Plating Efficiency Research Articles

Abstract 3078: The role of extracellular vesicles in chemotherapy resistance in gastric cancer

Abstract Gastric adenocarcinoma (GAd) is the fourth most common cancer and the second cause of cancer death in the world. Chemotherapy has been largely used to treat this disease however acquired drug resistance is a common event and is associated to the development of new more aggressive clones of tumor cells. Herein, a gastric adenocarcinoma cell line (AGS) resistant to the 5-fluorouracil (rAGS-FU) was generated in order to establish mechanisms associated to acquire drug resistance. In the presence of 5-FU (10μM) resistant cells show a 2-3 times higher proliferation ratio than parental cells, a plating efficiency of 38% compared to 1% of parental cells and a higher invasive phenotype. Remarkable, when compared to parental cells, rAGS-FU resistant cells secreted 2-3 times more extracellular vesicles, EVs (85-175nm). When parental cells were treated with EVs from rAGS-5-FU resistant cells they acquired the same phenotype of the latter cells regarding higher proliferation, plating efficiency and invasion. The proteomic analysis of parental and rAGS-FU cells showed 306 differentially expressed proteins, while 66 differentially expressed proteins were found when EVs secreted from these cells were compared. Our data suggest that cells resistant to chemotherapy have a more aggressive phenotype and are able to transfer these characteristics to non-resistant cells. These findings point to mechanisms of chemoresistance mediated by components that are carried from cell to cell by EVs, what may indicate novel approaches that can be addressed to impair drug resistance and improve treatment response. Supported by FAPESP Citation Format: Edson Cassinela, Michele C. Landemberger, Gabriela P. de Oliveira, Vilma Regina Martins. The role of extracellular vesicles in chemotherapy resistance in gastric 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 3078. doi:10.1158/1538-7445.AM2017-3078

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation.

BackgroundNo optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions.MethodsHepatocytes were preserved in the following conditions, and their outcomes were observed. First, the effectiveness of TD was investigated. Second, hepatocyte medium (HM) and organ preservation solutions with or without fetal bovine serum (FBS) were compared. Third, as supplementations, FBS and human serum albumin (HSA) were compared. Fourth, low, room and high temperature were compared. And finally, hepatocytes preserved in various conditions were transplanted into the subrenal capsule space of nonalbumin rats and engrafted areas were assessed.ResultsThe survival rate of hepatocytes preserved in TD tended to be higher and their viability and function were maintained significantly greater than those of non-TD group. Irrespective of FBS supplementation, the survival rate of HM group was significantly higher than those of organ preservation solution group while viabilities and plating efficiency were similar among them. Although survival rates of groups without FBS were extremely low, results of HSA supplemented group were not inferior to FBS supplemented group. Hepatocytes preserved at high temperature had the worst results. The engrafted area of TD group tended to be higher than those of other groups.ConclusionsTD is effective for short-term hepatocyte preservation. HSA is a useful substitute for FBS, and preserving in HM at low temperature is recommended.

Abstract 856: Notch-1 promotes a mesenchymal phenotype in colon cancer cells via Notch-3 and Smad-3 activation

