Growth Curve Experiments Research Articles

In vitro growth kinetics and gene expression analysis of the turkey adenovirus 3, a siadenovirus

Turkey adenovirus 3 (TAdV-3) belongs to the genus Siadenovirus, family Adenoviridae. Previously, nucleotide sequencing and annotation of the Virginia avirulent strain (VAS) of TAdV-3 genome, isolated in our laboratory, indicated the presence of a total of 23 genes and open reading frames (ORFs). The goals of this study were 1) to delineate the growth kinetics of the virus using a qPCR-based infectivity assay, and 2) to determine the virus gene expression profile during the early and late phases of infection in target B lymphocytes. The one-step growth curve experiment demonstrated 3 phases of virus replication cycle: a lag phase lasted for 12–18 h post-infection (h.p.i.), in which the virus titer declined; a log phase from 18 to 120 h.p.i., in which the number of infectious virus particles increased over 20,000 folds, and a brief decline phase thereafter. Southern blot analysis indicated that the synthesis of new viral DNA started by 8 h.p.i. Gene-specific RT-PCR analysis revealed the expression of mRNAs from the 23 TAdV-3 genes/ORFs. According to the temporal transcriptional profiling of TAdV-3 genome, genes could be divided into 3 groups based on the time of transcription initiation: group 1 showed detectable levels of transcription at 2 h.p.i and included 7 genes, i.e., hyd, III, pX, pVI, II, 100 K, and 33 K; group 2 included 12 genes whose mRNAs were detected for the first time at 4 h.p.i., i.e., ORF1, IVa2, pol, pTP, pIIIa, EP, DBP, E3, U exon, IV, ORF7, and ORF8; group 3 of transcripts were detectable starting 8 h.p.i. and included only 4 genes, i.e., 52 K, 22 K, pVII, and pVIII. Our data suggest that the transcriptional kinetics of genus Siadenovirus differ from that observed in other adenoviral genera; however, a few TAdV-3 genes showed similar expression patterns to their adenoviral homologs.

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli

ObjectiveTo evaluate the influence of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on biofilm formation and virulence factors of Escherichia coli clinical isolates. MethodsSub-MICs of CIP were determined using growth curve experiments. The biofilm-forming capacity of E. coli clinical isolates and E. coli ATCC 25922 treated or untreated with sub-MICs of CIP was assessed using a crystal violet staining assay. The biofilm structure of E. coli isolate was assessed with scanning electron microscopy (SEM). The expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were measured using quantification RT-PCR (qRT-PCR) in E. coli isolates and E. coli ATCC 25922. ResultsBased on our results, the sub-MICs of CIP were 1/4 MICs. Sub-MICs of CIP significantly inhibited biofilm formation of E. coli clinical isolates and E. coli ATCC 25922 (p<0.01). SEM analyses indicated that the biofilm structure of the E. coli changed significantly after treatment with sub-MICs of CIP. Expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were also suppressed. ConclusionsThe results revealed that treatment with sub-MICs of CIP for 24h inhibited biofilm formation and reduced the expression of virulence genes and biofilm formation genes in E. coli.

BAP18 Is Involved in Upregulation of CCND1/2 Transcription to Promote Cell Growth in Oral Squamous Cell Carcinoma

