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This paper applies a theory of persuasion using the elaboration likelihood model to explain how communication messages from mobile banking services are interpreted by receivers to increase their trust in mobile banking. We explain how users’ security and privacy concerns moderate the central route persuasion relationship between argument quality and trust. A questionnaire survey is employed to test a research model. The findings indicate that central and peripheral route persuasion cues enforce users’ trust in mobile banking services. Users with strong privacy and security concerns are more likely to rely on the quality of arguments of the messages. Theoretical and practical implications are discussed in the paper.

Organisations today must deal with a volume, pace, and variety of data that is unprecedented. This has brought about the growth of big data and the requirement for efficient data analytics approaches to get insightful information. In order to handle current difficulties and take advantage of opportunities across multiple disciplines, this research paper examines the importance of big data and data analytics. It examines how big data analytics might be used to improve operational effectiveness, decision-making processes, and consumer experiences. This study also looks at the procedures and equipment used to gather, store, analyse, and analyse big data. The paper shows the advantages, drawbacks, and ethical issues related to big data and data analytics through an extensive assessment of the literature that has already been written and case studies. Additionally, it covers how big data analytics will affect data governance, security, and privacy. This study seeks to provide light on the transformative potential of big data and data analytics in influencing company strategies and fostering innovation. To do so, it examines existing trends and future prospects. The study's findings help us grasp the potential and problems that big data presents, and they also lay the groundwork for future studies in this quickly developing area

Monolayer cell cultures, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, a complex 3-dimensional (3D) structure resemble in vivo tumor growth more closely thereby allowing results obtained with spheroids relating to proliferation, cell death, differentiation, metabolism, and various anti-tumor therapies to be more predictive of in vivo outcomes. The protocol herein presents a rapid and high throughput method for the generation of single spheroids using various cancer cell lines including (U87 MG, SEBTA-027, SF188) brain cancer cells, (DU-145, TRAMP-C1) prostate cancer cells, and (BT-549, Py230) breast cancer cells in a 96-round bottom well plates. The proposed method is associated with significantly low costs (ca. £1) per plate without the need for refining or transferring and homogeneous compact spheroid morphology was evidenced as early as 1 day after following this protocol. By using confocal microscopy and the IncuCyte live imaging system, proliferating cells were traced in the rim while dead cells were found to be located inside the core region of the spheroid. H&E staining of spheroid sections was utilized to investigate the tightness of the cell packaging and Western blotting analyses revealed that these spheroids adopted a stem cell-like phenotype. This method was also used to obtain EC50 of the anti-cancer dipeptide carnosine on U87 MG 3D culture. The affordable easy-to-follow 5 step-protocol allows for robust generation of various uniform spheroids which show 3D morphology characteristics.

Introduction: Monolayer cell cultures, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more closely resemble in vivo tumour growth thereby allowing results obtained with spheroids relating to proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. Methods: The protocol herein presents a rapid and high throughput method for the generation of single spheroids whose applicability was demonstrated on various cancer cell lines including (U87 MG; SEBTA-027; SF188) brain cancer cells, (DU-145, TRAMP-C1) prostate cancer cells, and (BT-549, Py230) breast cancer in green coded 96-round bottom well plates. Results: Homogeneous compact spheroid morphology was evidenced as early as 24 hours after following the protocol. By using confocal microscopy and IncuCyte live imaging, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. H&E staining of spheroid slices and Western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. Conclusions: This 5 step-protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals and encourage 3D modelling experiments from early-stage research.