Abstract A role for cancer initiating cells (CIC) in tumor growth, drug resistance, relapse, and metastasis via epithelial to mesenchymal transition (EMT) has been supported by experimental evidence. It is well-documented that Notch-1 receptor signaling participates in stem cell maintenance and lineage commitment in various organs, including the colon, and can regulate EMT. The function of Notch signaling in colorectal cancer (CRC) is not clear. We have reported that Notch-1 activation in the human colon tumor cell line HCT-116 resulted in upregulation of Jagged-1, Smad-3, CD44, and Slug proteins. These changes were accompanied by acquisition of a mesenchymal cell phenotype that included increased anchorage independent growth and migratory activity. Experiments with a γ-secretase inhibitor (DAPT) and soluble Jagged1-Fc protein demonstrated that Notch-1 signaling activates CD44 and Slug via other Notch receptors. In subsequent experiments, we observed an increase in Notch-3 expression in the presence of activated Notch-1. Herein, we tested the hypothesis that the stem cell-like mesenchymal phenotype induced by Notch-1 signaling in colon cancer cells is mediated by the Notch-3 receptor and Smad-3. The human colon tumor cell line HCT-116 was transduced with constitutively active Notch-1, and a Notch-3 shRNA to produce a Notch-3 null cell line with activated Notch-1 (ICN1-shN3 cells). ICN1-shN3 cells exhibited a 2.5-fold lower plating efficiency than the HCT-116/ICN1 cell line (p<0.01) that expresses constitutively active Notch-1 in the presence of Notch-3. In addition, Notch-3 null cells with constitutively active Notch-1 (ICN1-shN3 cells) were slower by 29% in completing wound healing as compared with the HCT-116/ICN1 cell line (p<0.05). Consistent with the lower plating efficiency, a 37% decrease in colosphere formation was observed with ICN1-shN3 cells as compared with the HCT-116/ICN1 cell line (p<0.001). These data indicated that loss of Notch-3 abrogated the Notch-1 induced mesenchymal cell phenotype. Western blot analysis demonstrated that the HCT-116/ICN1 cells with intact Notch-3 exhibited increased levels of the EMT associated proteins Smad-3, CD44 and Slug as compared to the parental HCT-116 cell line. Expression of CD44 and Slug in Notch-3 receptor null ICN1-shN3 cells was highly reduced compared with HCT-116/ICN1 cells and was similar to that detected in the parental HCT-116 cells. Treatment of HCT-116/ICN1 cells with a Smad-3 inhibitor resulted in significant reduction of the CD44 and Slug proteins. Meanwhile, Notch-3 receptor null ICN1-shN3 cells were also more sensitive to chemotherapy treatment than the HCT-116/ICN1 cell line (p<0.05). Collectively, our data provide evidence for a novel pathway in which Notch-1 signaling in colon tumor cells promotes a mesenchymal phenotype via activation of Jagged-1, Notch-3 and Smad-3, followed by expression of CD44 and Slug. Citation Format: George Sigounas, Fred E. Bertrand, Douglas A. Weidner, Kaitlyn E. Vinson, Alexander G. Clark, Azeem Khan. Notch-1 promotes a mesenchymal phenotype in colon cancer cells via Notch-3 and Smad-3 activation [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 856. doi:10.1158/1538-7445.AM2017-856

Long-term growth comparison studies of FBS and FBS alternatives in six head and neck cell lines.

Fetal bovine serum (FBS) is depended upon by investigators as an indispensable supplement in cell and tissue culture systems. Due to increased demand and limited availability, the price of FBS has increased by greater than 300% in the past few years. In addition, there are ethical and scientific controversies about the collection and use of FBS in culture systems. In response to the shortage of FBS, many FBS alternative serum products have been developed. Although many have claimed comparable performance to FBS, their support of long-term cell growth and effects on cell phenotype have not been revealed. In this study, we examined the performances of six bovine calf serum-based FBS alternatives in six head and neck cell lines and compared them with FBS. The results indicate that some of these sera had growth promoting capabilities comparable or superior to that of FBS. Additionally, these alternative sera supported long-term (30 passages) growth of tested cells and exhibited plating efficiencies comparable to that of FBS. Cells cultured in alternative sera also exhibited comparable anchorage-independent growth and similar drug inhibition responses in FBS. Still, caution should be taken in choosing suitable sera given that changes in cell morphology and variations in chemotactic responses were noted for cells maintained in certain sera. These FBS alternatives are more readily available, cost less, and are associated with less ethical concerns, thus making them attractive alternatives to FBS in cell culture systems.

Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications

We present (1) a fast and automated method for large scale production of HUVEC spheroids based on the hanging drop method and (2) a novel method for well-controlled lateral deposition of single spheroids by drop-on-demand printing. Large scale spheroid production is achieved via printing 1536 droplets of HUVEC cell suspension having a volume of 1 μl each within 3 min at a pitch of 2.3 mm within an array of 48 × 32 droplets onto a flat substrate. Printing efficiencies between 97.9% and 100% and plating efficiencies between 87.3% and 100% were achieved. Harvested spheroids (consisting of approx. 250 HUVECs each) appear uniform in size and shape. After incubation and harvesting, the spheroids are deposited individually in user-defined patterns onto hydrogels using an automated drop-on-demand dispenser setup. Controlled by an image detection algorithm focusing the dispenser nozzle, droplets containing exactly one spheroid are printed onto a substrate, while all other droplets are discarded. Using this approach an array of 6 × 3 HUVEC spheroids with intermediate distances of 500 μm embedded in fibrin was generated. Successful progress of spheroid sprouting and merging of neighboring sprouts was observed during the first 72 h of incubation indicating a good viability of the deposited spheroids.

