Triplicate Cultures Research Articles

CRISPR-Based Epigenome Editing and Genome Wide Screening Define Mediators of Chemotherapy Response in Glioblastoma

Alkylating chemotherapies exhibit survival benefit for patients with glioblastoma (GBM), the most common malignant primary brain tumor. CRISPRoff is a programmable epigenetic memory writer that stably and heritably silences any gene through DNA methylation. Epigenetic silencing of MGMT via promoter methylation is predictive of response to alkylating agents as well as prognostic for progression free and overall survival. Here we performed epigenome editing using CRISPRoff to stably silence MGMT through induced promoter methylation, as a therapeutically tractable approach for potentiating GBM to temozolomide (TMZ) or lomustine (CCNU). We then used genome-wide engineered CRISPR/Cas9 systems to broadly define sensitizers of GBM cells to alkylating agents, as a platform to discover novel sensitizing targets. Targeted epigenome editing was performed through electroporation of modified mRNAs encoding CRISPRoff machinery comprising deactivated Cas9 fused to a DNA methyltransferase complex, combined with sgRNAs, into MGMT unmethylated GBM cell lines (LN18, T98G) and then treated with either vehicle, TMZ, or CCNU. Parallel experiments were performed through electroporation of sgRNA/Cas9 ribonucleoproteins. Gene silencing was assessed using bisulfite targeted sequencing, RT-qPCR, and western blot. Drug sensitization was determined using luminescent cell viability assays. Genome-wide CRISPR interference (CRISPRi) screens were performed in triplicate cultures. Epigenomic silencing of the MGMT promoter through CRISPRoff-induced methylation reduced MGMT transcript levels by 96.7% and generated up to 88-fold sensitization to TMZ mediated cell death in GBM cells, with IC50 superior to GBM cells with baseline methylated MGMT. In addition, CRISPRoff of MGMT induced 20-fold sensitization to CCNU. CRISPRoff methylation of MGMT was equivalent to CRISPR/Cas9 homozygous deletion of the MGMT gene for drug sensitization and was superior to polyclonal Cas9 mediated deletion of MGMT by a factor of 10. To define additional mediators of chemotherapy response in GBM, CRISPRi screens revealed 185 and 266 genetic sensitizers to TMZ treatment in LN18 and T98G cells, respectively, in addition to validating MGMT. One hundred eighty and 238 sensitizers to CCNU were identified in LN18 and T98G cells, respectively. TMZ sensitizing genes conserved across cell lines were enriched for the ATR (i.e., BCRA2), DNA repair (i.e., REV1), and cell cycle pathways (i.e., PSMD13), while CCNU sensitizing genes were enriched for the Fanconi anemia pathway (i.e., FANCI, FANCD2). In contrast, gene hits that resulted in resistance to alkylating agents were enriched for the mismatch repair pathway (i.e., MSH2, PMS2). We integrate targeted epigenome editing with unbiased genome-wide approaches to build a novel discovery and therapeutic platform in glioblastoma, a framework that is well suited for targeting diseases with known or suspected epigenetic vulnerabilities.

Mycobacterium smegmatis strains genetically resistant to moxifloxacin emerge de novo from the moxifloxacin-surviving population containing high levels of superoxide, H2O2, hydroxyl radical, and Fe (II).

