Standard Colony Research Articles

Comparison of Performance of Rapid Petrifilm Test Method and Standard Test Method for Enumeration of Aerobic Microorganisms, Coliforms and E.coli in Food

Assuring food safety is currently a global challenge. Frequent testing of food samples for the presence of microorganisms should be performed as part of any food safety program. The conventional tests are in general laborious and time consuming. In this context, the use of alternative rapid tests becomes important. The objective of this study was to compare the performance of Petrifilm test method and standard colony count method for enumerating aerobes, Coliforms and Escherichia coli . The Aerobic Plate Count (APC), Coliform Plate Count (CPC) and E. coli obtained using Rapid Petrifilm test method were compared with the corresponding standard methods using 24 samples of 3 different foods, namely poultry, ready to serve drink (RTS) and milk powder. The counts obtained using Rapid Petrifilm method were not significantly (p E. coli were 0.9817, 0.9969 and 0.9983, respectively. The study shows that the Rapid Petrifilm Test method can effectively be used for APC and CPC for foods. Tropical Agricultural Research Vol. 23 (4): 363-369 (2012) DOI: http://dx.doi.org/10.4038/tar.v23i4.4872

227 Association of K-Ras Genotype with Xenograft Tumor Response to the Investigational Proteasome Inhibitor MLN9708

The investigational drug MLN9708 is a potent, reversible and specific inhibitor of the proteasome. MLN9708 is currently being evaluated in clinical trials of hematologic malignancies (Phase 1, 2, and 3) as well as solid tumors (Phase 1 and 2). Upon exposure to aqueous solutions or plasma, MLN9708 immediately hydrolyzes to MLN2238, the biologically active form. In vitro, MLN2238 is potent across a broad range of solid tumor cell types evaluated with standard cell viability and colony formation assays. However, not all the cells which respond to MLN2238 in vitro are responsive in vivo when grown as xenografts in immunocompromised mice. In order to understand the contributing factors that determine sensitivity to MLN2238, we evaluated tumor PK, tumor PD (proteasome inhibition), and sequencing of commonly mutated gene in a panel of xenograft tumors. Tumor PK and PD did not show any difference between MLN2238 sensitive and resistant models. A survey of a number of NSCLC and colon xenografts, including both cell line-derived and primary human xenografts, showed a striking correlation between KRas genotype and MLN2238 response. Tumors with WT Kras showed increased sensitivity to MLN2238 when compared to KRas mutant tumors. This relation was not evident with any other common gene mutations included in Oncocarta panel. To confirm our finding that KRas mutation impacts MLN2238 activity, we used SW48 isogenic colon cell lines, in which KRas-G13D mutation was introduced in SW48 cells (KRas WT) to generate stable SW48-KRas-G3D cells. Although in vitro sensitivity to MLN2238 was similar between the paired cell lines, in vivo studies showed a difference in sensitivity. MLN2238 dosed at MTD (13mg/kg IV BIW) showed significant antitumor activity in SW48 xenografts (T/C =0.45), but no antitumor activity in SW48-KRasG13D xenografts (T/C =1.08), thus confirming the association of KRas status and response to MLN2238. In order to understand the mechanism of resistance, we are evaluating the regulation a variety of markers related to KRas signaling. Recent literature has shown that KRas mutant cells exhibit altered glucose metabolism and glycolysis and can proliferate and survive under low glucose conditions by changing glucose transporter expression. We observed significantly higher protein levels of the glucose transporter Glut4 in MLN2238 resistant xenografts compared to the MLN2238 sensitive xenografts. Work is ongoing to understand if Glut4 expression may account for the differential survival of KRas WT vs. mutant xenograft tumors exposed to MLN2238. Ras mutation status will be evaluated in patient samples from future MLN9708 solid tumor trials.

Evaluation of the new OxyPlate™ Anaerobic System for the isolation of ocular anaerobic bacteria.