Background: As a reader of histone H3K4me3, BPTF associated protein 18 kDa (BAP18) is a subunit of histone methyltransferase complex. BAP18 is involved in modulation of androgen receptor action in prostate cancer. However, the function of BAP18 on Oral squamous cell carcinoma (OSCC) and its molecular mechanism remains to be elusive. Methods: OSCC derived cell lines carrying silenced BAP18 were establish by Lentiviral vector transfection. RNA sequence analysis, quantitative RT-PCR, western blot, ChIP were performed to detect gene transcription regulation and the possible mechanism. Colony formation, cell growth curve and xenograft tumor experiments were performed to examine cell growth and proliferation. Findings: Our study demonstrated that BAP18 was highly expressed in OSCC samples compared with that in benign. BAP18 depletion obviously influenced the expression of a series of genes, involved in multiple biological processes. We thus provided the evidence to demonstrate that BAP18 depletion significantly decreases CCND1 and CCND2 (CCND1/2) transcription. In addition, BAP18 facilitated the recruitment of core subunits of MLL1/WDR5 complex to the promoter regions of CCND1/2, consequently increasing histone H3K4me3 levels. Furthermore, BAP18 depletion delayed G1-S phase transition and inhibited cell growth in OSCC derived cell lines. Interpretation: These study suggest that BAP18 involved in modulation of CCND1/2 transcription promotes OSCC progression and could be a potential therapeutic target for OSCC treatment. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China [31871286 for Yue Zhao, 81872015 for Chunyu Wang, 31701102 for Shengli Wang, 81702800 for Renlong Zou]; Foundation for Special Professor of Liaoning Province for Yue Zhao (the 5th batch). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Animal works were approved by the Animal Ethics Committee of China Medical University. The collection of tissue samples was approved by the Human Research Ethics Committee of China Medical University. All samples’ patient informed consents were already obtained.

993. Combining Key Residues of the Russian and US Live-Attenuated Influenza Viruses for a More Attenuated Virus

BackgroundThe Live Attenuated Influenza Virus (LAIV) used in the United States is based on the cold-passaged A/AnnArbor/6/60 strain (AA). An alternative LAIV (Len), developed from the cold-passaged A/Leningrad/134/17/57 strain, has also been used in some countries outside the United States. Recent concerns with the efficacy and safety of the current US LAIV warrant the development of an improved LAIV.MethodsWe used in vitro minireplicon and multicycle viral growth assays to analyze the combined effects of polymerase mutations from LAIV (AA) and LAIV (Len) on the phenotype of PR8. Mini-replicon assays were performed in HEK-293T cells with firefly luciferase under the control of the influenza virus NP promoter; we controlled for cell density with a constitutively active Renilla luciferase. Multicycle growth curve experiments were performed at 33°C, 37°C, and 39°C in MDCK cells with an m.o.i. of 0.001. Mean values for triplicate infections at 12, 24, 48, and 72 hours were plotted as TCID50/mL.ResultsControl experiments showed replication of PR8 (AA) and PR8 (Len) in MDCK cells was significantly decreased as compared with WT PR8 at 37°C and 39°C at 24–48 hour time points, but not at 33C (the temperature of nasal passages). We found that polymerase activity was up to 3 logs more temperature-sensitive (ts) at 37°C and 39°C with the combined Len and AA mutations using the mini-replicon assay. In the growth curve experiments, the combined Len and AA mutations conferred up to a 4-log decrease in replication levels at 37°C as compared with PR8 (Len) and an even greater decrease compared with PR8 (AA).ConclusionOur findings suggest combining the AA and Len LAIV polymerase mutations decreases LAIV replication at body temperature (37°C), as compared with either LAIV alone. This could be useful in developing an improved LAIV that is safer in vulnerable hosts (e.g., children under the age of 2 who may be vulnerable to wheezing), while also permitting dose escalation that might result in greater efficacy.Minireplicon assay. Polymerase activity of combination mutants. Multicycle replication kinetics of combination mutants (red) against PR8 Len (black) at 33C (solid), 37C (dashed) and 39C (dotted). Disclosures All authors: No reported disclosures.

PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression.