The antibiotic-exposed bacteria often contain the reactive oxygen species (ROS), hydroxyl radical, which inflicts genome-wide mutations, causing the de novo formation of antibiotic-resistant strains. Hydroxyl radical is generated by Fenton reaction of Fe (II) with the ROS, H2O2, which, in turn, is formed by the dismutation of the ROS, superoxide. Therefore, for the emergence of bacterial strains genetically resistant to antibiotics, increased levels of superoxide, H2O2, hydroxyl radical, and Fe (II) should be present in the antibiotic-exposed bacteria. Here, we verified this premise by finding out whether the in vitro cultures of M. smegmatis, exposed to MBC of moxifloxacin for a prolonged duration, contain significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Biological triplicate cultures of M. smegmatis, were exposed to MBC of moxifloxacin for 84 h. The colony-forming units (CFUs) of the cultures were determined on moxifloxacin-free and moxifloxacin-containing plates for the entire 84 h at a regular interval of 6 h. The cultures were analyzed at specific time points of killing phase (KP), antibiotic-surviving phase (ASP), and regrowth phase (RGP) for the presence of superoxide, H2O2, hydroxyl radical, and Fe (II) using the ROS- and Fe (II)-detecting fluorescence probes. The experimental cultures were grown in the presence of ROS and Fe (II) quenchers also and determined the levels of fluorescence corresponding to the ROS- and Fe (II)-specific probes. This was performed to establish the specificity of detection of ROS and Fe (II). Biological triplicate cultures, unexposed to moxifloxacin but cultured for 84 h, were used as the control for the measurement of ROS and Fe (II) levels. The CFUs of the cultures were determined on moxifloxacin-free and moxifloxacin-containing plates for the entire 84 h at regular intervals of 6 h. Flow cytometry analyses were performed for the detection and quantitation of the levels of fluorescence of the ROS-and Fe (II)-specific probes. The experimental cultures were grown in the presence of thiourea and bipyridyl as the ROS and Fe (II) quenchers, respectively, for the determination of the levels of fluorescence corresponding to the ROS- and Fe (II)-specific probes. Paired t-test was used to calculate statistical significance (n = 3). The moxifloxacin-exposed cultures, but not the cultures unexposed to moxifloxacin, showed a triphasic response with a KP, ASP, and RGP. The cells in the late KP and ASP contained significantly elevated levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Thus, high levels of the ROS and Fe (II) were found in the small population (in the ASP) of M. smegmatis cells that survived the moxifloxacin-mediated killing. From this moxifloxacin-surviving population (in the ASP), moxifloxacin-resistant genetic resisters emerged de novo at high frequency, regrew, divided, and populated the cultures. The levels of these ROS, Fe (II), and the high moxifloxacin resister generation frequency were quenched in the cultures grown in the presence of the respective ROS and Fe (II) quenchers. The cultures unexposed to moxifloxacin did not show any of these responses, indicating that the whole response was specific to antibiotic exposure. Significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II) were generated in the M. smegmatis cultures exposed to moxifloxacin for a prolonged duration. It promoted the de novo emergence of genetic resisters to moxifloxacin at high frequency.

QT effects of bedaquiline, delamanid, or both in patients with rifampicin-resistant tuberculosis: a phase 2, open-label, randomised, controlled trial

Bedaquiline and delamanid are the first drugs of new classes registered for tuberculosis treatment in 40 years. Each can prolong the QTc interval, with maximum effects occurring weeks after drug initiation. The cardiac safety and microbiological activity of these drugs when co-administered are not well-established. Our aim was to characterise the effects of bedaquiline, delamanid, or both on the QTc interval, longitudinally over 6 months of multidrug treatment, among patients with multidrug-resistant or rifampicin-resistant tuberculosis taking multidrug background therapy. ACTG A5343 is a phase 2, open-label, randomised, controlled trial in which adults with multidrug-resistant or rifampicin-resistant tuberculosis receiving multidrug background treatment were randomly assigned 1:1:1 by centrally, computer-generated randomisation, by means of permuted blocks to receive bedaquiline, delamanid, or both for 24 weeks. Participants were enrolled at TASK in Cape Town and the South African Tuberculosis Vaccine Initiative in Worcester, both in South Africa, and Hospital Maria Auxiliadora in Peru. Individuals with QTc greater than 450 ms were excluded. HIV-positive participants received dolutegravir-based antiretroviral therapy. Clofazimine was disallowed, and levofloxacin replaced moxifloxacin. ECG in triplicate and sputum cultures were done fortnightly. The primary endpoint was mean QTcF change from baseline (averaged over weeks 8-24); cumulative culture conversation at week 8-24 was an exploratory endpoint. Analyses included all participants who initiated study tuberculosis treatment (modified intention-to-treat population). This trial is registered with ClinicalTrials.gov, NCT02583048 and is ongoing. Between Aug 26, 2016 and July 13, 2018, of 174 screened, 84 participants (28 in each treatment group, and 31 in total with HIV) were enrolled. Two participants did not initiate study treatment (one in the delamanid group withdrew consent and one in the bedaquiline plus delamanid group) did not meet the eligibility criterion). Mean change in QTc from baseline was 12·3 ms (95% CI 7·8-16·7; bedaquiline), 8·6 ms (4·0-13·1; delamanid), and 20·7 ms (16·1-25·3) (bedaquiline plus delamanid). There were no grade 3 or 4 adverse QTc prolongation events and no deaths during study treatment. Cumulative culture conversion by week 8 was 21 (88%) of 24 (95% CI 71-97; bedaquiline), 20 (83%) of 24 (65-95; delamanid), and 19 (95%) of 20 (79-100; bedaquiline plus delamanid) and was 92% (77-99) for bedaquiline, 91% (76-99), for delamanid, and 95% (79-100) for bedaquiline plus delamanid at 24 weeks. Combining bedaquiline and delamanid has a modest, no more than additive, effect on the QTc interval, and initial microbiology data are encouraging. This study provides supportive evidence for use of these agents together in patients with multidrug-resistant or rifampicin-resistant tuberculosis with normal baseline QTc values. Division of AIDS, National Institutes of Health.

