Low-melting Agarose Research Articles

Targeted Inhibition of p21 Promotes the Growth of Breast Cancer Cells and Impairs the Tumor-Killing Effect of the Vaccinia Virus.

Vaccinia virus is widely used as an oncolytic agent for human cancer therapy, and several versions of vaccinia virus have demonstrated robust antitumor effects in breast cancer. Most vaccinia viruses are modified by thymidine kinase (TK) deletion. The function of the cyclin-dependent kinase inhibitor p21 in breast cancer remains controversial. We explored the impact of p21 gene knockdown (KD) on breast cancer cells and whether p21 KD interferes with the antitumor effect of TK-negative vaccinia virus. p21 KD MDA-MB-231 and p21 KD MCF-7 cells were prepared, and cell proliferation and migration rates were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch healing assays. The tumor growth of xenografts originating from p21KD MDA-MB-231 cells and control cells was compared in a mouse model. The colony formation and sphere-forming abilities of p21 KD breast cancer cells were also determined using low-melting agarose and serum-free culture. The tumor-killing effect of the vaccinia virus was determined in breast cancer cells and mouse models using an MTT assay and tumor cell xenografts. p21 KD increased the growth and migration of MDA-MB-231 and MCF-7 cells and promoted the cell growth of MDA-MB-231 cells in mice, while decreasing the colony formation and sphere formation abilities. Expression of TK was reduced in p21 KD MDA-MB-231 cells. Oncolytic effects of both wild-type and TK-deleted vaccinia viruses were attenuated in p21KD MDA-MB-231 cells. The tumor-killing effect of TK-deleted vaccinia virus was also weakened in xenografted mice bearing p21 KD MDA-MB-231 cells. Targeted inhibition of p21 accelerates the proliferation and migration of breast cancer cells and impairs the tumor-killing effect of vaccinia virus, suggesting that p21 levels in cancer cells interfere with vaccinia virus oncolytic therapy.

A Microfluidic Approach for Probing Heterogeneity in Cytotoxic T-Cells by Cell Pairing in Hydrogel Droplets.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.

A temperature-responsive selenium nanohydrogel for strawberry grey mould management.

Grey mould is a fungal disease caused by Botrytis cinerea (B. cinerea), which can cause serious damage to a variety of crops. Herein, we developed iprodione (Ipr) reagent-loaded mesoporous selenium nanoparticles (MSe NPs), combined them with low-melting agarose (LA), and obtained a temperature-responsive selenium particle nanogel (Ipr@MSe@LA NPs) using a simple method. Importantly, Ipr@MSe@LA could capture B. cinerea and quickly be softened to realize the controlled release of Ipr, and effectively inhibit and kill B. cinerea. Plate-based antibacterial tests showed that the colony area of the Ipr@MSe@LA NPs was 4.27 cm-2, which was much smaller than that of the control (25 cm-2). In addition, the Ipr@MSe@LA NPs showed good biocompatibility, and they could improve the photosynthetic efficiency of plants and promote plant growth. Measurement of the fluorescence parameters showed that the maximum photochemical efficiency (Fv/Fm) of the plant leaves of the inoculated group (B. cinerea) is 0.58, but the Fv/Fm value of the Ipr@MSe@LA group is higher than 0.8. In particular, Ipr@MSe@LA NPs could prolong the storage time of strawberries, thereby preserving their freshness. Overall, Ipr@MSe@LA NPs exhibit excellent effects in terms of controlling strawberry gray mould and prolonging the fruit storage time, and this is expected to become a promising strategy for developing intelligent pesticide formulations.

Epigenetic modifications of tumor necrosis factor-alpha in joint cartilage tissue from osteoarthritis patients - CONSORT.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

Synthesis and Characterization of Porous, Electro-Conductive Chitosan-Gelatin-Agar-Based PEDOT: PSS Scaffolds for Potential Use in Tissue Engineering.