To investigate the protective function of pigment epithelium-derived factor (PEDF) against oxidative stress (OS) in ARPE-19 cells, ARPE-19 cells were divided into different OS groups and treated with various concentrations of H2O2 (0, 75, 150 and 200 µmol/l) for 24 h. To establish the protective group, 200 ng/ml of PEDF was administered to ARPE-19 cells. Cell Counting Kit-8 assays and cell growth curve experiments were performed to determine levels of cell viability; lactate dehydrogenase and propidium iodide (PI) staining assays were also performed. The expression levels of genes associated with apoptosis as well as uncoupling protein 2 (UCP2) were detected by reverse transcription-quantitative, or semi-quantitative polymerase chain reaction. Furthermore, an OS injury animal model was established in both C57BL/6 and BALB/c mice via injection of 5 µg of PEDF in the vitreous cavity and subsequent injection of 150 µM H2O2 following a 24 h time interval. Hematoxylin and eosin (H&E) staining, as well as UCP2 immunofluorescent labeling were also performed. One-way analysis of variance was used to determine statistically significant differences, followed by multiple comparison analysis using the Newman Keuls method. The results of cell viability assays demonstrated that the numbers of apoptotic cells were increased following treatment with H2O2 in a dose-dependent manner; however, this effect was reversed following treatment with PEDF. The expression levels of caspase 3 and B cell lymphoma (Bcl2) associated X genes associated with apoptosis were inhibited, whereas levels of the anti-apoptotic gene Bcl2 were enhanced following treatment with PEDF in different passages of ARPE-19 cells. Significant differences were demonstrated in the levels of UCP2 gene expression between the PEDF+ H2O2 treated group and cells treated with H2O2 alone. Labeling of the UCP2 detector in the confocal images demonstrated decreased UCP2 protein staining in the retinal pigment epithelium (RPE) cells and RPE layers following H2O2 injury; however, this effect was inhibited following treatment with PEDF. H&E staining was performed to investigate the thickness of the RPE layers, and the results revealed that thicknesses were significantly increased in sections treated with PEDF during OS, due to increased numbers of RPE cells. Furthermore, PEDF was demonstrated to increase UCP2 gene expression in ARPE-19 cells and animal RPE layers under OS, which suggested that PEDF may protect RPE cells and tissues during oxidative injury.

Abstract 379: Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer

Abstract Purpose: Approximately 20% of breast cancers (BC) among US women overexpress HER2, an oncoprotein that stimulates increased cellular proliferation and survival. HER2 acts through two major signaling pathways, PI3K/Akt and Raf/MAPK, and its overexpression is associated with aggressive disease, resistance to therapy, and poor prognosis. Our laboratory has previously demonstrated in preclinical models that calorie restriction (CR) modulates HER2 BC pathogenesis, decreasing incidence and increasing latency in transgenic MMTV-HER2/neu mice. Our current study aims to determine the impact of CR on HER2 signaling and cancer cell phenotypes, including proliferation, cell cycle progression, and apoptosis. Methods: In vitro experiments were performed using a BC cell line derived from the MMTV-HER2/neu mouse model. To mimic CR, cells were treated with nutrient restricted media containing either reduced glucose (1mM), reduced serum (1%) or both (1mM/1%). The impact of CR on proliferation and survival was measured using growth curve experiments, and flow cytometric analysis of cell cycle and apoptosis. HER2 signaling proteins were assessed by western blotting. Results: Relative to cells grown in control media (25mM glucose/10% BCS): a) cells grown in glucose restricted (1mM) + serum restricted (1%) media grew 87% slower (p&amp;lt;0.01); b) cells grown in serum-restricted media (1%) with control glucose levels (25mm) grew 86% slower (p&amp;lt;0.01); and c) cells grown in glucose restricted media (1mM) with control serum levels (10%) grew 67% slower (p&amp;lt;0.05). Serum restricted groups, regardless of glucose levels, also exhibited increased G1 cell cycle arrest following 24 hours of treatment and increased percentage of apoptotic cells following 72 hours of treatment compared to control. In contrast, glucose restriction alone did not significantly affect apoptosis compared to control; however, it induced a G2/M arrest, possibly explaining reduction in growth. Western blotting analysis revealed that serum restricted cells had reduced levels of pAkt (Ser473) but no differences in Akt, pERK, or ERK, compared to the control and 1mM groups following 24 hours of CR treatment. Conclusions: In vitro models of CR, specifically reduced serum proteins alone or in combination with glucose, reduced activation of PI3K/Akt in HER2 BC cells. This altered signaling was associated with reduced tumorigenic potential, as evidenced by decreased growth and increased apoptosis. These findings suggest that combining a nutrient restriction regimen or pharmacologic mimetic of CR with HER2-targeted therapy, which also inhibits PI3K/Akt signaling, may act synergistically. Future experiments will test the hypothesis that CR modulates the efficacy of HER2 targeted therapies through in vitro and in vivo models of HER2 BC. Citation Format: Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Ciara H. O'Flanagan, Laura W. Bowers, Stephen D. Hursting. Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer [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 379.

