Paper-based Culture Research Articles

Combined electrical-electrochemical phenotypic profiling of antibiotic susceptibility of in vitro biofilm models.

More than 65% of bacterial infections are caused by biofilms. However, standard biofilm susceptibility tests are not available for clinical use. All conventional biofilm models suffer from a long formation time and fail to mimic in vivo microbial biofilm conditions. Moreover, biofilms make it difficult to monitor the effectiveness of antibiotics. This work creates a powerful yet simple method to form a target biofilm and develops an innovative approach to monitoring the antibiotic's efficacy against a biofilm-associated infection. A paper-based culture platform can provide a new strategy for rapid microbial biofilm formation through capillary action. A combined electrical-electrochemical technique monitors bacterial metabolism rapidly and reliably by measuring microbial extracellular electron transfer (EET) and using electrochemical impedance spectroscopy (EIS) across a microbe-electrode interface. Three representative pathogens, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, form their biofilms controllably within an hour. Within another hour their susceptibilities to three frontline antibiotics with different action modes (gentamicin, ciprofloxacin, and ceftazidime) are examined. Our antibiotic susceptibility testing (AST) technique provides a quantifiable minimum inhibitory concentration (MIC) of those antibiotics against the in vitro biofilm models and characterizes their action mechanisms. The results will have an important positive effect because they provide immediately actionable healthcare information at a reduced cost, revolutionizing public healthcare.

Continuous flow delivery system for the perfusion of scaffold-based 3D cultures.

The paper-based culture platform developed by Whitesides readily incorporates tissue-like structures into laboratories with established workflows that rely on monolayer cultures. Cell-laden hydrogels are deposited in these porous scaffolds with micropipettes; these scaffolds support the thin gel slabs, allowing them to be evaluated individually or stacked into thick constructs. The paper-based culture platform has inspired many basic and translational studies, each exploring how readily accessible materials can generate complex structures that mimic aspects of tissues in vivo. Many of these examples have relied on static culture conditions, which result in diffusion-limited environments and cells experiencing pericellular hypoxia. Perfusion-based systems can alleviate pericellular hypoxia and other cell stresses by continually exposing the cells to fresh medium. These perfusion systems are common in microfluidic and organ-on-chip devices supporting cells as monolayer cultures or as 3D constructs. Here, we introduce a continuous flow delivery system, which uses parts readily produced with 3D printing to provide a self-contained culture platform in which cells in paper or other scaffolds are exposed to fresh (flowing) medium. We demonstrate the utility of this device with examples of cells maintained in single cell-laden scaffolds, stacks of cell-laden scaffolds, and scaffolds that contain monolayers of endothelial cells. These demonstrations highlight some possible experimental questions that can be enabled with readily accessible culture materials and a perfusion-based device that can be readily fabricated.

Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.

Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.

Selecting the appropriate indirect viability assay for 3D paper-based cultures: a data-driven study.

Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins. Direct viability measures count each cell to provide an accurate readout. This approach can prove analytically challenging and time-consuming when cells are maintained in 3D structures akin to tissues or solid tumors. While less labor-intensive, indirect viability measures can be less accurate due to the heterogeneous structural and chemical microenvironment that arises when cells are maintained in tissue-like architectures and in contact with extracellular matrices. Here we determine the analytical figures of merit of five indirect viability assays in the paper-based cell culture platform we continue to develop in our laboratory: calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay. We also determined the compatibility of each indirect assay with hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. Our results show that each assay has benefits and drawbacks to consider when choosing the appropriate readout to answer a particular research question. We also highlight that only one indirect readout is unaffected by hypoxia, a commonly overlooked variable in cell culture that likely yields inaccurate viability measures.