Herein we report the synthesis and characterization of electro-conductive chitosan–gelatin–agar (Cs-Gel-Agar) based PEDOT: PSS hydrogels for tissue engineering. Cs-Gel-Agar porous hydrogels with 0–2.0% (v/v) PEDOT: PSS were fabricated using a thermal reverse casting method where low melting agarose served as the pore template. Sample characterizations were performed by means of scanning electron microscopy (SEM), attenuated total reflectance–Fourier transform infrared spectroscopy (ATR–FTIR), X-ray diffraction analysis (XRD) and electrochemical impedance spectroscopy (EIS). Our results showed enhanced electrical conductivity of the cs-gel-agar hydrogels when mixed with DMSO-doped PEDOT: PSS wherein the optimum mixing ratio was observed at 1% (v/v) with a conductivity value of 3.35 × 10−4 S cm−1. However, increasing the PEDOT: PSS content up to 1.5 % (v/v) resulted in reduced conductivity to 3.28 × 10−4 S cm−1. We conducted in vitro stability tests on the porous hydrogels using phosphate-buffered saline (PBS) solution and investigated the hydrogels’ performances through physical observations and ATR–FTIR characterization. The present study provides promising preliminary data on the potential use of Cs-Gel-Agar-based PEDOT: PSS hydrogel for tissue engineering, and these, hence, warrant further investigation to assess their capability as biocompatible scaffolds.

Tools to Image Germplasm Dynamics During Early Zebrafish Development.

During the first day of zebrafish development, ribonucleoprotein (RNP) complexes called germplasm form large aggregates that initially segregate asymmetrically during cleavage stages. After zygotic genome activation, the granules break into smaller fragments that associate with the nuclear membrane as perinuclear (germ) granules toward the end of gastrulation. The mechanisms underlying the highly dynamic behavior of germ granules are not well studied but thought to be facilitated by the cytoskeleton. Here, we present efficient mounting strategies using 3d-printed tools that generate wells on agarose-coated sample holders to allow high-resolution imaging of multiplexed embryos that are less than one day post-fertilization (dpf) on inverted (spinning disk confocal) as well as upright (lattice light-sheet and diSPIM) microscopes. In particular, our tools and methodology allow water dipping lenses to have direct access to mounted embryos, with no obstructions to the light path (e.g., through low melting agarose or methyl cellulose). Moreover, the multiplexed tight arrays of wells generated by our tools facilitate efficient mounting of early embryos (including cleavage stages) for live imaging. These methods and tools, together with new transgenic reporter lines, can facilitate the study of germ granule dynamics throughout their lifetime in detail, at high resolution and throughput, using live imaging technologies.

Low bias multiple displacement amplification with confinement effect based on agarose gel.

Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30μm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing.

Aggrecan and COMP Improve Periosteal Chondrogenesis by Delaying Chondrocyte Hypertrophic Maturation.

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by hypertrophic differentiation of the engineered cartilaginous tissue caused by endochondral ossification. Since a healthy cartilage matrix contains high amounts of Aggrecan and COMP, we hypothesized that their supplementation in the biogel used in the generation of subperiosteal cartilage mimics the composition of the cartilage extracellular matrix environment, with beneficial properties for the engineered cartilage. Supplementation of COMP or Aggrecan was studied in vitro during chondrogenic differentiation of rabbit periosteum cells and periosteum-derived chondrocytes. Low melting agarose was supplemented with bovine Aggrecan, human recombinant COMP or vehicle and was injected between the bone and periosteum at the upper medial side of the tibia of New Zealand white rabbits. Generated subperiosteal cartilage tissue was analyzed for weight, GAG and DNA content and ALP activity. Key markers of different phases of endochondral ossification were measured by RT-qPCR. For the in vitro experiments, no significant differences in chondrogenic marker expression were detected following COMP or Aggrecan supplementation, while in vivo favorable chondrogenic marker expression was detected. Gene expression levels of hypertrophic markers as well as ALP activity were significantly decreased in the Aggrecan and COMP supplemented conditions compared to controls. The wet weight and GAG content of the in vivo generated subperiosteal cartilage tissue was not significantly different between groups. Data demonstrate the potential of Aggrecan and COMP to favorably influence the subperiosteal microenvironment for the in vivo generation of cartilage for the optimization of cartilage regenerative approaches.