Anaerobic bacteria can cause ocular infections. We tested the OxyPlate™ Anaerobic System (OXY) to isolate pertinent anaerobic bacteria that can cause ocular disease. OXY, which does not require direct anaerobic conditions (i.e. bags, jars), was compared to conventional isolation of incubating culture media in anaerobic bags. Standard colonies counts were performed on anaerobic ocular bacterial isolates under aerobic and anaerobic conditions (anaerobic bags) using agar media: 1) OXY (aerobic only), 2) 5% sheep blood (SB), 3) Chocolate, and 4) Schaedler. The bacteria tested were de-identified ocular isolates cultured from endophthalmitis and dacryocystitis that include 10 Propionibacterium acnes and 3 Actinomyces species. The colony counts for each bacteria isolate, on each culturing condition, were ranked from largest to smallest, and non-parametrically compared to determine the best culturing condition. All anaerobic conditions were positive for all of the anaerobic isolates. SB and Schaedler's agar under aerobic conditions did not support the growth of anaerobic bacteria. Sparse growth was noted on chocolate agar with Propionibacterium acnes. As an anaerobic system, SB in an anaerobic bag isolated higher colony counts than OXY (P=0.0028) and chocolate agar (P=0.0028). Although OXY did not test to be more efficient than other anaerobic systems, it appears to be a reasonable alternative for isolating anaerobic bacteria from ocular sites. The use of an agar medium in a specially designed plate, without the requirement of an anaerobic bag, rendered OXY as an advantage over other anaerobic systems.

Asymptomatic presence of Nosema spp. in Spanish commercial apiaries

Nosemosis is caused by intracellular parasites (Nosema apis and Nosema ceranae) that infect the midgut epithelial cells in adult honey bees. Recent studies relate N. ceranae to Colony Collapse Disorder and there is some suggestion that Nosema spp., especially N. ceranae, induces high mortality in honey bees, a fact that is considered as a serious threat for colony survival.604 samples of adult honey bees for Nosema spp. analysis were collected from beekeeping colonies across Spain and were analysed using PCR with capillary electrophoresis. We also monitored 77 Andalusian apiaries for 2years; the sampled hives were standard healthy colonies, without any special disease symptoms.We found 100% presence of Nosema spp. in some locations, indicating that this parasite was widespread throughout the country. The two year monitoring indicated that 87% of the hives with Nosema spp. remained viable, with normal honey production and biological development during this period of time. The results of these trials indicated that both N. ceranae and N. apis could be present in these beehives without causing disease symptom and that there is no evidence for the replacement of N. apis by N. ceranae, supporting the hypothesis that nosemosis is not the main reason of the collapse and death of beehives.

Abstract 1203: Targeting FGFR4 in colorectal cancer cells

Abstract Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in the regulation of cell proliferation, differentiation and survival as well as tumor development and therefore constitute prominent targets for cancer therapy. Over-expression of FGFR4 has been described in various cancer types and was also related to tumor aggressiveness. Reports concerning the role of FGFR4 in colorectal cancer (CRC) are still under dispute. In our study we found FGFR4 expression to be significantly up-regulated in human CRC tissue compared to normal mucosa. CRC cell line models over-expressing FGFR4 were created by transfecting SW480, HCT116 and HT29 colon carcinoma cells with a FGFR4 expression vector. This resulted in an increase of cell proliferation and colony formation under standard tissue culture conditions and colony outgrowth in soft agar was stimulated. In xenografted SCID mice tumor growth was significantly increased by FGFR4 over-expression. siRNA mediated knock down of FGFR4 in the high FGFR4 expressing cell lines HCT116 and HT29 resulted in decreased cell proliferation, viability and colony outgrowth in 2-dimensional growth assays. Additionally a dominant negative FGFR4 adenoviral construct inhibited 3-dimensional anchorage independent growth and suppressed tumor growth in vivo almost completely. Ongoing experiments analyze the effects of a soluble FGFR4 variant on 2-dimensional and 3-dimensional growth. FGFR4 up-regulation resulted in activation of survival pathways via FRS2 and PI3K leading to phosphorylation of GSK3β and S6. Signaling effects by blockage of FGFR4 dependent signaling via siRNA and the dominant negative adenoviral construct are currently being investigated. Based on our results we identified the FGFR4 as an important oncogene in the carcinogenesis which is involved in the tumor growth regulation of CRC in vitro and in vivo. Consequently FGFR4 should be regarded as a new therapeutic target in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1203. doi:1538-7445.AM2012-1203

