microRNAs (miRNAs) are short, noncoding RNAs that inhibit translation by binding primarily to the 3′‐untranslated region (3′‐UTR) of mRNA. The enzyme TOP2α induces covalent complexes with DNA and produces transient double‐strand DNA breaks crucial for processes such as replication and normal chromosomal dysjunction at mitosis. TOP2α is an important target for clinically effective anticancer agents, such as etoposide (VP‐16) since these drugs stabilize the otherwise short‐lived enzyme‐DNA covalent complexes, thereby inducing cytotoxic DNA damage. However, the efficacy of these agents is limited by chemoresistance. Our lab has characterized acquired resistance to VP‐16 in human leukemia K562 cells. This cloned resistant cell line, K/VP.5, contains reduced levels of TOP2α compared to parental K562 cells. The goal of this project is to test the hypothesis that TOP2α levels are decreased in K/VP.5 cells, in part, through miRNA‐mediated mechanisms.Pooled miRNA qPCR profiling experiments were performed to investigate the expression levels of ~500 miRNAs in K562 and K/VP.5 cells. hsa‐miR‐9‐3p and ‐5p (miR‐9‐3p and ‐5p) were overexpressed in K/VP.5 cells compared to K562 cells. The TOP2α 3′‐UTR harbors putative miRNA recognition elements (MRE) for these miRNAs. Therefore, these miRNAs were chosen for further study. To assess post‐transcriptional regulation of TOP2α by miRNAs, a dual luciferase reporter plasmid harboring the entire 3′‐UTR of TOP2α mRNA (998 bp) was constructed (psiTOP2α/UTR). Transfection with psiTOP2α/UTR demonstrated decreased luciferase expression in K/VP.5 compared with K562 cells (p<0.001), suggesting altered post‐transcriptional regulation in resistant cells. K562 cells that were co‐transfected with psiTOP2α/UTR and miR‐9‐5p or ‐3p mimic resulted in a decrease in luciferase expression only for miR‐9‐5p (p<0.001). Mutating the putative miR‐9‐5p seed sequence prevented the decrease in luciferase activity, demonstrating a direct interaction of this miRNA with the MRE of TOP2α. Immunoblotting for TOP2α in K562 cells transfected with miR‐9‐3p or ‐5p mimic resulted in decreased TOP2α protein compared to mock transfected K562 cells (miR‐9‐3p; p<0.05, miR‐9‐5p; p=0.01). In contrast, immunoblotting for TOP2α in K/VP.5 cells transfected with miR‐9‐3p or ‐5p inhibitor resulted in an increase of TOP2α protein (p<0.05), strongly suggesting a role for both miRNAs in acquired resistance to VP‐16.Our findings indicate that miR‐9‐3p and ‐5p reduce TOP2α expression levels. In addition, results presented here contribute to the elucidation of chemoresistance mechanisms and have the translational potential for circumvention of drug resistance by modulation of miRNA concentrations.Support or Funding InformationPatrick and Jane O'Neill Endowed Scholarship, Honors and Scholars Enrichment Grant, Research Scholars AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA topoisomerase IIα (170 kDa, TOP2α/170) is a key homodimeric enzyme resolving DNA topologic entanglements during chromosome dysjunction by introducing transient DNA double‐strand breaks (DSB). TOP2α/170 is also a prominent clinical target for anticancer drugs, such as etoposide (VP‐16), whose clinical efficacy is often compromised due to chemoresistance. We recently demonstrated that parental K562 cells and cloned K562 cells (K/VP.5) with acquired resistance to VP‐16 express a novel truncated TOP2α/90 (90 kDa) isoform dramatically overexpressed in drug‐resistant K/VP.5 cells (JPET 360(1): 152–163, 2017). Recent results also demonstrated overexpression of TOP2α/90 in isogenic HL‐60 AML cells resistant to the TOP2α‐targeting agents, mAMSA and mitoxantrone. TOP2α/90 is the C‐terminal truncated translation product of TOP2α mRNA lacking the active‐site Tyr805 required for the generation of DSBs. TOP2α/90 mRNA retains a portion of intron 19 indicating altered RNA processing and an exon‐intron read‐through. TOP2α/90 was detectable in the nucleus and heterodimerizes with TOP2α/170. Forced expression of TOP2α/90 in K562 cells decreased VP‐16–induced DNA damage and cytotoxicity while siRNA knockdown of TOP2α/90 in K/VP.5 cells increased VP‐16 activity (Mol. Pharmacol. 93: 515–525, 2018). Together results suggest that TOP2α/90 is a resistance determinant/biomarker through a dominant negative effect. qPCR analyses of paired/matched AML patient samples (pre‐treatment & relapse) revealed that, in 3 of 4 patients, there was a statistically significant increase in the ratio of expression of TOP2α/90 mRNA compared to TOP2α/170, after relapse. We hypothesize that an intrinsically weak exon 19/intron 19 (E19/I19) splice site (ss) in TOP2α pre‐mRNA results in partial I19 retention and formation of truncated TOP2α/90. We further posit that therapeutic genome to modify I19 will provide a tractable strategy to circumvent TOP2α‐mediated drug resistance . To investigate the influence of a weak TOP2α E19/I19 ss (GAG/gtaaac)in I19 retention, a minigene (MG1+) expression construct with a consensus I19 5′ ss (CAG/gtaagt) was generated. Transfection of K/VP.5 cells with MG1+ resulted in a reduction of I19 retention (assessed by qPCR), accompanied by an increase in the levels of a properly spliced amplicon indicative of TOP2α/170 mRNA. These findings set the stage for CRISPR/Cas9 experiments to determine whether editing the I19 5′ ss of the TOP2α gene will result in decreased I19 retention, diminished TOP2α/90 generation, and increased VP‐16 sensitivity in K/VP.5 cells. To date, CRISPR/Cas9 targeting has resulted in successful targeting/mutation of E19/I19 to a consensus ss sequence. Selection of an edited cell clone is in process. Our results contribute to a better understanding of alternative TOP2α mRNA processing leading to the development of resistance to TOP2α poisons. Characterization of alternative RNA processing of the TOP2α mRNA will lead to strategies to circumvent acquired drug resistance. These and future results may also allow for evaluation of TOP2α/90 as a biomarker for drug resistance, prognosis, and/or guide TOP2α‐targeted therapies. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