Efficacy of Amphotericin B in Corneal Preservation Media After Extended Frozen Storage.

To investigate the antimycotic activity of amphotericin B deoxycholate that has been previously frozen for 28 days before supplementation of Optisol-GS. Triplicate Optisol-GS samples were inoculated with 10 colony-forming units (CFU) of Candida albicans. Each set of triplicate cultures was supplemented with 2.5 μg/mL of amphotericin B that was either freshly resuspended and never frozen, frozen overnight at -20°C and thawed, or frozen at -20°C for 4 weeks and thawed. The cultures were stored at 4°C, with aliquots taken at 0, 6, 24, and 72 hours for quantification. The efficacy of each preparation of amphotericin B in reducing C. albicans growth was assessed at these time points. Six hours after antifungal supplementation, there was a 1.33 log10 CFU reduction with freshly resuspended amphotericin B, compared with a 1.31 log10 reduction with amphotericin B that was frozen overnight (P = 0.20) and a 1.18 log10 reduction with amphotericin B that was frozen for 4 weeks (P = 0.05). After 72 hours, there was a 2.72 log10 CFU reduction with freshly resuspended amphotericin B, a 2.64 log10 CFU reduction with amphotericin B that was frozen overnight (P = 0.45), and a 2.18 log10 CFU reduction with amphotericin B that was frozen for 4 weeks (P = 0.05). Previously frozen amphotericin B remains highly effective against C. albicans. Optisol-GS supplemented with 2.5 μg/mL amphotericin B that was frozen for 4 weeks at -20°C resulted in >90% CFU reduction by 6 hours and >99% reduction by 72 hours.

The 3D reconstructed skin micronucleus assay: considerations for optimal protocol design.

Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations.

Nutrigenomic Effect of Saturated and Unsaturated Long Chain Fatty Acids on Lipid-Related Genes in Goat Mammary Epithelial Cells: What Is the Role of PPARγ?

A prior study in bovine mammary (MACT) cells indicated that long-chain fatty acids (LCFA) C16:0 and C18:0, but not unsaturated LCFA, control transcription of milk fat-related genes partly via the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, in that study, the activation of PPARγ by LCFA was not demonstrated but only inferred. Prior data support a lower response of PPARγ to agonists in goat mammary cells compared to bovine mammary cells. The present study aimed to examine the hypothesis that LCFA alter the mRNA abundance of lipogenic genes in goat mammary epithelial cells (GMEC) at least in part via PPARγ. Triplicate cultures of GMEC were treated with a PPARγ agonist (rosiglitazone), a PPARγ inhibitor (GW9662), several LCFA (C16:0, C18:0, t10,c12-CLA, DHA, and EPA), or a combination of GW9662 with each LCFA. Transcription of 28 genes involved in milk fat synthesis was measured using RT-qPCR. The data indicated that a few measured genes were targets of PPARγ in GMEC (SCD1, FASN, and NR1H3) while more genes required a basal activation of PPARγ to be transcribed (e.g., LPIN1, FABP3, LPL, and PPARG). Among the tested LCFA, C16:0 had the strongest effect on upregulating transcription of measured genes followed by C18:0; however, for the latter most of the effect was via the activation of PPARγ. Unsaturated LCFA downregulated transcription of measured genes, with a lesser effect by t10,c12-CLA and a stronger effect by DHA and EPA; however, a basal activation of PPARγ was essential for the effect of t10,c12-CLA while the activation of PPARγ blocked the effect of DHA. The transcriptomic effect of EPA was independent from the activation of PPARγ. Data from the present study suggest that saturated LCFA, especially C18:0, can modulate milk fat synthesis partly via PPARγ in goats. The nutrigenomic effect of C16:0 is not via PPARγ but likely via unknown transcription factor(s) while PPARγ plays an indirect role on the nutrigenomic effect of polyunsaturated LCFA (PUFA) on milk fat related genes, particularly for CLA (permitting effect) and DHA (blocking effect).