Abstract 5646: Allele-specific shRNA to Nanog family members inhibits 3-D growth of colorectal carcinoma through the intrinsic apoptotic pathway

Abstract Nanog and NanogP8 modulate stemness of human colorectal cancer (CRC) as measured by directly affecting spherogenicity, tumorigenicity and metastatic potential. As a result, we tested whether targeting NanogP8 (the dominant Nanog member expressed in human CRC lines and liver metastases) by specific lentiviral (LV) shRNA inhibits the growth of CRC in 3-D spheroids in suspension culture. We first designed allele-specific shRNAs to NanogP8 and Nanog, genes that differ only in 5 nucleotides in a 915 nt ORF, and demonstrated that the shRNA to NanogP8 (shNp8-1) and Nanog (shNG-1) specifically inhibited their target in PA-1, a human embryonal carcinoma line expressing only Nanog, or CX-1, a CRC that expresses predominantly NanogP8. When Clone A or CX-1 CRC cells transfected with Luc2 were cultured for 24 hours in suspension in serum-free medium (SFM), transduced with LV shNP8-1, shNG-1 or a control shRNA (shNeg) at a multiplicity of infection (MOI) 8 and cultured for another 72 hours in SFM, all of the LV vectors transduced 40 - 75% of the cells in 3-D constructs. shNp8-1 transduction significantly reduced the mass of cells and luciferase activity by 50%. Apoptosis was induced because CRC treated with shNp8-1 had significantly more annexin V+ cells than did shNEG or untreated parental cells. Previous work in our lab demonstrated that caspase 8 was important in anoikis - the apoptosis caused by suspension of cells whose growth generally requires adherence. Caspase 3, 8 and 9 activity was measured by fluorogenic substrates in Clone A and CX-1 cells at 4 days of suspension culture, 3 days after LV treatment. Caspase 3 activity was increased by shNp8-1 by 2.5 to 3-fold in both CX-1 and Clone A compared to untreated cells. shNp8-1 treatment increased Caspase 9 activity by ∼50% in both CRC lines compared to untreated cells whereas shNEG and shNG-1 transduction did not increase Caspase 9 activity significantly. Treatment with a Caspase 9 peptidomimetic blocked apoptosis and increased CX-1 survival significantly. When cells were harvested at the end of the 4 day apoptosis assay and replated in a standard colony forming assay, CX-1 cells transduced with shNp8-1 LV produced 33% fewer colonies than did the shNEG and shNG-1 controls. These results indicate that shNp8-1 transduction may activate apoptosis through a Caspase 9-dependent pathway and that this treatment may have long term effects that inhibit tumor regrowth after treatment. Supported by Center for Cancer Research funds and Department of Defense IDEA grant W81XWH-11-1-0327. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5646. doi:1538-7445.AM2012-5646