Effects of siRNA Targeting Interference of APE1 Gene on Proliferation and Apoptosis of Glioma Cells

This paper explored the application of RNA in cancer gene therapy. Second, the effect of the recombinant plasmid APE1 siRNA on the expression of APE1 protein was observed. In addition, the proliferation of cells in apoptosis and the radiosensitivity of human glioma U251 cells were also discussed. U251 cells were divided into control group, negative group and APE1siRNA group for culturing. There were two holes in each group, and independent experiments were repeated three times. The expression of APE1 protein in U251 cells was detected by Western blot experiments for siRNA transfection. The siRNA cells were transfected, while another group of FAM fluorescent labeled non-sense sequence siRNA was transfected to determine the transfection rate and extract the total protein of the cell. BCA protein assay kit was used for determining the concentration. Meanwhile, 50g sample and SDS-polypropylene diamine gel were prepared. Coomassie blue staining was used to observe the protein electrophoresis, after which the protein on the gel was transferred to the PVDF membrane and then developed in a chemiluminescent detection system with specific antibodies. The β -action test was used as an internal controller, while the optical density scanner was used to detect the gray value of the target strip. The relative expression of target protein was equal to the target band gray value or the same sample reference value of gray. MTT method was used to detect the cell growth curve experiments, while AO or EB method was used to detect the apoptosis rate of glioma cells. SPSS17.0 was used for statistical analysis. The results show that the APE1 siRNA expression plasmid was successfully introduced into human glioma cells by RNA interference, which can reduce the expression of APE1 in cells. It was confirmed that the reduction of APE1 protein could inhibit proliferation and promote the apoptosis of glioma cells. Third, APE1 targeting interference may be an effective gene therapy strategy for human glioma, which can provide an experimental basis for the clinical gene therapy of glioma.

Efficacy of potential phage cocktails against Vibrio harveyi and closely related Vibrio species isolated from shrimp aquaculture environment in the south east coast of India

A diverse set of novel phages infecting the marine pathogenic Vibrio harveyi was isolated from shrimp aquaculture environments in the south east coast of India. Based on initial screening, three phages with a broad host range revealed that the growth inhibition of phage is relatively specific to V. harveyi. They were also able to infect V. alginolyticus and V. parahemolyticus that belonged to the Harveyi clade species from shrimp pond and sea coast environment samples. However, the impact of these phages on their host bacterium are well understood; a one-step growth curve experiment and transmission electron microscope (TEM) revealed three phages grouped under the Myoviridae (VHM1 and VHM2); Siphoviridae (VHS1) family. These phages were further molecular characterized with respect to phage genomic DNA isolates. The randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) digestion with HindIII, and major structural proteins were distinguished by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that all the phage isolates were different, even when they came from the same source, giving an insight into the diversity of phages. Evaluation of microcosm studies of Penaeus monodon larvae infected with V. harveyi (105 CFU mL−1) showed that larvae survival after 96 h in the presence of phage treatment at 109 PFU mL−1 was enhanced when compared with the control. The resolution in over survival highly recommended that this study provides the phage-based therapy which could be an innovative and eco-friendly solution against Vibrio disease in shrimp aquaculture and in the natural environment.