Isolation and Pathogenic Characterization of Vibrio bivalvicida Associated With a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile.

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P < 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.

Transcriptomic responses of Serratia liquefaciens cells grown under simulated Martian conditions of low temperature, low pressure, and CO2-enriched anoxic atmosphere

Results from previous experiments indicated that the Gram-negative α-proteobacterium Serratia liquefaciens strain ATCC 27592 was capable of growth under low temperature (0 °C), low pressure (0.7 kPa), and anoxic, CO2-dominated atmosphere–conditions intended to simulate the near-subsurface environment of Mars. To probe the response of its transcriptome to this extreme environment, S. liquefaciens ATCC 27592 was cultivated under 4 different environmental simulations: 0 °C, 0.7 kPa, CO2 atmosphere (Condition A); 0 °C, ~101.3 kPa, CO2 atmosphere (Condition B); 0 °C, ~101.3 kPa, ambient N2/O2 atmosphere (Condition C); and 30 °C, ~101.3 kPa, N2/O2 atmosphere (Condition D; ambient laboratory conditions). RNA-seq was performed on ribosomal RNA-depleted total RNA isolated from triplicate cultures grown under Conditions A-D and the datasets generated were subjected to transcriptome analyses. The data from Conditions A, B, or C were compared to laboratory Condition D. Significantly differentially expressed transcripts were identified belonging to a number of KEGG pathway categories. Up-regulated genes under all Conditions A, B, and C included those encoding transporters (ABC and PTS transporters); genes involved in translation (ribosomes and their biogenesis, biosynthesis of both tRNAs and aminoacyl-tRNAs); DNA repair and recombination; and non-coding RNAs. Genes down-regulated under all Conditions A, B, and C included: transporters (mostly ABC transporters); flagellar and motility proteins; genes involved in phenylalanine metabolism; transcription factors; and two-component systems. The results are discussed in the context of Mars astrobiology and planetary protection.

Effect of TiO2 Nanofiller Concentration on the Mechanical, Thermal and Biological Properties of HDPE/TiO2 Nanocomposites

The necessity for advanced and effective biomimetic tissue engineering materials has increased massively as bone diseases such as osteoporosis and bone cancer have become a major public health problem. Therefore, the objective of this study is to develop titanium dioxide (TiO2) nanoparticles-enriched high-density polyethylene (HDPE) nanocomposites that could serve as potential biomaterials. HDPE/TiO2 nanocomposites with varying TiO2 nanoparticles content were fabricated by using injection molding technique and were subjected to mechanical, thermal and biological characterization. SEM–EDS analysis confirmed even dispersion of TiO2 nanoparticles into the HDPE matrix. It was observed from the mechanical testing that the addition of TiO2 nanoparticles to HDPE noticeably improved the stiffness (from 345 to 378 MPa) while maintaining almost similar yield strength of pure HDPE. The thermal analyses revealed that TiO2 nanoparticles inclusion to HDPE matrix enhanced the thermal stability of nanocomposites, as the overall rate of crystallization increased by almost 4%. Furthermore, biocompatibility of nanocomposites was also studied by means of various cell culture experiments; human osteoblasts (hFOB) were seeded on the HDPE/TiO2 nanocomposites and were visualized through SEM after 72 h of incubation; surface morphology revealed normal cell growth and spreading with more attachment on PNC-10 that contains 10 wt.% of TiO2. Moreover, cell viability assays (i.e., MTT and cell attachment) revealed consistent increase in cell count and metabolic activity when triplicate cultures were incubated for 1, 3 and 7 days.