Expression and Applications of HriCFP in E. coli: A Novel Biosensing Fluorescent Protein

Metalloids and heavy metal contamination in the environment have become a global problem. Therefore, there is a dire need to develop effective and inexpensive approaches that can facilitate efficient monitoring of the hazardous level of these environmental pollutants. Microbial cell-based and fluorescent protein-based biosensors offer relatively convenient and inexpensive tools for the analysis of environmental pollutants as opposed to traditional instrumental approaches. Small size fluorescent proteins can withstand exposure to denaturants, high temperature and a wide pH range. These characteristics, along with their potential of sensing different toxic analytes, makes them a suitable candidate for developing on-site detection biosensors. The current study exploits the biosensing potential of a novel fluorescent protein called HriCFP. HriCFP was expressed in the prokaryotic system (gram-negative E. coli), which showed stable and discreet expression in bacterial cells. Whole-cell biosensors (WCB) were developed by immobilization of HriCFP expressing non-pathogenic E. coli via nitrocellulose membrane, low melting agarose and sodium silicate gel. These immobilized biosensors were tested for their sensitivity of detection for environmental pollutants, i.e., heavy metals (Cu(II), Hg(II), As(III)). These WCBs exhibited profound fluorescent quenching when exposed to a range of heavy metals. These biosensors remained active for 12 days at 4°C, demonstrating their potential for long-term stability and storage. This study implies that HriCFP may have a significant advantage over other larger and multimeric proteins as it has a minimal impact on host strain metabolism and hence, increasing its sustainability for a longer period.

Development of a 3D human osteoblast cell culture model for studying mechanobiology in orthodontics.

Mechanobiology phenomena constitute a major element of the cellular and tissue response during orthodontic treatment and the implantation of a biomaterial. Better understanding these phenomena will improve the effectiveness of our treatments. The objective of this work is to validate a model of three-dimensional (3D) culture of osteoblasts to study mechanobiology. The hFOB 1.19 cell line was cultured either traditionally on a flat surface or in aggregates called spheroids. They were embedded in 0.8% low-melting agarose type VII and placed in a polyethylene terephthalate transwell insert. Compressive forces of 1 and 4 g/cm2 were applied with an adjustable weight. Proliferation was evaluated by measuring diameters, monitoring glucose levels, and conducting Hoechst/propidium iodide staining. Enzyme-linked immunosorbent assays focusing on the pro-inflammatory mediators interleukin (IL)-6 and IL-8 and bone remodelling factor osteoprotegerin were performed to evaluate soluble factor synthesis. quantitative reverse transcription-polymerase chain reaction was performed to evaluate bone marker transcription. The 3D model shows good cell viability and permits IL dosing. Additionally, three gene expression profiles are analysable. The model allows analysis of conventional markers; larger exploration is needed for better understanding osteoblast mechanobiology. However, it only allows an analysis over 3 days. The results obtained by applying constant compressive forces to 3D osteoblastic cultures validate this model system for exploring biomolecule release and analysing gene transcription. In particular, it highlights a disturbance in the expression of markers of osteogenesis.