Abstract 610: Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response

Abstract Background: For in vitro cancer research, cancer microenvironment is being increasingly recognized as a key factor. In the past, several 3D cell culture models have been tested as the powerful method for reproducing cancer microenvironment. However, in some cases, the growth and drug sensitivity of cells grown on anchorage-independent 3D culture models have been different to that of cells grown in vivo. Further, development of the 3D culture model including the physical (the cell-ECM interaction (scaffold)) and chemical factor (cytokine) are essential for mimicking cancer microenvironment in vitro. Therefore, we explored the effect of the growth factor dependent proliferation and drug sensitivity in scaffold/scaffold-free culture models, and try to understand which method is mimicking in vivo response due to stimulation and suitable for anti-cancer drug development. Methods: We cultured lung cancer, breast cancer, pancreatic cancer and ovarian cancer cell line on monolayer, Matrigel (ECM-embedded 3D culture, scaffold type), NanoCuluture Plate (NCP, a gel-free scaffold type 3D culture plate) and Low adhesion round-bottom plate (scaffold-free 3D culture plate). For growth curve experiment upon addition of growth factors (EGF, HB-EGF, FGF, HRG), we evaluated cell viabilities by ATP assay in several cancer cell lines. We examined drug sensitivities of cancer cell lines against clinically active anti-cancer agents by addition of growth factors. Results: Growth factor promoted the proliferation of cancer cell lines cultured on NCP while it was not enhanced the cell growth when cultured on 2D, Matrigel and Low adhesion round-bottom plate. In drug response, the cells grown on NCP by growth factor stimulation was more sensitive to drug than 2D and other 3D culture models. Conclusion: Differences of the cell growth and drug sensitivity were appeared by effect of scaffold and growth factor. Growth factor dependent cell proliferation and drug sensitivity on NCP were similar to in vivo behavior (1). These results demonstrated that scaffold and growth factor are the novel approach to be mimicking the cancer microenvironment in vitro. Therefore, it is obvious that NCP can be used as a suitable 3D culture model for mimicking in vivo response and anti-cancer drug development. 1. J Cell Sci. 2009 Dec 1;122(Pt 23):4277-86. Citation Format: Norio Masuda, M. Mamunur Rahman, Kazuya Arai, Atsushi Mizuno, Manabu Itoh. Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 610.

Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves.

BackgroundPlate readers can measure the growth curves of many microbial strains in a high-throughput fashion. The hundreds of absorbance readings collected simultaneously for hundreds of samples create technical hurdles for data analysis.ResultsGrowthcurver summarizes the growth characteristics of microbial growth curve experiments conducted in a plate reader. The data are fitted to a standard form of the logistic equation, and the parameters have clear interpretations on population-level characteristics, like doubling time, carrying capacity, and growth rate.ConclusionsGrowthcurver is an easy-to-use R package available for installation from the Comprehensive R Archive Network (CRAN). The source code is available under the GNU General Public License and can be obtained from Github (Sprouffske K, Growthcurver sourcecode, 2016).

Modulating Acinetobacter baumannii biofilm development with molecules containing 3,4,5-trimethoxy-N,N',N'-trimethylbenzohydrazide moiety.

In recent years, Acinetobacter baumannii has emerged as a major cause of nosocomial infections, including infections of implanted medical devices. The treatment of infections caused by A. baumannii has been severely hampered due to their frequent resistance to currently available antibiotics, and most importantly the ability of A. baumannii to form biofilms, which plays a significant role in both persistence and antibiotic resistance. The inherent resistance of A. baumannii biofilms to host defenses and antimicrobial agents necessitates the search for novel approaches to deter biofilm formation. Here, we report our findings on nine compounds identified from structure-activity relationship (SAR) studies on an antibiofilm compound LP3134 that was reported earlier by Biofouling2014, 30, 17. Compounds were evaluated for antibiofilm and anti-adherence activities against A. baumannii. The ability of the compounds to prevent biofilm development on urinary catheters was studied. Growth curve experiments indicated that compounds did not affect the planktonic growth of A. baumannii. All compounds inhibited A. baumannii biofilm development as well as impacting early adhesion on abiotic surfaces. Seven compounds were able to deter biofilm development on silicone catheters. Due to the continued rise of emerging multidrug-resistant A. baumannii, results from this study provide foundation for further development of these molecules to treat A. baumannii infections in wounds and medical devices.