Abstract 47: A clinically curative combination therapy acts by low cross-resistance between drugs, not by synergistic interactions between drugs

Abstract Introduction: The development of optimal combination therapies is one of the central challenges of research to improve the treatment of cancers. The discovery of drug synergy is a common goal of preclinical development of combination therapies, but it is unclear whether this pharmacologic synergy, as experimentally measured in the potency of a dose response, is a property of clinically effective combination therapies. Furthermore, the historical motivation for combination therapy was not synergistic interactions per se but the use of different mechanisms of drug action to overcome subclonal cancer heterogeneity. Here we tested which of these interaction mechanisms are properties of a clinically exemplary combination, the chemo-immunotherapy regimen R-CHOP, which cures most cases of diffuse large B-cell lymphoma (DLBCL). Methods: Pharmacodynamic interactions (synergy, additivity, antagonism) between the drugs in RCHOP were measured by Isobologram Analysis in a DLBCL cell culture system that facilitates in vitro activity of R-CHOP. Subclonal cross-resistance between drugs was measured using high-complexity DNA barcoding (ClonTracer) to label a million DLBCL subclones. Labelled subclones were expanded to billions and split into numerous replicate cultures. Each culture received different single drug treatment (each drug tested in triplicate cultures), and deep sequencing of DNA barcodes before and after treatment measured each subclone's resistance or sensitivity to each drug in the combination. Cross-referencing resistant subclones between drugs quantified distributions of drug-drug cross-resistance. Results: Isobologram analysis of RCHOP revealed many antagonistic and at best additive interactions between drugs, despite its curative clinical effects. DNA barcoding experiments revealed that every drug in R-CHOP is resisted by thousands of subclones; hundreds of subclones can resist pairs of different drugs; tens of subclones can resist three different drugs; but not one subclone of a million has resistance to each drug in this combination. Conclusion: The profound “clinical synergy” of RCHOP—cure—derives from overcoming within-tumor heterogeneity by combining different drugs with low cross-resistance, with no need for pharmacologic synergy, such as is measured by bulk cell inhibition assays. These experiments show that synergy of the pharmacologic variety is not essential to the clinical efficacy of a drug combination, and demonstrate the utility of lineage tracing by DNA barcoding as a method to identify drug combinations that can collectively overcome drug resistance. We propose that these high-throughput measurements of subclonal drug resistance and cross-resistance should be a complement to the measurement of drug interactions such as synergy in the preclinical development of combination therapies. Citation Format: Adam C. Palmer, Peter K. Sorger. A clinically curative combination therapy acts by low cross-resistance between drugs, not by synergistic interactions between drugs [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 47.

Inhibitory effect of benthic diatom species on three aquaculture pathogenic vibrios

Diatoms can produce inhibitory compounds against bacteria, constituting an alternative to the use of chemicals to control pathogenic bacterial growth. Antibacterial activity has been detected in co-cultures of microalgae-bacteria and extracts. Thus, we evaluated the ability of 6 benthic diatom species to inhibit the growth of Vibrio alginolyticus, V. campbellii, and V. harveyi, which are bacterial species that are pathogenic to mollusks, crustaceans, and fish. Triplicate cultures of benthic diatoms were inoculated with each individual Vibrio species, and the density of microalgae cells, vibrios, and heterotrophic bacteria was estimated at baseline (0h), 24, 48, 72, 96, 168h post-inoculation, and when the diatom cultures reached the stationary growth phase. Significant differences in the growth of each diatom species were observed, depending on the Vibrio species that was inoculated, and in all cultures, the density of Vibrio cells decreased, even to undetectable levels (<0.01Vibrio cellsmL−1), after 4, 7, and 16days post-inoculation. Initial bacterial counts in the diatom cultures were on the order of 103 (Colony Forming Units)CFUmL−1, reaching 106 to 107CFUmL−1 by the end. The concentration of bacteria in the diatom cultures was similar between co-cultures and control treatments, indicating that the inhibitory effects of the diatom strains were specific for Vibrio species. We conclude that the diatom species that we tested control the concentration of pathogenic vibrios, presumably through the production of antibacterial compounds. These findings should encourage the use of diatom cultures as a feeding source and to control the density of pathogenic Vibrio species in culture systems, adding diatom species to culture water as a “green water” technique.

Optimal ratios of essential amino acids stimulate β-casein synthesis via activation of the mammalian target of rapamycin signaling pathway in MAC-T cells and bovine mammary tissue explants.