Torch Relay Route Optimization by Using the Artificial Intelligence

Ceremonial fire is an important symbol for an athletic game. Through the torch relay process, the atmosphere of peace and union is delivered and people are encouraged to participate in the athletic games together. Based on the data obtained from the global position system (GPS), in the study, various artificial intelligence approaches were used to solve the torch relay routing problem. The objective of the torch relay routing problem was to minimize the total distance of torch relay route. By reasonably combining genetic algorithms (GA) and particle swarm optimization (PSO), we developed a fast and easily implemented hybrid algorithm (HA) for solving the considered problem. The effectiveness and efficiency of the proposed HA were demonstrated and compared with those of standard ant colony optimization (ACO), PSO and GA by numerical results of the simulated instance with 10 spots and the real torch relay routing problems of National Intercollegiate Athletic Games in 2010. Numerical results indicate that the total distance of torch relay routes by HA was 335 km, which was 11.68% shorter than the original routes adopted by National Intercollegiate Athletic Games in 2010. It implies that the proposed HA can use the GPS information to schedule the torch relay routes, and it can reduce the cost of reconnoitering and management in an athletic game. Therefore, the proposed HA approach is an effective approach, and it can improve the efficiency for an athletic game.

Piezoelectric tuning fork based mass measurement method as a novel tool for determination of antibiotic activity on bacterial biofilm

Abstract In this work an application of quartz tuning forks (QTF) in Pseudomonas aeruginosa biofilm growth monitoring is presented. QTF-based mass measurement method was examined regarding its applicability in P. aeruginosa biofilm mass evaluation during antibiotic treatment. Two antibiotics (gentamicin and ciprofloxacin) were tested on their antibacterial activity. Three independent methods were also applied for the experiments as a control for the performed QTF measurements: standard colony count of living cells, crystal violet colorimetric method for biofilm monitoring and microscopy visualization. Using quartz tuning forks the inhibition of Pseudomonas aeruginosa biofilm formation during antibiotic treatment was observed in correlation with other standard tests. These several experiments proved suitability and potential application of the presented technique in biofilm growth measurement for antibacterial compound activity evaluation.

Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G(2)/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.

Zygomycetes from “Reserva Biológica de Mogi Guaçu”, São Paulo State, Brazil

The taxonomic composition of zygomycetes from a reserve of Brazilian Cerrado was analyzed. Soil and leaf litter samples were collected at five sampling dates. Cultures of Absidia, Backusella, Circinella, Rhizopus and Conidiobolus were obtained from canopy- and soil-plates. Due to the scarcity of detailed taxonomic information for Brazilian zygomycetes, additional information such as descriptions, standard colony colour codes, illustrations, geographical coordinates and vouchers are provided for Absidia spinosa var. spinosa, Backusella lamprospora, Circinella simplex, Rhizopus stolonifer var. stolonifer and Conidiobolus coronatus.

Inhibition of STAT-3 results in greater cetuximab sensitivity in head and neck squamous cell carcinoma

ObjectiveThe inhibition of epidermal growth factor receptor (EGFr) with the monoclonal antibody cetuximab reduces cell proliferation and survival which correlates with increased DNA damage. Since the signal transducer and activator of transcription-3 (STAT-3) is involved in the EGFr-induced signaling pathway, we hypothesized that depletion of STAT-3 may augment cetuximab-induced processes in human head and neck cancer cells. Materials and methodsHuman head and neck squamous carcinoma cells (UM-SCC-5) were transfected with short hairpin RNA (shRNA) against STAT-3 (STAT3-2.4 and 2.9 cells). A mutated form of this shRNA was transfected for a control (NEG4.17 cells). Radiosensitivity was assessed by a standard colony formation assay. Proliferation was assessed by daily cell counts following treatment and apoptosis was assessed by an annexin V-FITC assay. The alkaline comet assay was used to assess DNA damage. ResultsThe STAT-3 knockdown cells (STAT3-2.4 and STAT3-2.9 cells) demonstrated enhanced radiosensitivity compared to control NEG4.17 cells, which correlated with increased apoptosis. Also, the STAT-3 knockdown cells demonstrated decreased proliferation with cetuximab treatments compared to control cells (NEG4.17). The increased cetuximab sensitivity of the STAT-3 knockdown cells correlated with increased apoptosis and DNA damage compared to control cells (NEG4.17). ConclusionThese studies revealed that the greater anti-proliferative effects and increased cytotoxicity of cetuximab in the STAT3-2.4 and STAT3-2.9 cells compared to control NEG4.17 cells, may be a result of STAT3-mediated effects on cellular apoptosis and DNA damage.