Deterioration of Kaihua handmade paper: Evolution of molecular, supermolecular and macroscopic structures

Valued for its toughness and durability, Kaihua paper has been famous in China since the Qing Dynasty. It attracts great attention on the extension of paper life. Understanding of the complex degradation behavior of handmade paper under various aging conditions is an essential precondition for preparing long-life handmade paper. However, it is still difficult to explain the reactions sequence of cellulose microstructure and its relationship with the macroscopic deterioration of handmade paper. Herein, different types of Kaihua handmade papers, produced by various raw materials and crafts, were artificially aged. The evolution of their multi-scale structures in dry-heat (DH) and wet-heat (WH) conditions was explored systematically. Two-dimensional correlation spectroscopy (2D-COS) distinguished possible carbonyl vibrations involving hydrolysis and oxidation of cellulose and gave sequential changes of various carbonyl groups, illustrating different evolutionary behaviors of molecular structure of papers in DH and WH aging process. The energy and distance of hydrogen bond, crystal size and microfiber accessibility were quantitatively calculated during the degradation of cellulose. In addition, correlations between microstructural evolution and macroscopic deterioration of handmade papers involving reduced mechanical properties and yellowing were revealed. The current results will help to establish a platform for the multi-scale detection of handmade paper and improve the understanding of the degradation mechanism of handmade paper under different reaction paths. It also provides useful foundation on the preparation of long-life paper and the conservation of paper-based culture relics.

Spatially resolved quantification of drug metabolism and efficacy in 3D paper-based tumor mimics

Paper-based cultures are an emerging platform for preparing three-dimensional (3D) tissue- and tumor-like structures. The ability to stack individual sheets of cell-containing paper affords a modular means of assembling structures with defined cellular compositions and microenvironments. These layered stacks are easily separated at the end of an experiment, providing spatially resolved populations of live cells for further analysis. Here we describe a workflow in which cell viability, drug penetration, and drug metabolism are quantified in a spatially resolved manner. Specifically, we mapped the distribution of the drug irinotecan and its bioactive metabolite SN38 in a colorectal cancer cell-containing stacked structure with liquid chromatography-mass spectrometry (LC-MS). This paper provides the first example of a 3D culture platform that quantifies viability and drug metabolism in a spatially resolved manner. Our data show that cells at the bottom of the stack are more drug-resistant than layers in contact with the culture medium, similar to cells in the nutrient-poor center of a proliferating tumor being more drug-resistant than the rapidly dividing cells at its periphery. The powerful combination of quantitative viability and drug metabolism measurements will enable future studies to determine the exact mechanism(s) of drug resistance in different regions of a tumor.

Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells

This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.

Tracking the invasion of breast cancer cells in paper-based 3D cultures by OCT motility analysis.

3D paper-based cultures (PBCs) are easy-to-use and provide a biologically representative microenvironment. By stacking a sheet of cell-laden paper below sheets containing cell-free hydrogel, we form an assay capable of segmenting cells by the distance they invaded from the original cell-seeded layer. These invasion assays are limited to end-point analyses with fluorescence-based readouts due to the highly scattering nature of the paper scaffolds. Here we demonstrate that optical coherence tomography (OCT) can distinguish living cells from the surrounding extracellular matrix (ECM) or paper fibers based upon their intracellular motility amplitude (M). M is computed from fluctuation statistics of the sample, rejects shot noise, and is invariant to OCT signal attenuation. Using OCT motility analysis, we tracked the invasion of breast cancer cells over a 3-day period in 4-layer PBCs (160-300 µm thick) in situ. The cell population distributions determined with OCT are highly correlated with those obtained by fluorescence imaging, with an intraclass correlation coefficient (ICC) of 0.903. The ability of OCT motility analysis to visualize live cells and quantify cell distributions in PBC assays in situ and longitudinally provides a novel means for understanding how chemical gradients within the tumor microenvironment affect cellular invasion.

Developing a Drug Screening Platform: MALDI-Mass Spectrometry Imaging of Paper-Based Cultures.

Many potential chemotherapeutics fail to reach patients. One of the key reasons is that compounds are tested during the drug discovery stage in two-dimensional (2D) cell cultures, which are often unable to accurately model in vivo outcomes. Three-dimensional (3D) in vitro tumor models are more predictive of chemotherapeutic effectiveness than 2D cultures, and thus, their implementation during the drug screening stage has the potential to more accurately evaluate compounds earlier, saving both time and money. Paper-based cultures (PBCs) are an emerging 3D culture platform in which cells suspended in Matrigel are seeded into paper scaffolds and cultured to generate a tissue-like environment. In this study, we demonstrate the potential of matrix-assisted laser desorption/ionization-mass spectrometry imaging with PBCs (MALDI-MSI-PBC) as a drug screening platform. This method discriminated regions of the PBCs with and without cells and/or drugs, indicating that coupling PBCs with MALDI-MSI has the potential to develop rapid, large-scale, and parallel mass spectrometric drug screens.