Nanometer scale rods of superparamagnetic iron oxide have been encapsulated, along with the anti-cancer therapeutic carnosine, inside porous poly(lactic

Poly(butylene succinate) (PBSU) is a biodegradable and biocompatible synthetic aliphatic polyester, which has been used extensively in packaging, catering and agriculture, and more recently in drug delivery and bone and cartilage repair. PBSU-based mats created by electrospinning show promise as wound dressing materials because of their good mechanical properties, high surface area-to-volume ratio and increased levels of porosity. In this work, we present the creation of antimicrobial PBSU fibrous mats through the incorporation of natural food grade agents via blend electrospinning. Three types of edible gums (namely arabic, karaya and tragacanth), two essential oils (coriander and lavender), and one free fatty acid (linoleic acid) were added to PBSU containing a chain extender and their effect on six clinically relevant pathogens was evaluated. Mats containing essential oils at the highest concentration studied (7% w/v) showed some antimicrobial behaviour against S. aureus, E. hirae and P. aeruginosa, whereas the incorporation of linoleic acid at both concentrations tested (3% and 5% w/v) gave a strong reaction against S. pyogenes. Gum arabic was the only gum that had a considerable impact on S. aureus. Furthermore, the three gums enhanced the mechanical properties of the polymer mats and brought them closer to those of the human skin, whilst all agents maintained the high biocompatibility of the PBSU mats when contacted with mouse fibroblasts. This work, for the first time, shows the great promise of PBSU blended fibres as a skin substitute and paves the way towards bioactive and cost effective wound dressings from renewable materials.

The power of personalized medicine is based on a deep understanding of cellular and molecular processes underlying disease pathogenesis. Accurately characterizing and analyzing connections between these processes is dependent on our ability to access multiple classes of biomarkers (DNA, RNA, and proteins)-ideally, in a minimally processed state. Here, we characterize a biomarker isolation platform that enables simultaneous isolation and on-chip detection of cell-free DNA (cfDNA), extracellular vesicle RNA (EV-RNA), and EV-associated proteins in unprocessed biological fluids using AC Electrokinetics (ACE). Human biofluid samples were flowed over the ACE microelectrode array (ACE chip) on the Verita platform while an electrical signal was applied, inducing a field that reversibly captured biomarkers onto the microelectrode array. Isolated cfDNA, EV-RNA, and EV-associated proteins were visualized directly on the chip using DNA and RNA specific dyes or antigen-specific, directly conjugated antibodies (CD63, TSG101, PD-L1, GPC-1), respectively. Isolated material was also eluted off the chip and analyzed downstream by multiple methods, including PCR, RT-PCR, next-generation sequencing (NGS), capillary electrophoresis, and nanoparticle size characterization. The detection workflow confirmed the capture of cfDNA, EV-RNA, and EV-associated proteins from human biofluids on the ACE chip. Tumor specific variants and the mRNAs of housekeeping gene PGK1 were detected in cfDNA and RNA isolated directly from chips in PCR, NGS, and RT-PCR assays, demonstrating that high-quality material can be isolated from donor samples using the isolation workflow. Detection of the luminal membrane protein TSG101 with antibodies depended on membrane permeabilization, consistent with the presence of vesicles on the chip. Protein, morphological, and size characterization revealed that these vesicles had the characteristics of EVs. The results demonstrated that unprocessed cfDNA, EV-RNA, and EV-associated proteins can be isolated and simultaneously fluorescently analyzed on the ACE chip. The compatibility with established downstream technologies may also allow the use of the platform as a sample preparation method for workflows that could benefit from access to unprocessed exosomal, genomic, and proteomic biomarkers.

BACKGROUND: Modern day scientific endeavour strives towards global sustainability through the smart utilisation of renewable resources as base materials for chemicals. Until now, the most common commercial process to produce levulinic acid (a mass-produced platform chemical) depends on a two-stage mineral acid-catalysed reaction, which generates harmful environmental waste. In this work, an environmentally friendly levulinic acid production route using less harmful organic acids assisted by microwave heating from biomass feedstocks is reported for the first time. RESULTS: Using aluminum sulfate as a green Lewis acid catalyst and seven organic acids, levulinic acid was successfully produced from barley straw under microwave heating, with maleic acid giving the highest catalytic conversion. A Response Surface Methodology (RSM) approach was used to rapidly and effectively examine the effect of five reaction variables on the productivity of the levulinic acid. A wide range of different biomass wastes (barley straw, brewery waste, olive cake, spent tea leaves and potato, tomato, and mandarin peels) were subsequently screened to produce the levulinic acid. The highest yield of 86 wt% based on cellulose content from mandarin peel (a value comparable to a lengthier ‘non-green’ route) was achieved under the following optimized reaction conditions: 180 °C, 38 min, 2 M maleic acid concentration, 0.1 g Al 2(SO 4) 3 and 1:22 biomass: maleic acid ratio (g mL −1). CONCLUSIONS: The proposed method is a promising new route towards the green, high yield production of levulinic acid from a variety of agricultural and household lignocellulosic biomass wastes, without the need for pre-treatment.

