to antioxidant and antimicrobial studies against gram-negative and gram-positive bacteria, where AgNPs at a concentration of 20 mg/mL showed highest zone of inhibition. Synthesized AgNPs were evaluated for their antioxidant activity by 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH), H2O2, and superoxide inhibiting assays; increasing concentration has showed increase in scavenging ability. Cell toxicity was assessed on HepG2 cell lines, and synthesized nanoparticles at a concentration of 128 μg/mL produced significant reduction in viability of Hep cells (P < .05). The availability of Dharba throughout the year and the eco-friendly approach in the synthesis of AgNPs coupled with bioactivity has demonstrated its potential as a novel biomaterial which can be used for various biomedical applications.

Effects of erythropoietin on astrocytes and brain endothelial cells in primary culture during anoxia depend on simultaneous signaling by other cytokines and on duration of anoxia

Studies on animals revealed neuroprotective effects of exogenously applied erythropoietin (EPO) during cerebral ischemia/hypoxia. Yet, application of exogenous EPO in stroke patients often lead to haemorrhagic transformation. To clarify potential mechanism of this adverse effect we explored effects of EPO on viabilities of astrocytes and brain endothelial cells (BECs) in primary culture during anoxia of various durations, in the presence or absence of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1), which are cytokines that are also released from the neurovascular unit during hypoxia. Anoxia (2-48 h) exerted marginal effects on BECs' viability and significant reductions in viability of astrocytes. Astrocyte-conditioned medium did not exert effects and exerted detrimental effects on BECs during 2 h and 24 h anoxia, respectively. This was partially reversed by inhibition of Janus kinase (Jak)2/signal transducer and activator of transcription (STAT)5 activation. Addition of rat recombinant EPO (rrEPO) during 2 h-6h anoxia was protective for astrocytes, but had no effect on BECs. Addition of rrEPO significantly reduced viability of BECs and astrocytes after 48 h anoxia and after 24 h-48 h anoxia, respectively, which was attenuated by inhibition of Jak2/STAT5 activation. Simultaneous addition of rrEPO and VEGFA (1-165) caused marginal effects on BECs, but a highly significant protective effects on astrocytes during 24-48 h anoxia, which were attenuated by inhibition of Jak2/STAT5 activation. Simultaneous addition of EPO, VEGFA 1-165 and Ang1 exerted protective effects on BECs during 24 h-48 h anoxia, which were attenuated by addition of soluble Tie2 receptor. These data revealed that EPO could exert protective, but also injurious effects on BECs and astrocytes during anoxia, which depended on the duration of anoxia and on simultaneous signaling by VEGF and Ang1. If these injurious effects occur in stroke patients, they could enhance vascular damage and haemorrhagic transformation.

Valosin-Containing Protein/p97 as a Novel Therapeutic Target in Acute Lymphoblastic Leukemia

B acute lymphoblastic leukemia (B-ALL) cells are distinctively vulnerable to endoplasmic reticulum (ER) stress. Recently, inhibition of p97 was shown to induce ER stress and subsequently cell death in solid tumors and in multiple myeloma. We investigated the role of a novel, orally available, p97 inhibitor (CB-5083; Cleave Biosciences) in B-ALL. CB-5083 induced a significant reduction in viability in 10 human B-ALL cell lines, harboring the most common fusion-genes involved in pediatric and adult B-ALL, with IC50s ranging from 0.34 to 0.76 μM. Moreover, CB-5083 significantly reduced the colony formation of OP1 and NALM6 cells. Early and strong induction of apoptosis was demonstrated in BALL1 and OP1 cells, together with a robust cleavage of PARP. CB-5083 induced ER stress, as documented through: 1) prominent expression of chaperones (GRP78, GRP94, PDI, DNAJC3, and DNAJB9); 2) increased activation of IRE1-alpha, as demonstrated by the splicing of XBP1; and 3) activation of PERK, which resulted in a significant overexpression of CHOP, and its downstream genes. CB-5083 reduced the viability also in GRP78−/−, GRP94−/−, and XBP1−/− cells, suggesting that none of these proteins alone was strictly required for CB-5083 activity. Moreover, we showed that the absence of XBP1 (XBP1−/−) increased the sensitivity to CB-5083, leading to the hypothesis that XBP1 splicing counteracts the activity of CB-5083, probably mitigating ER stress. Finally, vincristine was synergistic with CB-5083 in both BALL1 and OP1 cells. In summary, the targeting of p97 with CB-5083 is a novel promising therapeutic approach that should be further evaluated in B-ALL.