A pH-Sensing Optode for Mapping Spatiotemporal Gradients in 3D Paper-Based Cell Cultures.

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pKa of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

3D cellular invasion platforms: how do paper-based cultures stack up?

Cellular invasion is the gateway to metastasis, which is the leading cause of cancer-related deaths. Invasion is driven by a number of chemical and mechanical stresses that arise in the tumor microenvironment. In vitro assays are needed for the systematic study of cancer progress. To be truly predictive, these assays must generate tissue-like environments that can be experimentally controlled and manipulated. While two-dimensional (2D) monolayer cultures are easily assembled and evaluated, they lack the extracellular components needed to assess invasion. Three-dimensional (3D) cultures are better suited for invasion studies because they generate cellular phenotypes that are more representative of those found in vivo. This feature article provides an overview of four invasion platforms. We focus on paper-based cultures, an emerging 3D culture platform capable of generating tissue-like structures and quantifying cellular invasion. Paper-based cultures are as easily assembled and analyzed as monolayers, but provide an experimentally powerful platform capable of supporting: co-cultures and representative extracellular environments; experimentally controlled gradients; readouts capable of quantifying, discerning, and separating cells based on their invasiveness. With a series of examples we highlight the potential of paper-based cultures, and discuss how they stack up against other invasion platforms.

Assessing chemotherapeutic effectiveness using a paper-based tumor model.

In vitro models for screening new cancer chemotherapeutics often rely on two-dimensional cultures to predict therapeutic potential. Unfortunately, the predictive power of these models is limited, as they fail to recapitulate the complex three-dimensional environments in tumors that promote a chemoresistant phenotype. In this study, we describe the preparation and characterization of paper-based cultures (PBCs) engineered to assess chemotherapeutic effectiveness in three dimensional, diffusion-limited environments. Similar environments are found in poorly vascularized tumors. Monotonic gradients develop across these cultures, which are assembled by stacking cell-laden paper scaffolds to yield thick tissue-like structures, and provide distinct chemical environments for each scaffold. After prolonged incubation, the scaffolds can simply be peeled apart and analyzed. Through fluorescence imaging, we determined that viable and proliferative cell populations were most abundant in scaffolds close to the nutrient-rich medium. By adjusting the cell density, we modulated the spatiotemporal evolution of oxygen gradients across the cultures and correlated these environmental changes with cellular sensitivity to SN-38 exposure. From these results, we showed that differences in the oxygen gradients produced cellular populations with significantly different chemosensitivities. Through this work, we highlight PBCs ability to serve as an analytical model capable of determining chemotherapeutic effectiveness under a range of chemical environments.

Electrochemical Detection of Bacteria Using Portable Paper-Based Culture Devices

Testing of food and water samples for viruses and bacteria in a rapid, portable, quantitative way is of growing interest as much for government, farmers, industrials, as end-consumers. In the past two decades, many research groups were involved in the development of various paper-based devices for quantitative or semi-quantitative detection of clinically relevant analytes and in particular pathogens. Some difficulties remain in analyzing samples with complex matrices (e.g., colored samples or muddy water), with enough sensitivity (e.g., detecting less than 10-100 cfu/mL) while minimizing culture time. Electrochemical detection can allow for rapid quantification over a wide dynamic range, with a low limit of detection, and without limitations related to presence of colors or particles in samples. Derda and co-workers validated growth of bacteria in portable paper-based culture devices [1, 2]. We added screen-printed electrodes onto those paper-based devices (Figure A-B) to allow for electrochemical detection of micro-organisms. The presence of bacteria in the device, and particularly on the electrode, bacteria affects the characteristics of the electrode. By measuring those changes by electrochemical impedance spectroscopy, for example, we could detect the bacteria (Figure E). The platform can be selective to bacteria by immobilizing on the electrode proximity antibodies specific to the bacteria to detect, and thus enhancing the presence of the bacteria on the electrode. Using the paper-based portable culture device instead of metal electrodes as reported previously [3] as a support as multiple advantages: in addition to allow for an easy- sampling at the point-of-care or in-field, if the sample has a low number of bacteria (Figure C), it can be cultured in the same device and tested again once more bacteria is present (Figure D) without additional handling of the sample. The use of paper in these devices made them user-friendly, portable, light-weight, low-cost, easy to manufactured and disposed of once contaminated (e.g., burning).