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Effect of the Abortive Infection Mechanism and Type III Toxin/Antitoxin System AbiQ on the Lytic Cycle of Lactococcus lactis Phages

To survive in phage-containing environments, bacteria have evolved an array of antiphage systems. Similarly, phages have overcome these hurdles through various means. Here, we investigated how phages are able to circumvent the Lactococcus lactis AbiQ system, a type III toxin-antitoxin with antiviral activities. Lactococcal phage escape mutants were obtained in the laboratory, and their genomes were sequenced. Three unrelated genes of unknown function were mutated in derivatives of three distinct lactococcal siphophages: orf38 of phage P008, m1 of phage bIL170, and e19 of phage c2. One-step growth curve experiments revealed that the phage mutations had a fitness cost while transcriptional analyses showed that AbiQ modified the early-expressed phage mRNA profiles. The L. lactis AbiQ system was also transferred into Escherichia coli MG1655 and tested against several coliphages. While AbiQ was efficient against phages T4 (Myoviridae) and T5 (Siphoviridae), escape mutants of only phage 2 (Myoviridae) could be isolated. Genome sequencing revealed a mutation in gene orf210, a putative DNA polymerase. Taking these observations together, different phage genes or gene products are targeted or involved in the AbiQ phenotype. Moreover, this antiviral system is active against various phage families infecting Gram-positive and Gram-negative bacteria. A model for the mode of action of AbiQ is proposed.

Charting Microbial Phenotypes in Multiplex Nanoliter Batch Bioreactors

High-throughput growth phenotyping is receiving great attention for establishing the genotype-phenotype map of sequenced organisms owing to the ready availability of complete genome sequences. To date, microbial growth phenotypes have been investigated mostly by the conventional method of batch cultivation using test tubes, Erlenmeyer flasks, or the recently available microwell plates. However, the current batch cultivation methods are time- and labor-intensive and often fail to consider sophisticated environmental changes. The implementation of batch cultures at the nanoliter scale has been difficult because of the quick evaporation of the culture medium inside the reactors. Here, we report a microfluidic system that allows independent cell cultures in evaporation-free multiplex nanoliter reactors under different culture conditions to assess the behavior of cells. The design allows three experimental replicates for each of eight culture environments in a single run. We demonstrate the versatility of the device by performing growth curve experiments with Escherichia coli and microbiological assays of antibiotics against the opportunistic pathogen Pseudomonas aeruginosa. Our study highlights that the microfluidic system can effectively replace the traditional batch culture methods with nanoliter volumes of bacterial cultivations, and it may be therefore promising for high-throughput growth phenotyping as well as for single-cell analyses.

Abstract 1305: Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation.