Amino acids are the building blocks of proteins and serve as key molecular components upstream of the signaling pathways that regulate protein synthesis. The objective of this study was to systematically investigate the effect of essential AA ratios on milk protein synthesis in vitro and to elucidate some of the underlying mechanisms. Triplicate cultures of MAC-T cells and bovine mammary tissue explants (MTE) were incubated with the optimal AA ratio (OPAA; Lys:Met, 2.9:1; Thr:Phe, 1.05:1; Lys:Thr, 1.8:1; Lys:His, 2.38:1; and Lys:Val, 1.23:1) in the presence of rapamycin (control), OPAA, a Lys:Thr ratio of 2.1:1, a Lys:Thr ratio of 1.3:1, a Lys:His ratio of 3.05:1, or a Lys:Val ratio of 1.62:1 for 12 h; the other AA concentrations were equal to OPAA. In some experiments, the cells were cultured with OPAA with or without rapamycin (100 ng/mL) or with mammalian target of rapamycin (mTOR) small interference RNA, and the MTE were exposed to OPAA with rapamycin for β-casein expression. Among the treatments, the expression of β-casein was greatest in the MTE cultured with OPAA. In MAC-T cells, the OPAA upregulated the mRNA expression of SLC1A5 and SLC7A5 but downregulated the expression of IRS1, AKT3, EEF1A1, and EEF2 compared with the control. The OPAA had no effect on the mTOR phosphorylation status but increased the phosphorylation of S6K1 and RPS6. When the MTE were treated with rapamycin in the presence of OPAA, the expression of β-casein was markedly decreased. The phosphorylation of RPS6 and 4EBP1 also was reduced in MAC-T cells. A similar negative effect on the expression of RPS6KB1 and EIF4EBP1 was detected when the cells were cultured with either rapamycin or mTOR small interference RNA. The optimal AA ratio stimulated β-casein expression partly by enhancing the transport of AA into the cells, cross-talk with insulin signaling and a subsequent enhancement of mTOR signaling, or translation elongation in both MAC-T cells and bovine MTE.

Identification of drug- and drug-metabolite immune responses originating from both naive and memory T cells

Identification of drug- and drug-metabolite immune responses originating from both naive and memory T cells

Detection of p-coumaric Acid from Cell Supernatant Using Surface Enhanced Raman Scattering

A standard protocol for analysis of microbial factories requires the screening of several populations in order to find the best performing ones. Standard analytical methods usually include high performance liquid chromatography (HPLC), thin layer chromatography (TLC) or spectrophotometry, which are expensive and time-consuming processes. Surface Enhanced Raman Spectroscopy (SERS), instead, is a highly sensitive spectroscopic technique for specific, fast and real-time sensing of biological samples. Here we demonstrate the use of SERS to discriminate between two different bacterial populations based on detection of p-coumaric acid (pHCA) in cell supernatant.SERS active substrates, based on leaning gold-capped silicon nanopillars, were used for detection. They were successfully used to detect culture medium spiked with pHCA, and the effect of medium dilution was studied. For analysis of biological production of pHCA, triplicate cultures of E. coli strains expressing a pHCA-forming enzyme (P) as well as of a non-producing strain (C) were grown. Then, supernatant samples were collected and their pHCA content was measured using SERS and HPLC for comparison. The intensity of the pHCA Raman mode at 1169 cm-1 (CH-rocking motion) showed different trends for P and C strains, similar to the results obtained using the HPLC method. Results illustrate that SERS can be used for quick and semi- quantitative discrimination of pHCA concentrations in cell supernatant medium.

Heme Induces NLRP3 Inflammasome Formation in Primary Human Macrophages and May Propagate Hemolytic Inflammatory Processes By Inducing S100A8 Expression