Abstract 1092: Insulin-like growth factors modulate chemosensitivity to cisplatin and 5-fluorouracil in human esophageal cell lines

Abstract Resistance to chemotherapy remains a major obstacle to survival of patients with esophageal adenocarcinoma (EADC). The aim of this study was to evaluate whether insulin-like growth factors (IGFs), recently implicated in the molecular pathogenesis of EADC, modulate chemosensitivity to Cisplatin (CDDP) and 5-Fluoruracil (5-Fu), two agents widely used in current clinical practice. Expression of IGF1, its receptor (IGF1R), and IGF2 (mRNA and protein) were studied using quantitative (real-time) PCR and Western blot in human esophageal cell lines: Het1A (derived from immortalized normal esophageal epithelium), OE33 and JHEsoAd1 (each derived from a primary EADC). For each cell line, the effect of various concentrations of IGFs, CDDP and 5-Fu, alone and in combination, on cell proliferation and clonogenicity were evaluated by standard MTT and colony formation assays, respectively. Changes in expression of 84 critical genes downstream from IGF1R were studied by PCR-array. Relative to Het1A, IGF1 and IGF2 mRNA were significantly (P<0.05) underexpressed by OE33; by contrast, JHEsoAd1 underexpressed IGF1 but overexpressed IGF2. Administration of either IGF1 (250ng/ml) or IGF2 (500ng/ml) to cell cultures resulted in increased cellular proliferation of JHEsoAD1 cells (after IGF1: 1.24+/-0.05 vs. 1.00+/-0.00 untreated, P<0.01; after IGF2: 1.15+/-0.06 vs. 1.00+/-0.06 untreated, P<0.05). As expected, cell proliferation and clonogenicity of all cell lines were inhibited by CDDP and/or 5-FU (at all dosages ranging from 1-4ug/ml). However, these cytotoxic effects were overcome by the administration of either IGF1 or IGF2 with the exception of: 1) JHEsoAd1 cells treated with CDDP, where IGF2 administration further reduced cellular proliferation (0.35+/-0.06 vs. 0.53+/-0.03 for cells treated with CDDP alone; P<0.05), and 2) JHEsoAd1 cells treated with 5-Fu, where IGF1 administration significantly reduced clonogenicity (0.07+/-0.07 vs. 0.49+/-0.06 for cells treated with 5-Fu alone; P<0.05). PCR-array analysis revealed up-regulation (Het1A and JHEsoAd1) and down-regulation (OE33 and JHEsoAd1) of selected PI3K/AKT pathway genes following IGF1 and IGF2 therapy. These pre-clinical studies confirm a central role for the IGF axis in esophageal tumor biology, with potential for IGFs to enhance the cytotoxic efficacy of standard chemotherapeutic agents in esophageal cell lines. The identification of novel molecular regulatory pathways downstream from IGF1R modulated by IGF therapy may well inform future cytotoxic and targeting strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1092. doi:10.1158/1538-7445.AM2011-1092

Abstract 2508: Identification of potential relevant pathways and genes for resistance to chemoradiotherapy in colorectal cancer cells

Abstract The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, the tumor response to multimodal treatment has varied greatly and ranging from complete resistance to complete pathologic regression. The prediction and treatment of these observed heterogeneous response to therapy in patients is, therefore, an important clinical need. A strategy for studying the molecular basis of this heterogeneous tumor response is to establish the clinical parameters as an in vitro model. For this purpose we used 12 colorectal cancer cell lines and exposed them to 3 µM of 5-fluorouracil and 2 Gy of radiation, which reflect the parameters used in clinical patient treatment. The sensitivity differences of the cell lines to chemoradiotherapy, measured as surviving fractions by a standard colony formation assay, were then correlated with the pretherapeutic gene expression profiles of these cell lines. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. In the 12 colorectal cancer cell lines we observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q < 0.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for the treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. We anticipate that this analysis of a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2011-2508