ABSTRACT Pseudomonas aeruginosa causes chronic pulmonary infections in patients with cystic fibrosis (CF). P. aeruginosa mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both in vitro and in vivo . Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H 2 O 2 ). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H 2 O 2 stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on lys , encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of P. aeruginosa mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung. IMPORTANCE P. aeruginosa mucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities of P. aeruginosa demonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversified P. aeruginosa communities in vivo , as these variants can differentially shield one another from components of the host response.

Aim: Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28. We describe a robust Myc and Lin28-free IPSC reprogramming approach using reprogramming molecules. Methods: IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. Results: While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28-free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28. Conclusion: This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

Since the development of fiber optic technology in the 1960s, fiber optic cables have been used extensively as surgical field illumination devices. The lighting provided by functioning fiber optic cables has contributed to the success of many operational procedures. Current surgical fiber optic cables, however, are susceptible to damage during sterilization and over bending during storage. To increase the durability and life of surgical optical cables, a cost effective protective armor system is proposed. The protective armor was developed using computer aided design methods to restrict cable bending and pressure damage. Mechanical bending and compression tests demonstrated the durability of the protective armor. The introduction of an optical gel into the cable to increase the durability is also considered. The proposed armor, therefore, has the potential to increase the usefully life of surgical optical cables and significantly reduce replacement costs.

An electroporation system allowing control of pulse amplitude, frequency, and duration is proposed. The system was implemented using a programmable lock-in amplifier and switch module. A software data acquisition system was designed to allow the user to control the pulse parameters and data acquisition setting. The system was validated by applying 1 V p-p pulses at 45 kHz to an array of gold electrodes seeded with HCT-116 colorectal cancer cells. Unseeded naked electrodes were used as controls. The experimental results of this study demonstrated a transient response and recover of HCT-116 cells to the applied pulses. Small changes in the naked electrodes were also observed following the electroporation pulses.

A mechanical test and analysis system was developed to examine synthetic hydrogel matrix mechanical effects on cancer cells. Specifically, a stage apparatus was designed for studying the bulk mechanical properties of various hydrogel samples and their contributions to cancer cell phenotypic expression. The confined mechanical compression system was implemented for use with a commercial mechanical tester. Utilizing a “waterfall” design methodology, hardware suitable for the task was designed and built. Preliminary testing of the equipment was performed using formulations of 2-hydroxyethylmethacrylate with varying concentrations of methacrylic acid monomers. An overview of the equipment design and representative preliminary data gathered during process prove-out is presented. The measurement of storage and loss moduli with the system is discussed.

Nano-carrier systems for in-vivo targeted delivery have the potential to make important contributions to cancer therapy. The application of layer-by-layer methods for fabricating nano-carrier encapsulation systems is a particularly promising method. This study therefore investigated the quantification of layer-by-layer quantum dot encapsulation systems using fluorescence measurements, atomic force microscopy, and dynamic light scattering. Particle concentrations between 1010 and 1011 particles/mL showed relatively large changes in the fluorescence intensity. Particle sizing using atomic force microscopy and dynamic light scattering measurements were in qualitative agreement with each other and provided complimentary information. The strengths and weaknesses of atomic force microscopy and dynamic light scattering methods for quantifying nano-carrier systems are discussed.