Intravascular hemolysis results in the release of damaging hemoglobin and free heme into the circulation. A role for heme as a danger associated molecular pattern (DAMP), with a function in sterile inflammatory responses, is becoming increasingly recognized. Whilst heme has known effects on leukocytes, activating their migration, adhesion molecule expression and cytokine expression, more recent data demonstrate that this molecule can induce NLRP3 inflammasome formation in murine bone marrow macrophages, with consequent interleukin (IL)-1β processing and neutrophil recruitment (Dutra et al., Proc. Natl Acad Sci. 111: E4110, 2014). We aimed to investigate whether heme can also induce inflammasome activation in primary human macrophages (hMACs) and to further characterize the pathways by which heme-induced inflammatory responses may be amplified under sterile conditions. CD14+ cells were separated from human peripheral blood (using anti-CD14 magnetic beads) and differentiated into hMACs under M-CSF media supplementation and in the presence of 10% fetal bovine serum. In vitro results are expressed as means ± SEM for triplicate cultures and are representative of three independent experiments. Priming of hMACs with lipopolysaccharide (LPS; 100 ng/mL; 3h) alone induced low level secretion of IL-1β (14.11±9.2 pg/106 cells, as measured by ELISA), while heme (50 µM), in the absence of pre-stimulation with LPS, was unable to induce significant IL-1β secretion within 3h (2.46±1.4 pg/106 cells). In contrast, co-incubation of hMACs with both LPS and heme for 3h significantly enhanced hMAC IL-1β release (490.3±36.3 pg/106 cells; P<0.05 compared to LPS alone). The inflamassome pathway inhibitors, MCC950 (5 µM; a specific inhibitor of NLRP3) and YVAD (40 µM; a caspase-1 inhibitor) significantly inhibited IL-1β secretion in LPS-primed hMACs stimulated with heme (reduced to 35.12±3.9; 184±30.4 pg/106 cells, respectively; 3h; P<0.05 compared to LPS/heme). Co-incubation of the LPS-primed cells with varying concentrations of heme, under the conditions employed, did not induce TNF-α production (data not shown), consistent with the hypothesis that IL-1β processing in heme-induced LPS-primed hMAC was mediated by inflammasome formation. Interestingly, qPCR showed that incubation of hMACs (1x106 cells/mL) with heme (50 µM) for 24h stimulated an approximately 10-fold increase (P<0.01) in the expression of the gene encoding, S100A8, another DAMP known to act as a TLR-4 agonist and to contribute to ischemia/reperfusion injury. Priming of hMACs with 1 µg/ml recombinant S100A8 for 3h and subsequent activation with heme (50 or 100 µM, 14h) significantly augmented the release of IL-1β (42.1±0.4 and 89.4±32.4 pg/106 cells for 50 and 100 µM heme, respectively; P<0.05), compared with S100A8 alone (20.6±3.5 pg/106 cells), without any modulation in TNF-α secretion (P>0.05). Using a model of acute intravascular hemolysis, we confirmed an association between heme release and S100A8 secretion, in vivo. Plasma heme levels increased significantly from 26.3±5 µM (i.v. saline control; N=4) to 87±18 µM in C57BL/6 mice at 1h after receiving i.v. water (150 µl; N=4, P=0.04). A concomitant increase in plasma S100A8 levels was also observed within 1h of the hemolytic stimulus (986±102 pg/mL, compared to 694.2±102 pg/ml in control mice; N=4, P=0.05), which was maintained for 3h (P<0.05). Thus, we present data to demonstrate that heme can induce IL-1β processing in LPS-primed human macrophages under in vitro conditions, probably via formation of the NLRP3/caspase-1 inflammasome machinery. In the absence of LPS, heme-stimulated hMACs can express the S100A8 DAMP; furthermore, a hemolytic stimulus induced mouse S100A8 production in vivo. As such, S100A8 may amplify heme-dependent inflammasome formation in an autocrine fashion, even under sterile conditions. Data provide new insights into the mechanisms by which heme may induce and potentiate inflammatory responses in hemolytic diseases, such as sickle cell disease, and suggest S100A8, together with heme, as potential therapeutic targets for reducing inflammation in these diseases. DisclosuresFerreira:Bayer AG: Research Funding. Almeida:Jassen & Cilag: Other: Currently employed with. Conran:Bayer AG: Research Funding.

First Report of Vibrio tubiashii Associated with a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile.

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of this pathology. The pathogenic strain was identified as Vibrio tubiashii based on its phenotypic properties and the sequence analysis of its 16S rRNA and housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA). When triplicate cultures of healthy 10–day–old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.87 ± 3.33%, 34.32 ± 4.94%, and 0% were observed at 12, 24, and 36 h, respectively; whereas uninfected larval cultures showed survival rates of 97.4 ± 1.24% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.66%, significantly (P < 0.001) lower than the control group (97.4 ± 1.20%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.84% after 48 h of exposure. This is the first report of the isolation of V. tubiashii from the diseased larvae of the scallop A. purpuratus, occurring in a commercial culture in Chile, and it was demonstrated that the VPAP30 strain exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds.