Background The Resistance-Nodulation-Division (RND) family transporter AcrB plays a major role in the intrinsic and increased resistance of Escherichia coli to a large number of antibiotics. The distal binding pocket within this multidrug efflux transporter is very large, but the effort to define the roles of various residues facing this pocket through site-directed mutagenesis so far involved only the determination of minimal inhibitory concentrations of drugs in mutants. Methods We measured in intact E. coli cells the kinetics of efflux of two substrates, nitrocefin (a cephalosporin) that is predicted mainly to bind to the upper, “groove” domain of the pocket, and L-alanyl-β-naphthylamide (Ala-Naph) that is likely to bind to the lower, “cave” domain, in a number of site-directed mutants of AcrB, where a hydrophobic or aromatic residue was changed into alanine. Results The efflux of nitrocefin became attenuated by some mutations in the groove domain, such as I278A and F178A, but in some experiments a mutation in the cave domain, F628A produced a similar result. In some cases an increased value of KM was detected. The efflux of Ala-Naph was increased by mutations in the cave domain, such as F136A and I626A, but also by those in the groove domain (I277A, I278A, F178A). In most cases the increased Vmax values appeared to be responsible. F610A mutation had a profound effect on the efflux of both substrates, as reported earlier. Conclusions Our data show for the first time effects of various substrate-binding pocket mutations on the kinetics of efflux of two substrates by the AcrB pump. They also confirm interactions between substrates and drugs predicted by MD simulation studies, and also reveal areas that need future research.

Introduction: Extracellular vesicles (EVs) contain protein and nucleic cargo that has been shown to be reflective of physiological and pathological states, or their originating cells, in many diseases including cancer. Specifically, these EV-associated biomarkers have been shown to fluctuate with the change in pathological processes. As a result, characterization of blood-based circulating EVs have been widely investigated for diagnostic applications such as disease monitoring and treatment selection. The heterogeneity of blood-based EVs present significant challenges to current rapid isolation methods due to enrichment with unwanted sub-populations, e.g. apoptotic bodies, or contamination with lipids, e.g. APOB, for both capture-based chemistry-based isolation methods. We have developed a novel lab-on-a-chip technology for isolation and on-chip characterization of EVs from blood-based matrices using an AC electrokinetics (ACE) methodology. Methods: Blood samples were collected from 10 healthy volunteers and 10 donors with known cancer diagnosis into K2EDTA tubes under IRB approved protocols. Plasma was processed from the blood and stored at -80C. 120 µL of thawed plasma was applied to the microelectrode array flow cell (ExoVerita Flex, Biological Dynamics, San Diego, CA) and the EVs were isolated on the microelectrodes. Following isolation, the flow cell was washed, the isolated material was released from the array and eluted from the flow cell. For comparison, isolation of EVs from the same amount of plasma was performed using a chemistry-based method (ExoQuick Ultra, System Biosciences, Palo Alto, CA) according to manufacturer's instructions. Following isolation, the eluates were evaluated for the presence of EV-associated biomarkers using Western blotting capillary electrophoresis and mass spectrometry (Orbitrap Fusion Lumos, TFS). Nanoparticle tracking analysis (qNano Gold, Izon Science, New Zealand) was performed to determine EV concentration and size distribution. Quantitative qRT-PCR analysis of the purified plasma Evs was performed to confirm presence of EV-bound mRNA. Results: Western Blotting demonstrated that eluates from both ACE-based methodology and chemistry-based isolation contain the expected CD9 and CD63. The ACE-isolated EVs showed a single prominent band for CD63, whereas chemistry-based isolated EVs display multiple smaller CD63-reactive species, which was suggestive of proteolysis. Mass spectrometry analysis confirmed presence of CD9 and CD81 in EVs isolated by ACE method; but not in EVs purified by chemistry-based method. APOB contamination was present in chemistry-based EVs, but not in ACE-isolated EVs. For RT-PCR experiments, EVs were purified from 5 subjects with lung cancer and 3 subjects with melanoma patient plasma using ACE method. Quantitative PCR confirmed presence of mRNA, via successful amplification of the housekeeping genes PGK1 and β-actin, in all ACE-isolated EVs. Conclusions: The novel ACE-based platform successfully demonstrated direct isolation of EVs from plasma while preserving the integrity of protein and mRNA biomarkers. The compatibility of the eluted EVs with multiple downstream technologies, such as mass spectrometry, Western blotting and qRT-PCR, may enable novel biomarker discovery and use of EV-associated biomarkers in diagnostic assays. Citation Format: Rajaram Krishnan, Jean Lewis, David Searson, Orlando Perrera, Alfred Kinana, Heath Balcer, Iryna Clark, Juan Pablo Hinestrosa. Characterization of circulating extracellular vesicles isolated from plasma of cancer subjects using novel AC electrokinetics platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2850.