A low cost environmental chamber for supporting multiplexed live cell diagnostic and therapeutic testing is presented. The chamber is designed to be compatible with standard twenty-four and ninety-six well plates. In addition, an insert was designed to allow the chamber to interface to a micro-fluidic shear flow sensor. The chamber provides a low cost alternative to microscope enclosure and allows multiplexed optical and electrical impedance measurements to be performed on cells as a continuous function of time. The results of this work demonstrates the potential to reduce the barrier to entry to investigators performing multiplexed optical and impedance measurements. The ability to simultaneously compare optical microscopy images with cellular micro-impedance and other biosensor readings significantly improve the capacity to diagnosis and study new and existing pharmaceutical agents.

In vitro drug testing utilizing live cancer cell cultures has enormous potential in drug discovery and personalized medicine. Cancer grows as heterogeneous three dimensional clusters in vivo. As a result, traditional two dimensional cancer cell layer cultures are not physiologically representative of their in vivo growth. To provide better process control and more physiologically realistic cancer cell cultures, a modified hanging drop culture plate was designed. The culture plate allows three dimensional cancer cell spheroids to be cultivated and accurately placed over the working electrodes of multiplexed biosensor arrays. The results of this study are expected to demonstrate the formation of consistently sized cancer cell spheroids with proper positioning over working electrodes. Impedance measurements from biosensor arrays on 96-well plates will be obtained and compared to two dimensional cultures to demonstrate the utility of the hanging drop plate.

Understanding the interaction between cancer and endothelial cells is a key element to more effective chemotherapeutics. A microfluidic cellular co-culture biosensor capable of simultaneous optical and electrical impedance measurements is therefore proposed. Using micro-fabrication methods, an array of optically thin electrically conductive biosensors is designed capable of monitoring cancer and endothelial cell interactions. Computer aided design methods were used to layout out a microfluidic channel compatible with the working bio-electrode array. The micro-fluidic channel allowed cancer cell spheroids and pharmaceutical agents to be injected into an inlet port. Both the optical and electrical properties of the micro-fluidic device have been examined. The proposed system has a number of applications where cancer and endothelial cell interactions need to be studied. The results of this study demonstrate the feasibility of the multiplexed biosensor for studying cancer and endothelial cell interactions under static and dynamic shear flow conditions.

Optically based assays have traditionally been used to quantify cancer cell proliferation and cytotoxicity in response to chemotherapeutic agents. Cellular micro-impedance measurements provide complementary information that is a function of cellular adhesion and membrane properties. The ability to simultaneously acquire both optical and micro-impedance data has significant synergistic diagnostic potential. This study therefore proposes a multiplexed optical micro-impedance assay system compatible with routine pharmaceutical testing. Optically thin micro-impedance electrodes were designed for twenty-four and ninety-six well tissue culture plate readers. Electrode arrays were designed using micro-fabrication methods. The electrode arrays were then assembled into modified twenty-four and ninety-six well cell culture plates designed using computer aided design. Pharmaceutical research and personalized medicine applications are briefly discussed.

Optically thin micro-impedance electrodes have the potential to simultaneously provide optical microscopy measurements with electrical impedance data. Most optically thin electro-conductive films, however, do not provide the same sensitivity as industry standard gold electrodes. As a result, the electrode geometry must be carefully optimized to detect cellular adhesion and growth. By varying the working electrode and counter electrode geometries, the sensitivity to cellular attachment can be optimized. In addition, parasitic capacitances can be minimized by using small working electrodes and dielectric layers. Lead resistances were negligible. The results of this ongoing study demonstrate that one can manufacture optically thin electrodes that are capable of detecting cancer cell growth and adhesion.

Non-invasive fetal electrocardiogram measurements have the potential to monitor and reduce newborn clinical complications. Fetal electrocardiogram signals, however, are relatively weak and difficult to extract from the maternal electrocardiogram signal and other sources of interference. The goal of this project is to, therefore, successfully extract and process fetal electrocardiogram signals in real time from a competing background of maternal electrocardiogram signals and other forms of noise. To accomplish this goal, electrocardiogram instrumentation hardware has been designed, capable of processing multiple input data streams from electrocardiogram leads placed over the maternal abdomen. High-performance bio-potential amplification, filtering, processing, and safety issues are considered in the design. The results of this study outline a high precision and scalable electrocardiogram system for extracting fetal signal from a large background of maternal and other interfering signals.