Abstract Uterine leiomyomas are ovarian hormone-responsive tumors originating in the smooth muscle layer of the uterus called the myometrium. These benign tumors require surgery or other clinical intervention in ∼25% of all premenopausal women in the United States. Despite the high frequency and clinical impact of these tumors, the pathogenesis of uterine leiomyomas is not understood. The estrogen-responsive growth of uterine leiomyomas has been well established, however the role of G protein-coupled estrogen receptor (GPER) in leiomyoma cell regulation has not been studied. The membrane bound estrogen receptor GPER, formerly known as GPR30, is a 7-transmembrane G protein-coupled receptor that is activated by 17β-estradiol (E2). A role for GPER has been previously established in cancers that originate in E2-dependent tissues such as the breast and uterus. Other reports show that GPER is expressed in myometrial cells and activation of GPER modulates myometrial contraction suggesting an important regulatory role for this receptor in the myometrium. Based on these reports, we hypothesized that GPER regulates uterine leiomyoma cells and may represent a putative therapeutic target for this tumor type. The Eker rat model-derived uterine leiomyoma (ELT-3) cell line was used as a model system to study GPER in uterine leiomyoma cells. First, the expression of GPER in uterine leiomyoma cells was determined using RNA and protein samples from ELT-3 cells and real-time PCR and immunoblot analysis. Our findings indicate that GPER transcript and protein were expressed in ELT-3 cells. To determine if activation of GPER regulated uterine leiomyoma cell proliferation, ELT-3 cells were treated with G1, a GPER-specific ligand, or vehicle control and counted 5 days after treatment. Data from these experiments indicated that G1 treatment, and potentially GPER activation, reduced the number of cells over time compared to vehicle control. GPER activation is known to result in the accumulation of pERK and intracellular free Ca2+ and such accumulation is used as a biomarker for GPER activation. Upon G1 treatment, pERK and intracellular free Ca2+ were elevated compared to vehicle treatment in ELT-3 cells suggesting that GPER is functional in uterine leiomyoma cells and is activated by G1. In an effort to determine the mechanism of inhibition of uterine leiomyoma cell proliferation by G1 treatment, we hypothesized that treatment of estrogen-stimulated ELT-3 cells with G1 would inhibit the characteristic E2-induced proliferation of this cell type. Growth curve experiments were performed and data obtained in these experiments suggested that G1 treatment resulted in the inhibition of E2-induced ELT-3 cell proliferation. Taken together, these data suggest that GPER is an important regulator of uterine leiomyoma cell proliferation and is a putative target for novel molecular therapeutics for this tumor type. Citation Format: Maryann Castillo, Angelique M. Wimbley, Jacob J. Mayfield, Jenifer C. Lascano, Kevin D. Houston. Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2013-1305

Effect of Tumstatin T-7 Peptide on HepG-2 Cells and Human Umbilical Vein Endothelial Cells

The direct anti-tumor T-7 peptide of tumstatin was obtained to study its effect on human hepatoma cells (HepG-2) and human umbilical vein endothelial cells (ECV304). T-7 peptide was synthesized and its purity was up to 98.5%. MTT assay, growth curve, transmission electron microscopy(TEM) were used to detect the proliferation inhibition and pro-apoptotic function. MTT experiments and growth curve experiments showed that the survival of human hepatoma cells (HepG-2) decreased in a time- and dose-dependent way with the concentration of T-7 peptide increased. T-7 peptide can induce apoptosis of HepG-2 significantly. Apoptosis features were observed by TEM. However, the inhibition of T-7 peptide on human umbilical vein endothelial cells (ECV304) was weaker. Experiments showed thatT-7 peptide can affect HepG-2 cells. It had a certain therapeutic effect on human hepatocellular carcinoma. T-7 peptide have little effect on ECV304 cells, which means it didn’t influence the formation of tumor angiogenesis and normal cells

Isolation and Molecular Characterization of Pesticide (Fenitrothion) Resistant Bacteria from Agricultural Field

Elimination of pollutants from contaminated sites using microorganisms is a cheaper alternative to chemical technology. There is therefore a need to isolate, identify and characterize the microorganisms that exist and interact in contaminated environment and to isolate the genetic determinants of resistance, frequently located on plasmids and transposons. Investigations were carried out to isolate microbial strains from the fenitrothion polluted soil. Bacteria were isolated using enrichment culture technique containing fenitrothion as sole source of carbon and energy was isolated in pure culture and selected for their ability to degrade fenitrothion. Growth curve experiments showed that the bacterial consortium was able to grow in mineral medium containing fenitrothion as the only carbon source. The microorganisms were identified as Bascillus sp. The identity of the isolate was determined through biochemical and morphological characteristics. For more confirmation, 16s rRNA gene analysis was done using PCR with universal primers. Comparative analysis of the 16s rRNA gene sequences in the Gene Bank database revealed that this bacteria was related to the genus Basicllus substilis. The bacterial strains were found to harbor endogenous plasmids. KEYWORDSBacteria, Biochemical characters, Fenitrothion, Gene sequencing