Evaluation of silver nanospheres on viability and innate cellular parameters of gilthead seabream (Sparus aurata L.) head-kidney leucocytes

The increasing use of nanomaterials, e.g. nanosilver, has lead to concerns about environmental contamination and possible toxic effects on aquatic organisms. Here, we present evidence for the impact of silver nanospheres (AgNSs) on fish innate immune cells after in vitro exposure. AgNSs of 20, 50 or 100 nm in diameter were tested with the smallest ones (20 nm) clearly having the most deleterious effects, after an exposure period of 30 min, followed by the medium-sized ones; the NSs of 100 nm had no impact. The effective concentration was determined at 10 μg ml−1 while lower concentrations (1, 2.5 or 5 μg ml−1) were ineffective. Head-kidney mixed leucocyte population showed significant viability reduction which was attributable to diminished viability of macrophages/monocytes and lymphocytes only whereas granulocytes' viability was not affected at the above exposure regime. Furthermore, cellular respiratory burst activity, phagocytic capacity and phagocytic ability were all reduced, with the first two parameters exhibiting the sharper reductions. Finally, transmission electron microscopy revealed that the AgNSs' internalization was brought about via phagocytosis, pinocytosis, receptor-mediated endocytosis and macropinocytosis; also, that cell death could be effected in either an apoptotic or a necrotic manner. It is concluded that AgNSs are potentially very noxious for the teleost fish immune system as they can adversely affect the function and viability of the head-kidney leucocytes.

Mesenchymal stem cells as natural biofactories for exosomes carrying miR-124a in the treatment of gliomas.

MicroRNAs (miRs) are promising new therapeutics for glioblastoma. However, which miRs are most effective against glioblastomas and how these miRs should be delivered are major unanswered problems. To identify potent antiglioma miRs, we selected 8 miRs based on a literature search and screened them against a panel of glioma stem cell (GSC) lines, representing all of the glioblastoma subtypes defined by The Cancer Genome Atlas. To address delivery, we tested the hypothesis that ex vivo cultured bone marrow-derived mesenchymal stem cells (MSCs) can package miRs into exosomes and that these engineered exosomes can systemically deliver antiglioma miRs to glioblastomas. Of the screened miRs, we identified miR-124a as the most effective antiglioma agent against GSCs. We then transduced MSCs with lentivirus vectors containing miR-124a and isolated vesicles from the medium. Electron microscopy, western blotting, and Nanosight proved that the isolated vesicles were exosomes. Quantitative PCR documented that these exosomes contained high levels of miR-124a, which was not present in control exosomes. In vitro treatment of GSCs with exosomes containing miR-124a (Exo-miR124) resulted in a significant reduction in viability and clonogenicity of GSCs compared with controls. In vivo treatment of mice harboring intracranial GSC267 with systemically delivered Exo-miR124 resulted in 50% of animals living long term. No evidence of tumor was present on histological analysis of the survivors. Mechanistic studies showed that miR-124a acts by silencing Forkhead box (FOX)A2, resulting in aberrant intracellular lipid accumulation. MSCs can be used as natural biofactories to produce Exo-miR124, which is an effective antiglioma agent worthy of further clinical evaluation.

Ozonated Olive Oil with a High Peroxide Value for Topical Applications: In-Vitro Cytotoxicity Analysis with L929 Cells

ABSTRACTPrevious studies have shown that ozonated vegetable oils have been used topically for healing of cutenous wounds. The aim of this study is to evaluate the dose dependent use of ozonated olive oil with high peroxide value (OZ) on the viability of cells for preventing side effects in topical applications. To the best of our knowledge, there are no reports investigaing the effect of peroxide value of ozonated olive oil associated with its cytotoxic activity on mouse non-neoplastc fibroblast cell lines (L929). Therefore, the present study was carried out by using OZ alone and/or in combination with glycerol and olive oil. In our study OZ was prepared by using pure olive oil. Both olive oil and glycerol are non-toxic and can be mixed with OZ uniformly. The cytotoxic activity of samples against L929 fibroblasts was assessed using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. The peroxide value of synthesized OZ was found to be in the range of 2700–2900 mEq O2/kg oil. The OZ/olive oil group did not show any cell death at all concentrations tested (p > 0.05) however OZ/glycerol group showed statistically significant reductions in viability at higher concentrations (p = 0.004–0.006) compared to the control group. Conclusively, using OZ/olive oil with a peroxide value of 2700–2900 mEq O2/kg oil for short-term incubation was non-cytotoxic to the L929 fibroblast cell line.