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors.

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

Multilayer three-dimensional filter paper constructs for the culture and analysis of aortic valvular interstitial cells

Culturing aortic valvular interstitial cells in an environment that models the aortic valve is an essential step towards understanding the progression of calcific aortic valve disease. Here the adaption of a three-dimensional (3-D) stacked paper-based culture system is presented for analyzing valve cells in a thick collagen gel matrix. Filter paper layers, modeled after a 96-well plate design, were printed with a wax well-plate template and then seeded with valve cell and collagen mixtures that quickly gelled into 3-D cultures. Stacking these layers permitted extensive customization of culture thickness and cell density profiles to model the full thickness of native valve tissue. Aortic valvular interstitial cells seeded into the paper-based constructs consistently demonstrated high survival up to 14days of culture with significant increases in cell number through the first 3days of culture. After 4days following seeding, valve cells in single layer cultures showed reduced smooth muscle α-actin expression with a stabilized cell density, suggesting a transition from an activated phenotype to a more quiescent state. Valve cells in multilayer cultures demonstrated the ability to migrate from layer to layer and had the highest smooth muscle α-actin expression in areas with predicted low oxygen tensions. These results establish the filter-paper-based method as a viable culture system for analyzing valve cells in an in vitro 3-D model of the aortic valve.

An Introduction to Cyber Crime: General Considerations

Cyber crime reflects a peculiar type of techno-sophisticated criminality having different features. This criminality is posing the challenges to existing national legal systems and it appear to be difficult to control and combat these crimes within the existing framework of legal system. Specially, the problem of jurisdiction, identity crises and lack of legal recognition of most of acts make it difficult for legal systems to effective deal with the crime. The location and trans-national character of these crimes again added the flavor makes it too dangerous to imagine. This article has attempted to identify the characteristic features of cyber criminality and the its varied nature over the traditional criminality. Cyber crime may be categorized in two classes. One where the computer is instrument, and another where computer is incidental. It is not difficult to control the cyber crimes fall in former categories as here, computer is only an instrument and traditional legal system is better to coup up these problem by merely recognizing it, however, it is difficult to deal with the later class of criminality as computer technology is itself new to the legal system and it cannot be deal with the merely old principles which is territorially sensitive, jurisdictionally responsive, and susceptible to paper based culture and identity. This article has focused on the latter aspect of cyber criminality.

The need to ensure that the globalization of information and telematics does not destabilize health-care worldwide.

Modern telecommunication transcends institutional, organizational and cultural boundaries. In the context of health-care, this means that information, enquiries, demands and service provision can be routed anywhere, free of institutional or natural control. The Internet makes information available to anyone regardless of its quality, source or intention. Organizationally initiated applications such as telemedicine are usually intended to reinforce local systems, but their very design and intention indicate a potential rapidly to transcend inherited but electronically ineffectual boundaries. The consequences of such uncontrolled globalization of health-care activities will range from beneficial empowerment and quality improvement, to detrimental effects such as overloading of experts, and undermining of stable health-care systems. The major unplanned societal re-engineering effects in a paper-based culture are likely to be significant and global institutions need to respond by creating positive global frameworks and policies.