Exosomes carry signals between cells and may influence cancer development, metastasis, and drug resistance. Unfortunately, current tools for exosome isolation and analysis have been limited to examining a specific class of markers, such as DNA, RNA, and proteins. We have developed a microelectrode array chip that uses a novel Alternating Current Electrokinetic method to perform rapid, affinity-free isolation of exosomes. The chip runs on our proprietary ExoVeritaTM Flex platform. Isolated exosomes can then be analyzed in situ or can be lysed to obtain DNA and RNA for use in downstream assays, such as sequencing. In this study, we used the ExoVerita Flex to isolate exosomes directly from plasma specimens obtained from cancer subjects with lung, colorectal, or pancreatic cancers as well as healthy donors. Isolated exosomes were analyzed in situ for exosome-bound proteins and nucleic acids, and then purified following isolation to examine their nucleic acid expression profiles. Using the ExoVerita Flex with plasma specimens and antibodies targeting the exosome-bound proteins CEA, PD-L1, GPC-1 and CD63, we observed differences in protein expression levels among the different cancer types (N = 35) and healthy donors (N = 20), such as increased PD-L1 expression in non-small cell lung cancer subject samples and elevated levels of CEA in colorectal cancer subject samples (N = 10). Furthermore, we co-stained samples with antibodies and DNA- or RNA-selective dyes to simultaneously measure exosome-bound proteins and circulating nucleic acid levels in the same sample at the same time. RNA sequencing analysis was performed by extracting RNA from the isolated exosomes using a proprietary buffer, and then performing RT-ddPCR and miRNA-Sequencing. RT-ddPCR analysis of the PGK-1 housekeeping gene RNA transcript confirmed the presence of viable RNA following extraction. miRNA profiles from isolated exosomes were generated using a targeted RNA sequencing assay and used for repeatability and differential expression studies. The ExoVerita Flex platform offers a novel, unbiased method for isolation and characterization of exosome-bound proteins and circulating nucleic acids while being compatible with standard downstream analysis. The ability to perform two-dimensional protein & nucleic acid analysis in the same sample run, at the same time, is both unique and unprecedented and will enable promising future clinical studies in cancer looking at diverse classes of biomarkers directly from blood. Citation Format: Jean M. Lewis, David Searson, Alfred Kinana, Heath Balcer, Debrah Thompson, David Bodkin, Juan P. Hinestrosa, Rajaram Krishnan. Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3978.