Enhancement of antibiotic susceptibility ofStenotrophomonas maltophiliausing a polyclonal antibody developed against an ABC multidrug efflux pump

Stenotrophomonas maltophilia is an emerging nosocomial pathogen capable of causing healthcare-associated infections, including pneumonia and bacteremia. Intrinsic resistance in S. maltophilia is exhibited towards many broad-spectrum antibiotics, and treatment recommendations are controversial. One of the major causes of antimicrobial resistance is attributed to a robust array of efflux pumps that extrude drug compounds from the cell. Using checkerboard and growth kinetic assays, we evaluated the in vitro activity of a polyclonal antibody raised against an ATP-binding cassette efflux protein in S. maltophilia. Six clinical strains of S. maltophilia and one type strain were challenged with co-trimoxazole, ticarcillin-clavulanate, and ciprofloxacin, alone and in combination with antibody. One clinical strain was tested by growth curve experiments for each antibiotic-antibody combination. The use of antibody resulted in significantly increased susceptibility in 71.4% (15/21) of treatments tested, with 33.3% displaying synergy and 38.1% an additive effect. In growth kinetic studies, synergy was obtained for each antibiotic-antibody combination. Thus, the use of antibody raised against multidrug efflux pumps for the treatment of multidrug-resistant organisms warrants further investigation. Antibody targeting substrate recognition sites, or other functionally important epitopes, may lead to inhibition of multiple efflux pumps that share the same substrate and is an attractive area that should be explored.

Abstract 1419: Role of p70S6K in breast cancer recurrence

Abstract In Early Breast Cancer (EBC) a single dose of intraoperative radiotherapy with TARGIT (Targeted Intraoperative RadioTherapy) protocol is as effective as the standard therapy in the control of local recurrences (Vaidya et al. Lancet, 376: 91-102, 2010). We recently demonstrated that TARGIT treatment affects the tumor microenvironment, after breast conserving surgery, by altering the local production of several growth factors and cytokines. Eventually, these changes in the proteomic profile, result in a lower activation of the p70S6K pathway in breast cancer cells (Belletti et al. CCR, 14: 1325-32, 2008). To dissect the role of p70S6K in breast cancer cells proliferation, motility and invasion, we generated genetically modified human breast cancer cell lines expressing specific shRNA or carrying a dominant negative form of p70S6K (p70KR) and tested their ability to grow and migrate, first in vitro then in vivo. Growth curve experiments on MDA MB 231 and MDA MB 453 mammary carcinoma cells, demonstrated that expression of the p70KR mutant slightly reduced the growth rate of these cells. The same cells have been also characterized for their ability to migrate in 2D- and 3D-contexts, in response to the wound fluids harvested from EBC patients as chemoattractants, showing that p70KR reduced their ability to invade. Next, we moved to an in vivo model to evaluate more in detail the involvement of p70S6K in the local recurrence of breast cancer. To this aim, parental or p70S6K genetically modified breast cancer cells have been injected in female nude mice mammary fat pads. Two weeks later, the tumor was removed by surgical excision and the mice observed for the appearance of recurrences. Macroscopic and histo-pathological examination of the primary tumors revealed that despite formation of similar masses, only tumors derived from parental cells displayed lymph-vascular invasion. More importantly, only mammary pads injected with parental cells developed recurrent disease and, at pathological analysis, displayed nodal metastasis. Altogether these data highlight a crucial function of p70S6K in the formation of local recurrence in breast cancers, and support the hypothesis that, in EBC patients, an anti-p70S6K specific therapy may be useful for the prevention of local recurrences. 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 1419. doi:10.1158/1538-7445.AM2011-1419

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.