Fusion between M2-macrophages and cancer cells results in a subpopulation of radioresistant cells with enhanced DNA-repair capacity.

Cell fusion is a natural biological process in normal development and tissue regeneration. Fusion between cancer cells and macrophages results in hybrids that acquire genetic and phenotypic characteristics from both maternal cells. There is a growing body of in vitro and in vivo data indicating that this process also occurs in solid tumors and may play a significant role in tumor progression. However, investigations of the response of macrophage:cancer cell hybrids to radiotherapy have been lacking. In this study, macrophage:MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation, both hybrids and their maternal MCF-7 cells were treated with 0 Gy, 2.5 Gy and 5 Gy γ-radiation and examined by clonogenic survival and comet assays at three time points (0 h, 24 h, and 48 h). Compared to maternal MCF-7 cells, the hybrids showed increased survival fraction and plating efficiency (colony formation ability) after radiation. The hybrids developed less DNA-damage, expressed significantly lower residual DNA-damage, and after higher radiation dose showed less heterogeneity in DNA-damage compared to their maternal MCF-7 cells. To our knowledge this is the first study that demonstrates that macrophage:cancer cell fusion generates a subpopulation of radioresistant cells with enhanced DNA-repair capacity. These findings provide new insight into how the cell fusion process may contribute to clonal expansion and tumor heterogeneity. Furthermore, our results provide support for cell fusion as a mechanism behind the development of radioresistance and tumor recurrence.

Radiobiological effects of VPA-BSANPs on C6 and U87 glioma cells

Objective To investigate the radiobiological effects of VPA-BSANPs on C6 and U87 glioma cells in vitro. Methods C6 and U87 glioma cells were treated with different concentrations of VPA and VPA-BSANPs for 12 h and 24 h, and MTT assay was used to determine cell viability. C6 and U87 cells were treated with different concentrations of VPA and VPA-BSANPs combined with X-ray irradiation (0, 2, 4, 6, and 8 Gy), and colony formation assay was used to determine plating efficiency (PE). C6 and U87 glioma cells were treated with different concentrations of VPA and VPA-BSANPs for 12 h, followed by X-ray irradiation (0, 4, and 8 Gy), and flow cytometry using Annexin V-FITC/PI staining was used to examine cell apoptosis. Western blot was used to evaluate the effects of VPA and VPA-BSANPs on radiation-induced apoptosis protein expression. One-way ANOVA was used for comparison of means with homogeneity of variance between multiple groups, and the t-test was used for comparison of means between two groups. Results Without irradiation, VPA and VPA-BSANPs had no significant inhibitory effects on the proliferation of C6 and U87 cells (P=0.328, 0.920). The PE of cells treated with VPA-BSANPs combined with irradiation was significantly lower than that of cells treated with VPA combined with irradiation (P=0.000). In C6 and U87 cells, VPA-BSANPs combined with irradiation increased the expression of p53 and Bax (P=0.000, 0.000 and P=0.010, 0.002), but reduced the expression of Bcl-2(P=0.008, 0.000). Active caspase-3 fragments were only found in the cells treated with VPA-BSANPs combined with irradiation and VPA combined with irradiation, but were less in the former cells than in the latter cells (P=0.004). The active fragments of peroxisome proliferator-activated receptor were only found in the cells treated with VPA-BSANPs combined with irradiation. Conclusions VPA-BSANPs can increase the radiosensitivity of C6 and U87 glioma cells in vitro, possibly by promoting the apoptosis of tumor cells induced by radiation. Key words: Histone deacetylase inhibitors; Valproic acid; Apoptosis