Abstract 790: Are laboratory mice too cold to mount effective anti-tumor immune responses

Abstract During the course of experiments to determine the effects of elevated body temperature on anti-tumor immune responses, we unexpectedly observed that in control animals bearing tumors and maintained under standard room temperatures (IACUC-compliant, 21-23°C), the core temperature actually began to fall significantly as the tumors grew larger. Moreover, using thermal preference analyses, we found that animals bearing tumors exhibit considerable heat-seeking behavior. In an attempt to keep body temperature within the normal range (∼37°C), we began to house tumor bearing mice in a warmer environment (i.e., 28-30°C) considered to be the thermoneutral temperature for mice, as apposed to IACUC compliant temperatures which are considered mildly hypothermic for mice. Decisions that have resulted in the mildly cooler environmental temperature required in animal colonies have been generally based upon optimizing air-handling capacity of fumes and the thermal comfort of personnel rather than the optimal thermal comfort of animals (hence the requirements for multiple mice per cage to allow for huddling). When we analyzed tumor growth under thermoneutral conditions, syngeneic tumor growth was significantly delayed in animals maintained at 28-30°C compared to tumor growth in animals maintained at 21-23 oC. If the same comparison is made using SCID mice, no difference in tumor growth rate is observed. More specifically, BALB/c, C57BL/6 and SCID mice were housed in either standard room temperature (RT = 21-23°C) or in a warmer ambient temperature (WR = 28-30°C). Mice were then implanted with a syngeneic tumor (CT26 for BALB/c and SCID; B16.F10 for C57BL/6) subcutaneously and tumor growth was monitored. Tumor growth in C57BL/6 and BALB/c mice was inhibited when mice were housed in the warmer ambient temperature compared to animals maintained in standard room temperature. There was no difference in CT26 tumor growth in SCID mice. This finding suggested that the T cell-dependent anti-tumor immune response may be at least partly responsible for enhanced tumor growth control under conditions of warmer ambient temperature. Results of depletion studies in C57BL/6 mice bearing B16.F10 tumors, showed that both CD4+ and CD8+ T cells were required for inhibition of tumor growth in the warmer ambient temperature. In BALB/c mice bearing CT26 tumors, only CD8+ T cells were required to inhibit tumor growth. These findings suggest that the natural anti-tumor immune response exhibited by mice housed in standard colony conditions is sub-optimal and can be improved simply by housing mice under thermoneutral conditions. T cell dependent anti-tumor immunity appears to be at least one immune mechanism which is improved when animals are not cold stressed. Supported by NIH R01 CA 135368. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2011-790

Impact of oncogenic K-RASon YB-1 phosphorylation induced by ionizing radiation

IntroductionExpression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.MethodsIR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated.ResultsThe average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked repair of DNA-DSB. Likewise, YB-1 siRNA increased radiation sensitivity.ConclusionsIR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-Ras or IR enhances repair of DNA-DSB and postirradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways.

Piezoelectric Tuning Fork Mass Sensors as a Novel Tool for Determination of Antibiotic Activity on Pseudomonas Aeruginosa Biofilm

In this work an application of quartz tuning forks (QTF) in P. aeruginosa biofilm growth monitoring is presented. QTF-based mass measurement method was examined regarding its applicability in Pseudomonas aeruginosa biofim mass evaluation during antibiotic treatment. Two antibiotics (gentamicin and ciprofloxacin) were tested on their antibacterial activity. Three independent methods were also applied for the experiments: standard colony count of living cells, crystal violet colorimetric method for biofilm monitoring and atomic force microscopy visualization. Using quartz tuning forks the inhibition of Pseudomonas aeruginosa biofilm formation during antibiotic treatment was observed in correlation with other standard tests. These several experiments proved suitability and potential application of the presented technique in biofilm growth measurement for antibacterial compound activity evaluation.