Abstract P6-04-18: The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer

Abstract Introduction: Activating transcription factor-2 (ATF2), a member of the ATF and CREB group of bZIP transcription factors regulates gene transcription by forming homodimers or heterodimers with other AP-1 family members. We have previously shown that ATF2 is important for modulating the effect of tamoxifen, whereas Thr71-ATF2 phosphorylation predicts for improved outcome of ER-positive breast cancers receiving tamoxifen. In this study we further explore the role of ATF2 in modulating breast cancer endocrine therapy resistance. Methods: ATF2 silencing: The MCF7, TamR, LCC2, LCC9 breast cancer cell lines were maintained in their appropriate growth media. Cells were transfected using RNAiMAX (ThermoFisher Scientific) with two different siRNAs and a Silencer® negative control. Gene expression was assessed by quantitative real-time PCR (RT-qPCR) (TaqMan assays, ThermoFisher Scientific) and the protein levels were analysed by western blotting. Cell growth, invasion, migration: Sulforhodamine B assay was used for cell growth determination following manufactures’ protocol. Soft-agar colony formation was performed by seeding and incubating the cells in low-melting agarose plates. The resulting colonies were 5% MTT-stained and counted in a Gel Count™ (Oxford Optronix). Invasion/migration was performed using the transwell matrigel-coated (BD Biosciences) plates and the scratch wound healing assay. All the assays were performed in triplicates in three independent experiments. Microarray profiling: Human HT12_V4 microarray (Illumina) was performed to identify genes that were differentially regulated by ATF-2 silencing in TamR cells compared to MCF-7. Data analyses including log2 transformation and quantile normalisation were performed on R Bioconductor. Differentially-expressed genes were selected using FDR correction for multiple testing with p-value cut-off <0.05. Results: ATF-2 silencing significantly inhibited the proliferation (p<0.005), colony formation (p<0.005) and migratory ability (p<0.01) of TamR, LCC2 and LCC9 cells but had a no effect on the growth and migration of MCF-7 cells. Moreover, differential gene expression analysis in MCF7 versus TamR cells revealed a shift from the ER-enriched transcription factor network towards an ATF2-enriched and TGF2β signalling pathway (p=0.025) in TamR cells suggesting alternative hormone-independent growth mechanisms. We then considered differentially-expressed genes specific to the TamR cells (not differentially-expressed in MCF7) and found a reintroduction of ER and its signalling pathway upon ATF2 knockdown along with a reduction in MYC transcriptional activity (EnrichR). RT-qPCR and western blot confirmed the increased expression of ER and its targets TFF1, GREB1, NCOA3, in TamR cells after ATF2 silencing in both the gene and protein levels. Conclusions: In conclusion, for the first time, ATF-2 was identified as a novel transcription factor that can modulate endocrine resistance by regulating ER expression and activity. This data suggests that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity. Citation Format: Athina Giannoudis, Mohammed I Malki, Hisham Mohammed, Bharath Rudraraju, Suraj Menon, Simak Ali, Jason S Carroll, Carlo Palmieri. The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-18.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Abstract C42: Cell culture under perfusion conditions reduces cellular metabolic stress and mimics the in vivo physiologic environment in pancreatic cancer

Abstract Introduction: Tumor responsiveness to chemotherapy in PDAC varies significantly across patients, and some patients may succumb to very early recurrent disease (within 6 months). This reinforces the need to establish an accurate model that preserves the tumor microenvironment in the ex vivo, allowing a personalized drug screen for patients to identify optimal regime of therapy. Many aspects of tumor biology are difficult to reproduce in ex vivo, e.g., the tumor-host interaction and tumor vascularity. The current standard of 2D cell culture is that the medium is exchanged at defined time points (i.e., static culture). However, this is not a physiologically representative model as cells are maintained in either a nutrient and substrate “hyper-replete” or deplete environment, rather than an epistatic supply. Moreover, episodic media replacement creates significant and abrupt changes to the culture environment and availability of substrates, which induces cellular metabolic stress. We evaluated the role of perfusion culture to establish whether this technique reduced metabolic stress and created a physiologically representative model of the in vivo tumor environment by comparing in vitro cell responses to core biopsies from patients with pancreatic cancer. Methods: Cells were cultured and maintained either in static culture (serving as a control, with media exchange every 72 hours) or in a nutritionally replete condition with a constant low perfusion rate of media exchange over 7 days. Daily media extraction was performed in order to evaluate the metabolic activity and viability of cells in addition to quantify the availability of metabolic substrates. Core biopsies were temporarily fixed in low melting agarose gel prior to creating multiple tissue slices through the tumor. Tissue slices were then cultured either in static culture (as control) or under perfusion conditions. Tumors were also subjected to histologic assessment of the tissue morphology, cellular function, and components of the microenviroment. Results: Under conventional static culture, a decrease in the quantity of metabolic substrates was observed over time, indicating metabolic activity of cells in static culture was suppressed (noted by downregulation of mTOR pathway products, notably phosphorylated S6). Under perfusion conditions, glucose concentration was maintained over 7 days, indicating cells were able to maintain metabolic activity. Tissue sections were similarly evaluated for metabolic stress and will be presented. Conclusion: This platform highlights the considerable metabolic stress that cells undergo while under conventional static culture. Perfusion culture serves as a technique to reduce cellular metabolic stress in addition to creating a physiologically representative model ex vivo. Citation Format: Daniel Hughes, Frances Willenbrock, Zahir Soonawalla, Srikanth Reddy, Michael Silva, Somnath Mukherjee, Eric O'Neill. Cell culture under perfusion conditions reduces cellular metabolic stress and mimics the in vivo physiologic environment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C42.