There are significant limitations to the tools currently used to monitor clinical status and treatment response in late-stage non-small cell lung cancer (NSCLC) patients. Standard-of-care imaging is generally performed every few months, and emerging assays such as tumor mutational burden have yet to be clinically validated. Most of these tools are expensive, further limiting their use. We are exploring a promising new biomarker, high molecular weight cell-free DNA (hmw cfDNA, predominantly longer than 300 bp), which we assay using proprietary ACE (Alternating Current Electrokinetics) technology directly in plasma. In this study, we tested the hypothesis that changes in the level of hmw cfDNA in cancer patients correlate with traditional, imaging-based clinical assessments. We collected longitudinal blood specimens from subjects diagnosed with stage III and stage IV NSCLC and being treated with various therapies (chemo, immuno, targeted) at multiple sites across the US. Specimens were obtained within nine days of routine imaging and were processed to plasma prior to testing. For this analysis, using our hmw cfDNA assay and orthogonal PCR assays, we obtained validated results on samples from 62 subjects which included a total of 117 “deltas”, defined as the difference in hmw cfDNA levels between consecutive blood draws. The correlation between these deltas and clinical assessments, which were made independently by the physician responsible for the subject’s routine care and by a third-party radiologist, was determined. In post-hoc analysis of these results, we found significant correlation between deltas and clinical assessments across classes of therapies, according to receiver operator characteristic (ROC) analysis. This dataset produced an area under the ROC curve (AUROC) of 0.696, with a 95% confidence interval of 0.594 - 0.797. The time intervals between imaging dates varied, due to common practice being followed by physicians, and we found that exclusion of longer time intervals resulted in a progressive improvement in the correlation between hmw cfDNA delta and clinical assessment. Analyzing results with time intervals of less than 120 days, similar to NCCN guidelines, resulted in an AUROC of 0.784 (95% CI of 0.673 - 0.880). Analysis of data from a small subset of subjects (N=11) on immune checkpoint inhibitors resulted in an AUROC > 0.8. These results suggest that hmw cfDNA is a promising biomarker for monitoring treatment response of NSCLC patients, including those on immunotherapy. The ease and lower cost associated with obtaining, processing, and testing samples using this assay would enable it to be employed on a more frequent basis, and it would therefore complement current technologies based on imaging and sequencing. Additional studies to refine the application of this assay and to determine its impact on clinical outcomes will further inform its future use. Citation Format: Robert Kovelman, Patrick F. Desmond, Natalie J. Kennel, Juan P. Hinestrosa, Ellen B. Christie, Delia Ye, Michelle M. Nguyen, Alfred D. Kinana, Mark S. Schechter, David Chu, Chirag M. Shah, Osama Hlalah, John E. Doster, Rajaram Krishnan. Correlation of levels of cell-free DNA in plasma with imaging-based assessment in Stage III/IV Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1350.

By measuring quantitatively the active efflux of cephalosporins by the RND (resistance-nodulation-division) family efflux pump AcrB in intact cells of Escherichia coli, we found that the simultaneous presence of another substrate, such as chloramphenicol, benzene, cyclohexane, or Arg β-naphthilamide, significantly enhanced the extrusion of cephalosporins. The stimulation occurred also in a strain expressing the covalently linked trimer of AcrB, and thus cannot be ascribed to the enhanced assembly of the trimer from AcrB monomers. When Val139 of AcrB was changed into Phe, the stimulation by benzene was found to occur at much lower concentration of the solvent. A plausible explanation of these observations is that the AcrB pump is constructed to pump out very rapidly the solvent or chloramphenicol molecules, and thus the efflux of cephalosporins, which presumably bind to a different subsite within the large binding pocket of AcrB, can become facilitated. Computer simulations of ligand binding to AcrB, both by docking and by molecular dynamics simulations, produced results supporting and extending this hypothesis. Benzene and the cephalosporin nitrocefin can bind simultaneously to the distal binding pocket of AcrB, both in the wild type and in the V139F variant. Interestingly, while the binding position and strength of benzene are almost unaffected by the presence of nitrocefin, this latter substrate is significantly displaced toward the exit gate in both wild type and mutant transporter in the presence of benzene. Additionally, the cephalosporin efflux may be enhanced by the binding of solvents (sometimes to the cephalosporin-free protomer), which could accelerate AcrB conformational changes necessary for substrate extrusion.

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.

The AcrB protein of Escherichia coli , together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N -phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.