Tempo® most probable number technique for the enumeration yeasts and moulds in feed and food products

The large and diverse group of microscopic foodborne yeasts and moulds includes several hundred species. Both yeasts and moulds cause various degrees of deterioration and decomposition of food. They can invade and grow on virtually any type of food at any time, e.g. they invade field crops such as small grains, nuts, beans, tomatoes, and apples both in the field before harvesting and during storage. They also grow on processed foods and food mixtures. Their detectability in or on foods depends on food type, organisms involved and degree of invasion. Thanks to the research it was possible to evaluate how useful is automated TEMPO? system (bioM?rieux) for deterimining the total number of yeasts and moulds in feedand foodproducts. Twenty artificially contaminated foods were tested including dietetic products, seafoods and dairy products. Further, spices, food additives and animal feed were screened for natural contamination with yeasts and moulds. Statistical analysis of the results by linear regression proves the equivalence of the TEMPO? method and the standard colony count technique, at determination coefficient of 0,934. TEMPO? YM method can be considered as an effective, automated, accurate method for the enumeration of yeasts and moulds in feedand foodproducts. Traditional methods for enumeration of yeasts and moulds in food and feed products involve time - consuming plating techniques.

Lenalidomide Upregulates CXCR4 on CD34+ Hematopoietic Cells Resulting In Increased Binding to Bone Marrow Niche and Inhibiting Mobilization Into Peripheral Blood In Multiple Myeloma Patients

AbstractAbstract 4079 Background:In multiple myeloma (MM), lenalidomide has impressive clinical activity in patients with both relapsed/refractory and newly diagnosed disease. Nevertheless hematopoietic stem cell transplantation (HSCT) is still the “backbone” in the treatment of newly diagnosed MM patients and very often lenalidomide treatment (as induction therapy) is combined with HSCT in order to achieve high response rates. Clinical data have shown that in up to 43% of those patients, standard mobilization of CD34+ cells with G-CSF alone failed to mobilize significant numbers of hematopoietic progenitors into peripheral blood, raising concerns about potential stem cell toxicity of lenalidomide (Mazumder et al, Leukemia 2008). Interestingly mobilization of hematopoietic progenitors with AMD-3100 (Plerixafor) overcomes mobilization failures in almost all patients previously treated with lenalidomide. The fact that AMD-3100 antagonizes the binding of chemokine stromal- cell–derived factor-1α (SDF-1α) to CXC chemokine receptor 4 (CXCR4) suggests a potential role of the CXCR4/SDF-1α axis in mediating mobilization failure after lenalidomide treatment. Subsequently, the findings noted above raised questions on: 1) the stem cell toxicity of lenalidomide; 2) the underlying mechanism that mediates the development of G-CSF resistance; and 3) the mechanism of the modulation of the CXCR4/SDF-1α axis by lenalidomide. In our previous work we showed the following. 1) Lenalidomide neither inhibited colony formation in standard colony assays nor the development of cobble stone area forming cells (CAFC) in LTC-IC assays, suggesting that lenalidomide is not stem cell toxic (Koh et al, Blood 2005). 2) We further showed that lenalidomide significantly upregulated G-CSF secretion of CD34+ cells (600%), suggesting that the high levels of G-CSF may mediate a relative resistance towards G-CSF-induced mobilization (Pal et al, Blood, 2010). Results and Methods:We analyzed why blocking CXCR4 by AMD-3100 overcomes mobilization failure to G-CSF. We first examined the CXCR4 expression profile of CD34+ cells treated with lenalidomide. Lenalidomide treatment significantly upregulated the expression of CXCR4 on cell surface after 48h treatment measured by flow cytometry. Increased expression of CXCR4 onCD34+ cells remained high with continuous treatment. Western blot assay of the hydrophobic (membrane) and the hydrophilic (cytosol) cell fraction confirmed our data showing that lenalidomide increases the expression of CXCR4 on CD34+ cell surface. Confocal microscopy showed that lenalidomide inhibited SDF-1α induced CXCR4 internalization. In accordance with the increased CXCR4 surface expression, transwell migration assay revealed that the SDF-1α-induced migration of CD34+ cells in the presence of lenalidomide significantly increased by 52% in comparison to control. Quantitation of SDF-1α showed that CXCR4 had no significant effect on the secretion of SDF-1α by CD34+ cells and in human stromal cells. Result:In conclusion, our data show that lenalidomide is not toxic to hematopoietic progenitors. The strong increase of G-CSF secretion by CD34+ cells might contribute to a desensitization of CD34+ cells to G-CSF mobilization. Primarily our data indicate that increased expression of CXCR4 followed by blocked internalization, increases binding to SDF-1α secreted by the bone marrow niche. This subsequently prohibits the mobilization of CD34+ cells. These data suggest that blocking the CXCR4 receptor by AMD-3100 disrupts this circle and finally permits the mobilization of hematopoietic cells from the bone marrow niche into peripheral blood. This study provides novel insights into the effects of lenalidomide on CD34+ cells relevant for HSCT in MM. Disclosures:Roodman:Amgen: Consultancy; Celgene Corp: Consultancy; Acceleron: Consultancy; Millennium: Consultancy. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Impact of Prolonged Fraction Delivery Times Simulating IMRT on Cultured Nasopharyngeal Carcinoma Cell Killing