Development of an in vitro model to study tooth cystogenesis.

To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development. To form spheroid structures, various numbers (1×104 , 5×104 or 1×105 ) of epithelial cells (HaCaT and Cal27) were seeded in 96-well plates previously coated with 1.5% low-melting agarose. After 24h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24-well plates previously coated with polymerized collagen and kept for up to 21days. Images of spheroids were captured at each time-point (1, 5, 9, 15 and 21days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One-way Anova was used for statistical analysis. Both epithelial cell lines were able to generate regular and circular spheroids after 24h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P<0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst. This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Measurement of DNA damage by CellProfiler software in the liver of Caspian white fish exposed to environmental concentrations of fipronil

Pesticides are one of the most important factors threatening the ecosystem of the Caspian Sea. Due to prevalence of gastrointestinal cancer in Northern provinces of Iran and the possible involvement of pesticides in this case, genetic toxicity of fipronil insecticide on the liver of the Caspian white fish (Rutilus kutum) was studied. Twelve Caspian whitefish fingerlings (five groups including control and treatment groups) were exposed to environmental concentrations of fipronil (0.1, 1, 5 and 10 µg/L) to evaluate the effects of this pesticide on the DNA of fish liver cells. After 14 days of exposure, liver samples were taken. The samples were converted to separated cells after mincing, centrifugation and filtering through the cell strainer, and then dispersed on the slides coated with low melting agarose. After electrophoresis, the slides were stained with SYBRGreen. CellProfiler software was used to analyze the comets. The results showed that increasing the environmental concentration of fipronil increases the damage to DNA. In general, this study showed that fipronil has the potential of damage to DNA and can be considered as one of the factors reducing the survival rate of Caspian white fish fingerling and larva, and possibly expanding the cancer in Northern provinces of Iran.

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

Abstract 17162: Ambient Ultra Fine Particle Exposure Impairs Gut Vascular Barrier via Notch Signaling

Introduction: Ultrafine particles (UFP, d &lt; 0.1 μm), a major sub-fraction of particulate matter (PM 2.5 , d &lt; 2.5μm) in air pollutant, have been reported to increase cardiovascular morbidity. UFP promote endothelial dysfunction/permeability associated with Notch inhibition and degradation of tight junction protein. Gut vascular barrier (GVB) is a distinct protective barrier that inhibits translocations of antigens across the gut lumen, preventing infections by oral digestions. Here, we hypothesized UFP impair GVB by attenuating Notch signaling and disrupting tight junction protein. Method: The transgenic Tg( flk1:mCherry ) zebrafish embryos were immobilized in neutralized tricaine solution and mounted in 2% low melting agarose to perform micro-gavage at 5 days post fertilizations (5dpf). A mA mixture of 10 kD FITC conjugated dextran and phenol red dye wasere micro-gavaged on the intestinal bulb with or without UFP at 25 μg/mL. Anterior trunk and posterior cardinal vein (PCV) post micro-gavage were imaged under a confocal microscope. Notch signaling related genes including the Notch ligand, Dll4, and the target, HES1, and the level of tight junction protein genes zonula occludens-1 (ZO-1) and Occludin (OCLN) mRNA expression following different courses of exposure to UFP were assessed in Human Aortic Endothelial Cells (HAEC) with quantitative RT-PCR., Result: In the control group, the majority of FITC-dextran remained inside the gastrointestinal system. On the other hand, UFP exposure developed a significant accumulation of FITC-dextran in the intersomatic spaces (ISS) between the dorsal aorta (DA) and the PCV. This finding supports evidence of UFP exposure disrupted both the epithelial boundary and the endothelial vascular layer of the gut. In addition, Notch signaling related gene (HES1, DLL4) and the tight junction proteins genes (ZO-1, OCLN) expressions in HAEC were downregulated in concentration and time-course dependent manner by the exposure to UFP. Conclusion: The UFP exposure affected GVB homeostasis and increased endothelial permeability. This finding supports the link between ambient air pollutant exposure to the impairment in the gastrointestinal system.