Abstract 3445: ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro

Abstract Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain tumor of astrocytic origin characterized by very poor prognosis. Despite conventional therapeutic protocol the prognosis of GBM patients is very poor with median of overall survival ranging between 12 and 15 months from diagnosis. Therefore, many financial charges and lot of effort is spent in research of new therapeutic approaches that could prolong the survival of GBM patients. Long non-coding RNAs (lncRNAs) are a relatively new class of noncoding gene regulators playing critical roles in tumor biology, including GBM. From this perspective, lncRNAs seem to be promising therapeutic targets in GBM patients. Material and Methods: We performed next-generation sequencing analysis of fresh-frozen histopatologically confirmed 45 GBM tissues and 5 non-tumor brain tissues obtained from non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient before the treatment. rRNA depletion and cDNA library preparation were performed with GeneRead rRNA Depletion Kit (Qiagen) and NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), respectively. Sequencing was held using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). Statistical analysis evaluated 24 087 protein-coding and 8 414 non-coding RNAs and their sequential variants with non-zerou RPKM (Reads Per Kilobase of transcript per Million mapped reads) at least in one sample. We used CLC genomic workbench for the alignment and target counts. Targeted regulation of ZFAS1 level have been carried out by the transient transfection of specific siRNA in GBM stable cell lines (A172, T98G, U87MG, U251). Viability and migration were analyzed in vitro using MTT and scratch wound healing assay, respectively. Results: Statistical analysis has revealed 274 (P &amp;lt; 0.01) deregulated lncRNAs in GBMs in comparison with non-tumor brain samples. Moreover, the results have showed also 489 deregulated proten-coding RNAs with P value less than 0.001 and 26 protein-coding RNAs with P value less than 0.000001. For subsequent in vitro functional analyses was chosen one of the most upregulated lncRNAs in GBM samples ZFAS1. Targeted downregulation of this molecule led to the significant reduction of viability in all examined GBM cell lines. Decreasing of proliferation potential was observed only in A172 a U251 cell lines. Conclusion: We have demonstrated a deregulation of many lncRNAs and protein-coding RNAs in GBM tissue in comparison with non-tumor brain tissue. Moreover, ZFAS1, one of the most upregulated lncRNAs in GBM tissue, is involved in regulation of viability and migration of GBM cell lines in vitro. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Note: This abstract was not presented at the meeting. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Kamila Souckova, Leos Kren, Radim Lipina, Martin Smrcka, Ondrej Slaby. ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro [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 3445. doi:10.1158/1538-7445.AM2017-3445

Antibacterial properties of sophorolipid-modified gold surfaces against Gram positive and Gram negative pathogens

Sophorolipids are bioderived glycolipids displaying interesting antimicrobial properties. We show that they can be used to develop biocidal monolayers against Listeria ivanovii, a Gram-positive bacterium. The present work points out the dependence between the surface density and the antibacterial activity of grafted sophorolipids. It also emphasizes the broad spectrum of activity of these coatings, demonstrating their potential against both Gram-positive strains (Enteroccocus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes) and Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhymurium). After exposure to sophorolipids grafted onto gold, all these bacterial strains show a significant reduction in viability resulting from membrane damage as evidenced by fluorescent labelling and SEM-FEG analysis.

Evidence of emerging challenge of chlorine tolerance of Enterococcus species recovered from wastewater treatment plants

This study assessed the survival of some Enterococcus species recovered from the clarifier of two selected wastewater treatment plants in the Eastern Cape Province, South Africa, in the presence of different concentrations of chlorine disinfectant. The bacterial survival, lethal dose and inactivation kinetics at lethal doses were examined. Inactivation of the test bacteria (n = 20) at the recommended dose of 0.5 mg l−1 progressively increased from 3.0–5.8 log reduction within the range of chlorine residual of 0.14–0.45 mg l−1 after 30 min. The bactericidal activity of chlorine increased at higher chlorine dosages with a significant reduction in viability of the bacteria and complete sterilization of the bacterial population was achieved at a lethal dose of 0.75 mg l−1 and 1.0 mg l−1 in 30 min. For the inactivation kinetics, bactericidal activity of chlorine increased with time showing a 3.0–6.8 log reduction in 10 min, 4.0–8.3 log reduction in 20 min and above 7.9 log reduction in 30 min for all test Enterococcus isolates used in this study. Chlorine dose of 0.75 mg l−1–1.0 mg−1 showed a better disinfecting capacity to effectively reduce the tolerance of Enterococcus species.

A recombinant fungal compound induces anti-proliferative and pro-apoptotic effects on colon cancer cells.

Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly).We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein.rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models.Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.

Cadmium chloride treatment of rats significantly impairs membrane integrity of mesenchymal stem cells via electrolyte imbalance and lipid peroxidation, a possible explanation of Cd related osteoporosis.

Objective(s):Bone marrow mesenchymal stem cells (MSCs) play an important role in bone health. Cadmium causes osteoporosis, but the exact mechanisms of its effect on MSCs are not known.Materials and Methods:Rats were treated with cadmium chloride (40 mg/l) in drinking water for six weeks, and then the biochemical and morphological studies on MSCs were carried out as a cellular backup for osteoblasts. Viability and proliferation properties of the cells were evaluated using MTT assay, trypan blue, population doubling number, and colony forming assay. Morphology of the cells and biochemical parameters including activity of metabolic (ALP, AST, and ALT) and antioxidant enzymes (SOD, CAT, and POX) as well as the MDA level (as an indication of lipid peroxidation) were investigated. In addition, intracellular calcium, potassium, and sodium content were estimated. Data was analyzed statistically and P<0.05 was taken as the level of significance.Results:The results showed a significant reduction in viability and proliferation ability of extracted cells when compared to the controls. In addition, it was revealed that the cadmium treatment of rats caused a significant reduction in nuclear diameter and cytoplasm area. Also, there was significant increase in (ALT) and (AST) activity and intracellular calcium and potassium content but no change was observed with sodium content and ALP activity. The results showed [a] significant reduction in the antioxidant enzyme activity and increases in the MDA level.Conclusion:Based on the present study, reduction of viability and proliferation ability of MSCs might be a causative factor of osteoporosis in industrial areas.

Metabolic adaptations to targeted therapy in FLT3 mutated acute myeloid leukaemia

BackgroundAcute myeloid leukaemia has a dismal outlook. FLT3 tyrosine kinase (TK) activating mutations (FLT3mut) are present in 30% of cases and are predictive of a worse outcome. Although metabolic reprogramming has been recognised as a hallmark of cancer cells, little is known about its role in acute myeloid leukaemia. Published gene expression datasets show that glycolytic, citric acid cycle, and oxidative phosphorylation genes are upregulated in FLT3mut patient samples at diagnosis. We aimed to study metabolic changes in FLT3mut disease upon targeted inhibition of its TK activity in an attempt to unveil novel therapeutic vulnerabilities. MethodsLiquid chromatography coupled to mass spectrometry, using stable isotope-based carbon flux tracing, and an extracellular flux analyser (Seahorse, Agilent Technologies, Santa Clara, CA, USA) were used to assess metabolic changes in FLT3mut human and murine cells after FLT3 TK inhibition by the specific inhibitor AC220. Gene expression changes were measured in the same conditions by RNA sequencing. Fluorescence-activated cell sorting was used to measure changes in viability and reactive oxygen species after inhibition of TK activity in various culture conditions. FindingsWe confirmed in both human and murine cells that FLT3mut cells displayed an increased glycolytic (maximal glycolytic capacity, extracellular acidification rate of 80·64 mpH/min in FLT3mut vs 59·09 in FLT3wildtype, p<0·001) and respiratory capacity (maximal respiratory capacity, oxygen consumption rate of 326·4 pmoles/min in FLT3mut vs 180·9 in FLT3wildtype, p<0·001). Upon treatment with AC220, these metabolic phenotypes were reversed. However, whereas glucose uptake was significantly reduced upon TK inhibition (p<0·001), glutamine uptake was not affected. Metabolic flux analysis with [U−13C]glutamine suggested that glutamine was diverted to glutathione production upon AC220 treatment. FLT3mut cells displayed a large increase in concentration of reactive oxygen species upon AC220 treatment when grown in the absence of glutamine (fold increase of reactive oxygen species normalised to untreated cells 4·59 without glutamine vs 2·74 with glutamine, p<0·001), and these changes correlated with a significant reduction in viability (relative viability normalised to untreated 34·6% without glutamine vs 62·5% with glutamine, p<0·001) in the same conditions. InterpretationOur data suggest that FLT3mut acute myeloid leukaemia cells have increased respiratory and glycolytic capacity that is reversed by TK inhibition. Upon AC220 treatment glutamine metabolism becomes a metabolic dependency of FLT3mut disease, as glutamine is channelled towards glutathione production and reactive oxygen species detoxification, and protects them from cell death. This process could be exploited therapeutically by a combination of glutaminolysis or glutathione biosynthesis inhibitors with FLT3 TK inhibitors. FundingWellcome Trust.