To determine the impact of prolonged fraction delivery times (FDTs) simulating intensity-modulated radiotherapy (IMRT) on cultured nasopharyngeal carcinoma (NPC) cell killing. Cultured NPC cell lines CNE1 and CNE2 were used in this study. The biological effectiveness of fractionated irradiation protocols simulating conventional external beam radiotherapy and IMRT (FDT of 15, 36, and 50 minutes) was estimated with standard colony assay, and the differences in cell surviving fractions after irradiation with different protocols were tested by use of the paired t test. The impact degree of prolonged FDTs (from 8 to 50 minutes) on cell killing was also assessed by the dose-modifying factors, which were estimated by comparing the effectiveness of intermittently delivered 2 Gy with that of continuously delivered 1.5 to 2 Gy. The cell surviving fractions of both CNE1 and CNE2 after fractionated irradiation simulating IMRT were higher than those simulating conventional external beam radiotherapy (p < 0.05). The dose-modifying factors for a fraction dose of 2 Gy increased from 1.05 to 1.18 for CNE1 and from 1.05 to 1.11 for CNE2 with the FDT being prolonged from 15 to 50 minutes. This study showed that the prolonged FDTs simulating IMRT significantly decreased the cell killing in both CNE1 and CNE2 cell lines, and these negative effects increased with the FDT being prolonged from 15 to 50 minutes. These effects, if confirmed by in vivo and clinical studies, need to be considered in designing IMRT treatments for NPC.

Friend leukaemia insertion (Fli)-1 is a prediction marker candidate for radiotherapy resistant oral squamous cell carcinoma

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), but its therapeutic effects are unpredictable. To determine which genes correlate with radiation resistance in oral cancer, the authors evaluated radiation sensitivity using a standard colony formation assay with a gene microarray system for seven OSCC cell lines. They found significant associations between dozens of gene-expression levels and radiation resistance of OSCC cell lines. Following analysis of the different radiosensitive cancer cell lines, the friend leukaemia insertion (Fli)-1 gene was selected as a prediction marker gene for OSCC radiotherapy resistance. Fli-1 expression was associated with radiation resistance in OSCC patients. These data help to predict the effects radiation therapy has on OSCC, in turn contributing to the development of alternative radiation therapies.