Exploration of Novel Magnesium Battery Electrolytes based on Inorganic Salts

The theoretical volumetric capacity of Mg-ion batteries is far higher than state-of-the-art Li-ion batteries (3833mAh/cm3 for Mg vs. 850 mAh/cm3for Li). The development of suitable Mg-conductive electrolytic solutions has been a major barrier to Mg-ion technology: at present there is a need for liquids with wide anodic stability windows, whose interfaces allow plating and stripping of Mg with high coulombic efficiency and low deposition overpotential. We have produced a number of Mg-conductive electrolytes by direct reaction of inorganic chloride salts with suspensions of Mg powder in nonaqueous solvents. The Mg-carrying cation in these electrolytes is presumably similar to the one that provides conductivity in the recently discovered magnesium aluminum chloride complex (MACC) electrolyte [1,2], but there is apparently no metal-complex anion, and no need for an auxiliary Lewis acid during synthesis. The chemically synthesized Mg-conductive electrolyte formed by reacting ZnCl2 with Mg in tetrahydrofuran is stable up to 3.0 V vs. Mg/Mg2+, and produces an ionic conductivity of 0.24 mS/cm at room temperature. Cyclic voltammetry shows reversible Mg plating and stripping on platinum. The deposition overpotential of freshly synthesized electrolyte is below 200mV, and the coulombic efficiency of plating and stripping approaches 100%. Over a period of a week, the coulombic efficiency of plating/stripping only decays by 5%; the deposition overpotential increases more substantially with aging, however. Electrochemical Impedance Spectroscopy (EIS) was used to analyze the transport properties of the solutions. Other properties, such as composition and structure of electrolytic solutions, the morphology of Mg deposits, and thermal stability will be also discussed to explore the precise mechanism by which this and Mg electrolytes formed by other chloride salts form, as well as their feasibility for future magnesium battery applications. Barile, Christopher J. et al. J. Phys. Chem. C 118.48 (2014): 27623-27630 Vardar, Gulin, et al. Chem. Mater. 28.21 (2016): 7629-7637 Figure 1

The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1.

In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadiiIMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

Characterizing responses to gamma radiation by a highly clonogenic fish brain endothelial cell line

PurposeThe clonogenic property and radiobiological responses of a fish brain endothelial cell line, eelB, derived from the American eel were studied. MethodsClonogenic assays were performed to determine the plating efficiency of the eelB cells and to evaluate the clonogenic survival fractions after direct irradiation to low-dose low-LET gamma radiation or receiving irradiated cell conditioned medium in the bystander effect experiments. ResulteelB had the second highest plating efficiency ever reported to date for fish cell lines. Large eelB macroscopic colonies could be formed in a short period of time and were easy to identify and count. Unlike with other fish clonogenic cell lines, which had a relatively slow proliferation profile, clonogenic assays with the eelB cells could be completed as early as 12 days in culture. After direct irradiation with gamma rays at low doses ranging from 0.1Gy to 5Gy, the dose-clonogenic survival curve of the eelB cell line showed a linear trend and did not develop a shoulder region. A classical radio-adaptive response was not induced with the clonogenic survival endpoint when the priming dose (0.1 or 0.5Gy) was delivered 6h before the challenge dose (3 or 5Gy). However, a radio-adaptive response was observed in progeny cells that survived 5Gy and developed lethal mutations. eelB appeared to lack the ability to produce damaging radiation-induced bystander signals on both eelB and HaCaT recipient cells. ConclusioneelB cell line could be a very useful cell model in the study of radiation impacts on the aquatic health.

Electrochemical deposition of iridium and iridium-nickel-alloys

To develop durable and reliable electronic contacts, precious metals are still very important for finish plating of contact surfaces. The lesser-known iridium might be an interesting alternative to substitute gold alloys, platinum or rhodium for applications with highest demands to wear and corrosion resistance such as sliding and plug contacts. As matters stand there is no commercial electrolyte for iridium plating. Initial investigation screened the parameter range for different iridium compounds when an iridium layer occurred on the substrates. This approach showed that the oxidation state of iridium is crucial to reach contenting deposits. Best results came from Ir(IV) electrolyte with high bromine concentration coming from the starting compound, while electrolytes made from Ir(III) compounds gave very poor deposits. In subsequent experiments different organic compounds were added to the electrolytes to improve plating efficiency and stability of the solutions. So found electrolytes gave crack-free deposits up to two microns with a micro-hardness of 600 HV. To reduce the iridium content in the layer, iridium-nickel-alloys were investigated, finding that a nickel-content of 10 wt% raised the layer hardness to more than 900 HV.

Effect of dose rate of X-ray on clonogenic formation in human lung cancer cell line A549