Recent studies have shown that solid state, optically thin, electrically conductive biosensors have the potential to quantify live cell-drug interactions. These sensors have many potential applications in pharmaceutical testing, cytotoxicity screening, personalized medicine, and many other research areas. The materials used to fabricate these novel sensors, however, do not necessarily promote the long term cellular attachment and growth for studies over several days to weeks. The objective of this study is, therefore, to improve the long term cellular attachment to these sensors using synthetic hydrogel coatings. The choice of charged and neutral monomers is carefully examined to produce long term cellular attachment on optically thin electro-conductive biosensors without impeding the biosensor performance. A combination of cellular attachment studies on different hydrogels, swelling measurements, and electrically conductive measurements are examined to determine the optimal hydrogel composition. The results of this study show that copolymerizing 2-hydroxyethylmethacylate and polyethylene glycol dimethacrylate with acidic and basic monomers has the potential to significantly improve the long term performance of solid state optically thin, electrically conductive cellular biosensors.

E-learning has become a mainstream educational opportunity, as noted in U.S. News & World Report. Further, differences among college students have been documented in various disciplines. An experiment was conducted to determine the effects of network latency on pedagogical efficacy based on the students who were classified as in either humanities programs or engineering and science programs. The findings indicate that tolerances to screen update latencies are discipline-dependent and that students in engineering and science have a lower tolerance for screen update latency than students in the humanities.

Expression of cyclooxygenases (COX) and lipoxygenases (LOX) has been linked to many pathophysiological phenotypes, including cell adhesion. However, many current approaches to measure cellular changes are performed only in a fixed-time point. Since cells dynamically move in conjunction with the cell matrix, there is a pressing need for dynamic or time-dependent methods for the investigation of cell properties. In the presented study, we used stable human colorectal cancer cell lines ectopically expressing COX-1, COX-2, and 15LOX-1, to investigate whether expression of COX-1, COX-2, or 15LOX-1 would affect cell adhesion using our opto-electric methodology. In a fixed-time point experiment, only COX-1- and COX-2-expressing cells enhanced phosphorylation of focal adhesion kinase, but all the transfected cells showed invasion activity. However, in a real-time experiment using opto-electric approaches, transmitted cellular morphology was much different with tight adhesion being shown in COX-2 expressing cells, as imaged by differential interference contrast microscopy (DICM) and interference reflection contrast microscopy (IRCM). Furthermore, micro-impedance measurements showed a continued increase in both resistance and reactance of COX- and LOX-transfected cells, consistent with the imaging data. Our data indicate that both COX- and LOX-expressing cells have strong cell-to-cell and cell-to-substrate adhesions, and that cell imaging analysis with cell impedance data generates fully reliable results on cell adhesion measurement.

A novel transcellular micro-impedance biosensor, referred to as the electric cell-substrate impedance sensor or ECIS, has become increasingly applied to the study and quantification of endothelial cell physiology. In principle, frequency dependent impedance measurements obtained from this sensor can be used to estimate the cell–cell and cell–matrix impedance components of endothelial cell barrier function based on simple geometric models. Few studies, however, have examined the numerical optimization of these barrier function parameters and established their error bounds. This study, therefore, illustrates the implementation of a multi-response Levenberg–Marquardt algorithm that includes instrumental noise estimates and applies it to frequency dependent porcine pulmonary artery endothelial cell impedance measurements. The stability of cell–cell, cell–matrix and membrane impedance parameter estimates based on this approach is carefully examined, and several forms of parameter instability and refinement illustrated. Including frequency dependent noise variance estimates in the numerical optimization reduced the parameter value dependence on the frequency range of measured impedances. The increased stability provided by a multi-response non-linear fit over one-dimensional algorithms indicated that both real and imaginary data should be used in the parameter optimization. Error estimates based on single fits and Monte Carlo simulations showed that the model barrier parameters were often highly correlated with each other. Independently resolving the different parameters can, therefore, present a challenge to the experimentalist and demand the use of non-linear multivariate statistical methods when comparing different sets of parameters.

The main aims of this study were to elucidate the effect of green tea catechins on Nudix-type motif 6 (NUDT6) suppression and to characterize NUDT6's biological activity. Our microarray data showed that the green tea component epicatechin-3-gallate suppressed NUDT6 expression, and this was confirmed by RT-PCR. Subsequently, the use of different catechins showed that the effect of epigallocatechin-3-gallate (EGCG) was stronger than that of other catechins. At the posttranscriptional level, EGCG decreased the RNA stability of NUDT6, indicating it as a potential mechanism of NUDT6 suppression. Further cloning of the 3′ untranslated region of human NUDT6 mRNA resulted in reduced luciferase activity by EGCG treatment. This effect was at least, in part, mediated by the extracellular-signal-regulated kinase and p38MAPK pathways. Finally, increased cell proliferation and cell growth in soft agar were observed in NUDT6-overexpressing cells. These findings provide a novel mechanism for the suppression of the proliferative gene NUDT6 by green tea catechins in human colorectal cancer.