714. Use of a Modular Workstation for Reduced Biosafety Risks Also Increased Cellular Yields

Cell culture processing in a room-air biologic safety cabinet (BSC), even in a cleanroom, creates multiple risks for both cell cultures and users. Humans can be exposed to genetically-modified cells, viruses, and other biological hazards during suboptimal (open) cell handling in a room air BSC. The cell cultures can also be exposed to suboptimal separation of air during cell handling in the BSC which creates a contamination hazard. In addition, even in the cleanest clean room, cells are exposed to room air temperatures and gas levels. Until cells re-equilibrate with the incubator after handling, cells are chilled, hyperoxic, and hypocapnic compared to physiologic conditions. A barrier isolator (closed cell processing workstation) creates a constant physical barrier between cells and users. This barrier reduces the risks of air mixing between cells and people without dependence upon laminar air flow in a BSC or a costly whole-room HVAC system. We undertook the following study to test the hypothesis that the use of a barrier isolator, during cell handling as well as incubation, could improve cell growth by providing unbroken physiologic conditions to cells. We used K562 cells, an undifferentiated leukemic cell line that has been used for T cell bioprocess optimization. We divided a single culture into two sets of triplicate cultures in T-75 flasks, three for growth in a barrier isolator and three for growth in a standard room-air incubator equipped with an inner chamber (C-chamber) for control of gasses. Both sets of cultures were incubated at 5%CO2, 5%O2, 37 C, and tracked for cell growth and viability over two weeks. For routine subculturing twice weekly, the cultures in the isolator were handled in the processing chamber of the isolator in physiologic conditions identical to the incubator. The cultures in the room-air incubator and C-chamber were handled in a standard room-air BSC (open). While cell viability was high in both sets of cultures, we found that handling cells under continuous physiologic conditions produced statistically higher cell yields over time (p=0.00086, Day 14, two-tailed paired T test, assuming unequal variances). No contamination events occurred and particle count data indicated no breach of functional separation between room air and isolator cell culture workspace. We concluded that cell handling in unbroken physiologically relevant conditions produced better cell growth over time while reducing risks to both cells and cell culturists. We predict that as cells become increasingly valuable, controlled workspaces for better cell growth and lower biosafety risks will become increasingly valuable as a part of a total quality approach to cell culture.

Lysine and novel hydroxylysine lipids in soil bacteria: amino acid membrane lipid response to temperature and pH in Pseudopedobacter saltans.

Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response of soil bacteria.

English

The research made comparative studies on five media for bacterial isolation. This study aimed at capturing important comparative data in the various types of media for growth efficiency and specific bacteria identification in clinical microbiology. The sources of the samples were urine, nasal swabs and stool. The totality of 15 samples was plated monthly and 120 samples were studied during an eight month period. The mean bacterial load from the cultures grown over the period from each source was calculated and used for comparative growth efficacy. Dominant colonies were characterized and identified based on morphological features and biochemical tests. A 0.1 ml of 10-3 of each bacteria isolate was evaluated for growth potential in triplicate on three different special purpose media. The mean bacteria load from the triplicate cultures was calculated. Salmonella-Shigella agar (SSA); a selective medium had the highest number of bacterial colonies of 2.98 x 105 CFU/ ml followed by the enrichment medium; blood agar that had 2.96 x 105 CFU/ ml and MacConkey agar (MCA) with 2.93 x 105 CFU/ ml. Biochemical identification and characterization of four dominant isolates confirmed the presence of Staphylococcus aureus, Escherichia coli, Salmonella and Shigella. Growth potential of each medium on the bacterial isolates showed that MacConkey agar recorded the highest growth potential of 8.9 x 105 CFU/ ml for E. coli followed by Blood Agar that gave 8.8 x 105 CFU/ml for Shigella. The third highest growth potential of 8.6 x 105 CFU/ ml was recorded in nutrient agar against S. aureus. Statistically, there exists a significant difference among the mean of the five media in their support for bacteria growth at &alpha; =0.05. Key words: Bacterial isolation, isolates, Escherichia coli, Shigella, Blood agar.

MeCP2 modulates gene expression pathways in astrocytes

BackgroundMutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.MethodsTo identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.ResultsA total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.ConclusionsThe first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.