Objective To explore the effects of different dose rates of X-ray under the same dose on cell clonogenic formation in non-small-cell lung cancer cell line A549 in order to provide experimental basis for clinical radiotherapy plan. Methods The A549 cells were cultured at low density and irradiated with X-rays at dose of 4 Gy and selected dose rates of 1, 2, 4 and 6 Gy/min, respectively, from a linear accelerator. The 8th day after irradiation, the cells were fixed and stained with Giemsa solution, and colonies containing at least 50 cells were counted. The plating efficiency and surviving fraction were calculated. Results The clonogenic number in non-irradiated cells was 88.6±4.6. The numbers were significantly reduced in irradiated cells at dose rate 1, 2, 4 and 6 Gy/min (12.3±3.4, 9.0±0.8, 5.6±1.0, 11.5±1.7, respectively) than that in non-irradiated control cells (F = 678.799, P < 0.05). The plating efficiencies were decreased in irradiated cells, especially in 4 Gy/min irradiated cells, which was lower than that in any of the other three dose rate groups (P < 0.05). Conclusions Though at same radiation dose, cancer cells have different clonogenic formation efficiency when irradiation with X-ray at different dose rates. Thus, treatment with optimal dose rate may improve the radiotherapy efficacy. Key words: Lung neoplasms; X-rays; Radiation dose rate; Cell clonogenic formation; Radiotherapy

In cancer cell lines inhibition of SCF/c-Kit pathway leads to radiosensitization only when SCF is strongly over-expressed

Background and purposeThe SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced tumorigenesis, proliferation and migration. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a significant radiosensitization and whether a respective biomarker may be identified. Material and methodsExperiments were performed with different cancer cell lines (NSCLC: H23, H520, H226, H1975 and PrCa: DU145) growing either under 2D or 3D conditions. Expression of SCF and c-Kit was determined by RT-PCR and Western blot, SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cell survival was determined by colony formation assay. ResultsThere is a profound variation in the expression of both c-Kit and SCF with no association between each other. Neither levels did correlate with the respective cellular radiosensitivity determined for 2D or 3D with only a trend seen for SCF. Knock-down of SCF was generally found to result in no or only minor reduction of plating efficiency or cellular radioresistance. A significant reduction was only obtained for H520 cells characterized by an extreme over-expression of SCF. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization. ConclusionGenerally, the SCF/c-Kit pathway does not have a dominant effect on both, cell survival and radioresponse and, as a consequence, knockdown of this pathway does not result in a strong effect on radioresistance, except when SCF is strongly over-expressed.

Phage "delay" towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages.

In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their “vigor” when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such “less-robust” mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically “running away” to more phage-free locations. Delay mechanisms—to the extent that they are less specific in terms of what phages are targeted—collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

Isolation, characterization and evaluation of virulent bacteriophages against Listeria monocytogenes

Bacteriophages are considered a potential antimicrobial food intervention due to their ability to inactivate target bacteria in different environments. This study was undertaken to isolate, characterize, and identify a mixture of phages that could be used for control of Listeria monocytogenes. A total of 37 phages were isolated from raw sewage sludge using L. monocytogenes serotypes 4b, 1/2a and 1/2b strains. The host range of these phages was determined by spot-testing. Ten lytic phages were selected for further evaluation with efficiency of plating (EOP) analysis and characterization by pulsed field gel electrophoresis (PFGE). According to the spot tests and EOP results, the isolated phages against L. monocytogenes 4b strains were found to be capable of lysing a wider range of ribotypes. Also, three phages specific for 1/2a and 1/2b serotypes had similar lysis capabilities over a range of target Listeria with high EOP values between 0.5 and 1.0. PFGE analysis identified 5 distinct restriction profiles of the 10 selected phages whose DNA was digested with either EcoRI or HinDIII. Transmission electron microscopy (TEM) pictures revealed that five selected phages belonged to the Myoviridae and one phage to the Podoviridae family. Based on screening results, a phage cocktail (LP1) comprised of six lytic phages was used to inactivate a mixed culture of five L. monocytogenes strains. LP1 was able to lyse bacterial cells within 30–60 min at multiplicity of infection values (MOI, PFU/CFU) of 1000 and maintained the target cells under detection levels for 24 to 48 h at 4, 10, and 30 °C. These results suggested the LP1 phage collection contained effective and strongly lytic phages that may serve as antibacterial preparation to control L. monocytogenes of different serotypes.