Purification of Yeast Artificial Chromosome DNA for Microinjection Using a Two-Gel Electrophoresis Procedure.

This protocol describes a method to purify yeast artificial chromosome (YAC) DNA for microinjection. YAC DNA solutions cannot be concentrated by standard DNA precipitation methods and resuspended into smaller volumes. Attempts to precipitate YAC DNA solutions will, unavoidably, break and destroy the DNA molecules. Therefore YAC DNA must always be kept in a solution that maintains the integrity of the YAC DNA molecules. In the method described here, YAC DNA is extracted from pulsed-field gel electrophoresis (PFGE) agarose plugs by using two gel electrophoresis steps. The first gel electrophoresis step is the PFGE itself. The region of agarose containing the YAC DNA of interest is excised and run on a standard electrophoresis gel setup at a 90° angle to the PFGE run. The YAC DNA migrates out of the agarose slice and enters into a thicker low-melting agarose gel, thereby promoting compaction of YAC DNA molecules. The aim of this method is to convert a slice of agarose into a cube of agarose of smaller volume. The volume of this cube will determine the final concentration of the YAC DNA precipitation.

Abstract 195: Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells

Abstract Background: Locoregional therapy is playing an increasingly central role in the management of hepatocellular carcinoma (HCC), where ablation is distinguished among these techniques by its capacity to effect cure. Electrochemical treatment (EChT), a technique that facilitates necrosis via the application of direct current at low voltages shows promise; however, widespread use has been limited as studies of the underlying biology and the mechanism of action have been limited. Methods: A novel assay for the study of EChT mechanism was designed, wherein HCC cells (2.5e5 cells/mL) were embedded in low-melting temperature agarose (1.5%). EChT was performed on these assays between nitinol cathodes (d = 0.5 mm) and platinum anodes (d = 0.5 mm) in a variety of geometries. Buffering capacity of the encapsulation assay was varied via the addition of HEPES buffer at final concentrations of 10mM, 50mM, or 200mM. Either during or immediately following EChT, the following measurements were recorded: cell viability, pH, ROS burden, transmembrane potential, and temperature. Results: Following EChT, acidic pH was appreciated surrounding the anodes and basic pH surrounding the cathodes in regions where cell death was observed by fluorescence microscopy. Upon increasing the buffering capacity of the assay, the total area of cell death decreased (p &amp;lt; 10-6). There was not sufficient heat generation nor transmembrane potential to account for the observed cell death. A pH-threshold was identified, where a pH &amp;gt; 10.8 or &amp;lt; 4.4 was found to cause cell necrosis in the three different buffer conditions studied. By varying the number of electrodes, their spacing, and length of ablation, it was possible to shape the zone of cell death observed to an arbitrary geometry. Conclusions: The mechanism of EChT-induced cancer cell death is through the spread of acidic and basic species generated by hydrolysis of water upon electron transfer at the electrodes. The diffusion of these species leads to cell death in a predictable, pH dependent manner. These results suggest that an electrochemical ablation device could treat complex HCC tumor geometries with appropriate electrode placement and charge delivery. Citation Format: Nicholas Perkons, Elliot Stein, Chike Nwaezeapu, Joseph Wildenberg, Daniel Ackerman, Gregory Nadolski, Stephen Hunt, Terence Gade. Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells [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 195.