The Role of Paper in a Wired World

For years, the idea of the “paperless office” put forth by “futurists” a decade or so ago has been laughed at in the computer printer industry, which has watched gleefully as office and consumer paper consumption soared year after year. But the advent of the Internet means that the paperless, or at least less-paper office, and perhaps lesspaper home, is no longer a joke. The Internet has changed the role of paper: it once was the document, but has become only a transient display medium for the document, which is now a computer file, email, or Web site. Even though paper consumption continues to rise, research shows that many, even most documents that cross a person's desk are no longer printed. And, as computer design and display technology improve, that percentage can only rise. At the same time, a horde of “dot-coms” are spending billions to automate processes in every industry that have long depended on inefficient paper-shuffling. In short, a paper-based culture that has lasted hundreds of years is under assault, an assault that will certainly affect the market for computer printers and printing.

The Text Is Dead, Long Live the Techst

The Text Is Dead; Long Live the Techst Edward M. Jennings Landow, George P. Hypertext: The Convergence of Contemporary Literary Theory and Technology. Baltimore and London: Johns Hopkins UP, 1992. [1] This is a review of George P. Landow’s book about a phenomenon almost as outlandish in a paper-based culture as scripture must appear to be when it arrives in societies without records. Hypertext: The Convergence of Contemporary Literary Theory and Technology is part of a series called “Parallax: Re-visions of Culture and Society.” Steven G. Nichols, Gerald Prince, and Wendy Steiner are the series editors. I think it is a marvelous book, and this essay is meant to prod you into reading it from cover to cover. Hypertext could be the keystone volume in a graduate curriculum where the rhetorics of networking and screen display are scrutinized right beside those of oral and scribal modes, of scroll and codex technologies. But at least four audiences may still be hostile to it: Curmudgeons who don’t know which upsets them more, critical theory or technology; closet word-processors for whom the concept “programming” still smacks of mind control; theorists for whom Barthes and Derrida and Lyotard are old wallpaper against which background some significant struggles are (at last) taking place; and technophiles ashamed of their access to tools that others cannot afford. The book itself is not a menace, but the technologies it celebrates—or the still unexplored opportunities offered by the hypertext technology—threaten assumptions so deeply held that most people will deny that they can be challenged. After all, these words mean what they mean, don’t they? Text. Author. Story. Knowledge. Landow himself issues no directly apocalyptic challenges. No foam around his mouth. His presentation is measured, experiential, lucid, moderate and sensible. He merely points out that the concept “hypertext” lets us test some concepts associated with critical theory, and gracefully shows how the technology is contributing to reconfigurations of text, author, narrative and (literary) education. As an advocate for the technology Landow describes so clearly, my goal in this review is to tell you enough about it so that you will feel compelled at least to read Hypertext, even if you don’t rush out and invest all at once in the electronic paraphernalia you would need to become acculturated. I will try to describe the phenomenon, and then try to suggest how hypertext demands that we re-place those four self-evident terms. As I perceive it, the technology undermines fundamental assumptions about authority and control of time. Just what is this “thing,” this “concept,” this technology that has acquired the label “hypertext”? Landow does a good job of explaining it, as do Bolter and Moulthrop and Slatin (emphasizing “Storyspace”), but it’s like trying to describe digital recording to Oscar Wilde or trying to help a fish understand “breathing.” Even readers of PMC need help, I suspect, in spite of their acquaintance with at least two other transforming technologies, word-processing and networking. Not everyone has easy access to the relatively expensive Macintosh platform where most of the writer-artist hypertext software performs. Please note: We are not discussing the ballyhooed “multimedia” here, nor the pseudo-hypertext built in to the “Help Menus” of commercial software applications. My own experience (limited) is with Eastgate Systems’ “Storyspace” (and a few hours with Ntergade’s “Black Magic,” and a few minutes with Knowledge Garden’s “Knowledge Pro”). George Landow, in sharp contrast, has designed and experienced entire “docuverses” in the “Intermedia” environment developed and installed at Brown University. He has practiced what he preaches, that is. What’s more, he and Paul Delany have already edited Hypermedia and Literary Studies (MIT, 1991), 17 essays whose cluster of perspectives supplements and qualifies the authoritative focus of his 1992 monotext being reviewed here. Once more, then: What “is” hypertext? It can be imagined as an endless electronic nesting of “footnotes,” each one enriching all the others, none of them secondary even though one had to be encountered first. You can place them whenever you want, in whichever typeface (or “tone”) you choose, and with whatever coloration you prefer. Another image is of a book’s...