Dysfunction of Bone Marrow Endothelial Progenitor Cells from Subjects with Poor Graft Function Following Allogeneic Hematopoietic Stem Cell Transplantation Can be Improved By Atorvastatin

BackgroundPoor graft function (PGF) is a serious complication post allo-HSCT. The definition is a hypo- or aplastic bone marrow with 2 or 3 of the following: (1) neutrophils ≤0.5×10E+9/L; (2) platelets ≤20×10E+9/L; and/or (3) hemoglobin concentration ≤70 g/L for at least 3 consecutive days after day +28 post-HSCT. The mechanisms of PGF remain poorly understood. Murine studies suggest that endothelial progenitor cells (EPCs) are preferential supporting cells for hematopoietic stem cells (HSCs) in the bone marrow (BM) microenvironment. In addition, our previous prospective, nested case-control study found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PGF after allo-HSCT. However, little is known about the functional role of BM EPCs and how to improve impaired BM EPCs in PGF.Atorvastatin is widely used in the treatment of dyslipidemia and associated vascular abnormalities. Furthermore, atorvastatin has been reported to improve the mobilization and function of circulating EPCs in a number of diseases, such as heart disease, diabetes and others. Nevertheless, no previous studies have focused on the roles of atorvastatin on bone marrow-derived EPCs in subjects with poor graft function following allo-HSCT.AimsTo evaluate the function of BM EPCs in subjects with PGF. Moreover, to investigate the effect of atorvastatin on the number and function of cultivated BM EPCs derived from subjects with PGF and its underling molecular mechanisms.MethodsThree cohorts were included: subjects with PGF (N=23), subjects with good graft function (N=23), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=23). Atorvastatin (0.5nM,50nM,500nM) was administrated to the 5 day cultivated BM EPCs from subjects with PGF until tested on day 7. The number and functions of CD34(+)/CD133(+)/KDR(+)EPCs were evaluated by flow cytometry, cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, tube formation test and apoptosis. Reactive Oxygen Species (ROS) level was evaluated by flow cytometry. Cell proliferation was determined by cell counting kit-8 assay. Protein expression for ERK,JNK, p38, Akt was measured by flow cytometry and western blots. EPCs-CD34+ co-culture system and Colony-forming unit (CFU) assays were used to evaluate the supporting effect that atorvastatin exposure on hematopoietic progenitors in vitro.ResultsHuman BM EPCs was characterized by the spindle shape and expression of CD34, CD309 and CD133 at day 7 of cultivation. Dysfunctional BM EPCs, which were characterized by impaired proliferation, migration, angiogenesis, and higher levels of reactive oxygen species and apoptosis, were revealed in subjects with PGF. Activation of p38 and its downstream transcription factor cAMP-responsive element-binding protein (CREB) were detected in BM EPCs from subjects with PGF. Furthermore, the number and function of BM EPCs derived from subjects with PGF were enhanced by atorvastatin treatment in vitro through down-regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. The colony-forming unit plating efficiency in CD34+ BM cells was also improved by atorvastatin when co-cultured with BM EPCs from subjects with PGF. Atorvastatin demonstrated similar improvement effects on the number and function of bone marrow EPCs as ROS inhibitor and p38 inhibitor from subjects with PGF.ConclusionIn summary, dysfunctional BM EPCs were observed in subjects with PGF. Atorvastatin treatment in vitro quantitatively and functionally improved BM EPCs derived from subjects with PGF through down-regulation of the p38 MAPK pathway. These data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in subjects with PGF post-allotransplant. DisclosuresNo relevant conflicts of interest to declare.

HOPX functions as a tumour suppressor in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is generalized term that encompasses a diverse group of cancers that includes tumours of the oral cavity (OSCC), oropharynx (OPSCC) and nasopharynx (NPC). Genetic alterations that are common to all HNSCC types are likely to be important for squamous carcinogenesis. In this study, we have investigated the role of the homeodomain-only homeobox gene, HOPX, in the pathogenesis of HNSCC. We show that HOPX mRNA levels are reduced in OSCC and NPC cell lines and tissues and there is a general reduction of HOPX protein expression in these tumours and OPSCCs. HOPX promoter methylation was observed in a subset of HNSCCs and was associated with a worse overall survival in HPV negative tumours. RNAseq analysis of OSCC cells transfected with HOPX revealed a widespread deregulation of the transcription of genes related to epithelial homeostasis and ectopic over-expression of HOPX in OSCC and NPC cells inhibited cell proliferation, plating efficiency and migration, and enhanced sensitivity to UVA-induced apoptosis. Our results demonstrate that HOPX functions as a tumour suppressor in HNSCC and suggest a central role for HOPX in suppressing epithelial carcinogenesis.

PM 7/128 (1) Xanthomonas axonopodis pv. allii

Specific scopeThis Standard describes a diagnostic protocol for Xanthomonas axonopodis pv. allii.This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2016‐09.

Life without Division: Physiology of Escherichia coli FtsZ-Deprived Filaments.

ABSTRACTWhen deprived of FtsZ, Escherichia coli cells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress.