bluuebunny/crossref_metadata_2025 huggingface dataset:
Three-dimensional culture
Authors: Kunz-Schughart L A , Mueller-Kliesert W
Published: November 2023

Three-dimensional (30) cell cultures have been widely used in biomedical research since the early decades of this century. Holtfreter (1) and later Moscona (2, 3) pioneered the field by their research on morphogenesis using spherical reaggregated cultures of embryonic or malignant cells. Numerous subsequent in vitro studies on organogenesis or expression of malignancy were based on these early investigations. Substantial novel input into research on cell aggregates came from fundamental studies of Sutherland and associates (4-6). They pioneered multicellular tumors spheroids (MCTS) as an in vitro model for studies on tumors cell response to therapy. MCTS have also been used to study basic biological mechanisms, including the regulation of proliferation, differentiation, cell death, invasion, angiogenesis, and the immune response (for reviews see refs 7-11). This chapter will mainly focus on techniques for growing spheroids, with limited information on 3D cultures other than spheroids.
Cytotoxicity and viability assays
Authors: Wilson Anne P
Published: November 2023

Drug development programmes involve pre-clinical screening of vast numbers of chemicals for specific and non-specific cytotoxicity against many types of cells. Both are important for indicating the potential therapeutic target and safety evaluation. Animal models have always played an important role in both contexts, and although cell culture systems have figured largely in the field of cancer chemotherapy, where the potential value of such systems for cytotoxicity and viability testing is now widely accepted, there is increasing pressure for a more comprehensive adoption of in vitro testing in safety evaluation. The impetus for change originates partly from financial considerations, since in vitro testing has considerable economic advantages over in vivo testing. There is also an increasing realization of the limitations of animal models in relation to human metabolism, as more and more metabolic differences between species come to be identified. Finally and as importantly, there is the moral pressure to reduce animal experimentation.
Fluorescence in situ hybridization(FISH)
Authors: Keith W Nicol
Published: November 2023

Classical karyotyping uses dyes that differentially stain the chromosomes. Thus, each chromosome is identified by its banding pattern. However, traditional banding techniques are limited to the identification of large and relatively simple changes. In situ hybridization is the most sensitive and specific means of identifying the location of genes on chromosomes. In addition, using chromosome- and gene-specific probes, numerical and structural aberrations can be analysed within individual cells. Thus, in situ hybridization has applications in the diagnosis of genetic disease and in the identification of gene deletions, translocations, and amplification (1-7).
Genetic modification
Authors: Hafezparast Majid
Published: November 2023

To explore the role of a gene, we need to analyse its regulation and expression during and after development, the function of its product, and interactions with other proteins. In most cases, it is necessary to be able to transfer the gene, or its manipulated form, into cells. However, since mammalian cells do not take up foreign DNA efficiently, the availability of effective methods for introducing genes into the cells is essential. Many gene transfer methods have been developed that are routinely used by scientists studying mammalian cells. Moreover, with the advancement of recombinant DNA technology, gene transfer techniques have become powerful tools in gene cloning and mapping, construction of transgenic animals, and gene therapy.
Spatial cues for cellular asymtnetry in polarized epithelia
Authors: Nelson W James , Yeaman Charles , Grindstaff Kent K
Published: November 2023

Transporting epithelia form permeability barriers between two compartments in the body, and vectorially transport ions and solutes between those compartments in order to maintain ionic homeostasis. In the kidney and gastrointestinal tract, the epithelium forms a closed monolayer of cells that surrounds a lumen, which topologically defines the ‘outside’ compartment of the organism; on the opposite side, the epithelium contacts the interstitium and blood supply, which topologically define the ‘inside’ compartment. To maintain the structural integrity of the epithelium, and to regulate the diffusion of ions and solutes from one compartment to the other through the paracellular space, the epithelium must form strong and specialized contacts between cells. Access to the paracellular space is regulated by the tight junction (zonula occludens ), which forms a gasket-like, semipermeable seal around each cell (Fig. 1). For vectorial ionic and solute transport across the epithelium, ion channels, exchangers, and transporters must be segregated into two structurally and functionally different domains of the plasma membrane, termed apical and basolateral, that face these different compartments. Typically, the apical membrane faces the luminal compartment, and the basolateral membrane faces the interstitium (Fig. 1).
Electrochemical assays: the oxygen electrode
Authors: Clark J B
Published: November 2023

Until about 45 years ago the methods of choice for the measurement of oxygen consumption or evolution in biological systems were Warburg manometry or chemical techniques. However, with the development of the membrane-covered, complete polarographic oxygen electrode by L. C. Clark (1) and its subsequent modification, commercially available, robust and sensitive oxygen electrodes became available for general use. Prior to this, the use of oxygen polarography had been limited to the experts who could construct their own electrodes and the specialized recording apparatus to go with them. The advent of a means of measuring oxygen instantaneously and continuously dramatically changed approaches to the study of biological respiratory systems and more recently to those enzyme systems which utilize or produce molecular oxygen. Oxygen electrode systems are now available commercially for a range of applications, from macrotechniques for assessing oxygen content in rivers, sewage, or industrial reactors under high pressure and temperature, to micro systems for measuring oxygen content of body fluids, or oxygen utilized by subcellular organelles, e.g. mitochondria.
Enzyme assays after gel electrophoresis
Authors: Rothe Gunter M
Published: November 2023

Enzyme visualization after electrophoresis involves five stages: (a) preparation of crude enzyme extracts, (b) electrophoresis, (c) enzyme visualization, (d) documentation and (e) interpretation of data. While different electrophoretic methods may lead to similar separation patterns, extraction of enzymes from a biological source affords special attention. Therefore, a brief summary concerning enzyme extraction is given in the following chapter. However, detailed methods to isolate subcellular particles from mammalian or plant tissues are not dealt with, and the reader is referred to reference (1), for example. Also, the preparation of gels for starch-, polyacrylamideand Cellogel-electrophoresis are not considered. Protocols to set up such gels are to be found in several text books (1–5).
Techniques for enzyme extraction
Authors: Price Nicholas C
Published: November 2023

This chapter discusses the techniques used to extract enzymes from cells. The principal aim of such procedures is to obtain the enzyme in as high a yield as possible consistent with the retention of maximal catalytic activity. The various procedures involved in subsequent purification of enzymes to homogeneity as a prelude to detailed structural and functional analysis are not discussed here; full details of the methods involved are given elsewhere (1, 2).
Electron microscopy in cell biology
Authors: Lucocq John
Published: November 2023

Electron microscopy is undergoing a strong revival as biologists strive to understand how molecular elements within cells are spatially and temporally integrated into functioning structures. The newer EM approaches have super-seded the descriptive EM studies of the 1950s and 1960s by providing more quantitative characterizations of structure and molecular composition that more closely correspond to the in situ ‘native’ state. This chapter illustrates examples of important techniques currently used in EM study of structures in cell biology. In particular I have included descriptions of methods for immuno electron microscopy and low temperature techniques of thawed cryosections, cryofixation, and embedding in resin. New approaches such as field emission scanning EM and SDS-cryofracture labelling along with quantitation of immuno labelling using stereology are also included. The protocols are presented as basic starter methods and for additional in-depth discussion see the more extensive treatises on EM methods (1, 2). Electron microscopy is a large subject to cover in a short chapter and I have out of necessity been very selective. The choice of protocols has been largely dictated by current trends in EM use, by personal preference, and the advice of colleagues. The chapter concentrates mainly on transmission EM (TEM) with a brief mention of scanning EM (SEM). Important but more specialized approaches such as electron tomography and cryoelectron microscopy of frozen hydrated specimens are not discussed here.
Cell lipids: from isolation to functional dynamics
Authors: Jan Veldman Robert , Pécheur Eve-Isabelle , van IJzendoorn Sven C D , Kok Jan Willem , Hoekstra Dick
Published: November 2023

The boundary of every cell as well as that of intracellular compartments, including intracellular transport vesicles, is provided by a membrane, the major components of which are proteins and lipids. Membrane lipids are amphipathic molecules that consist of a polar head group and usually two non-polar hydro carbon chains. The hydrocarbon chains are covalently attached to a lipid back bone structure such as glycerol (in the case of phospholipids and glycoglycero-lipids) or sphingosine (in the case of(glyco)sphingolipids) via ester, ether, amide, or C-C bonds. In biological membranes, a variety of lipid classes can be distinguished, the major lipids belonging to the class of the phospholipids. Although a minor class, the more complex (glyco)sphingolipids are biologically highly relevant (1).
Extracellular matrix protocols for the study of complex phenotypes
Authors: Roskelley Calvin D , Dedhar Shoukat
Published: November 2023

Cell adhesion assays are most often carried out on rigid planar substrata coated with ligands that facilitate cell binding and cell spreading. These ligands include: complex extracellular matrices (ECM; i.e. basement membrane), defined extra cellular matrix proteins (i.e. laminin), short peptide fragments of ECM proteins that bind integrins (i.e. contain RGD sequences) or antibodies that engage, and in some cases cluster, cell surface integrins. Under these conditions integrin dependent signalling events and changes to the cytoskeleton can be assessed, often concomitantly. These classical ‘stick-and-spread’ assays have proven particularly useful for identifying the mechanisms responsible for basic adhesion dependent phenotypes such as anchorage-dependent proliferation, membrane ruffling, or the initiation of cell motility (1-3). However, it is rarely technically possible to examine more complex differentiative phenotypes using these assays. For example, mammary epithelial cells spread on a rigid basement membrane substratum and begin to proliferate. In contrast, when these cells are placed on a malleable gel of the same matrix, morphogenesis and differentiation occur. Given the complexity inherent in the emergence of the latter phenotypes it is very difficult to determine the molecular mediators responsible. To overcome this problem we have developed specialized assays in which the entire process can be deconstructed such that individual morphogenic and differentiative events can be assayed and manipulated in isolation (4-6).
The cytoskeleton
Authors: Stradal Theresia B , Sechi Antonio S , Wehland Jürgen , Rottner Klemens
Published: November 2023

The cytoskeleton is a complex fibrillar system required for mechanical stability and phenomena as diverse and essential as cell division, anchorage, and cell locomotion. This system further provides highways for the directed transport of organelles or vesicles, driven by molecular motors. The tight spatial compartmentalization of cytoplasmic components by the cytoskeleton is a prerequisite for the high order of signalling that discriminates eukaryotic cells from pro-karyotes. The cytoskeleton is grossly divided into three distinct filamentous systems: actin filaments, microtubules, and the intermediate filament system. All three filament systems are formed by monomers from protein families that can each polymerize into filaments in a non-covalent manner. The dynamic turnover of these filaments via polymerization and depolymerization creates the high flexibility of the cell that is needed to respond to extracellular signals or to follow endogenous rhythms like the cell cycle.
Enzyme activities
Authors: Tipton Keith F
Published: November 2023

This account will concentrate on the behaviour of enzymes, the determination of their activities, and their responses to alterations in substrate concentration. Particular emphasis will be placed on studies of the effects of inhibitors, since these may be used as therapeutic drugs as well as tools to help elucidate their mechanisms of action and behaviour in the cell.
Regulation of the cell cycle
Authors: Gitig Diana M , Koff Andrew
Published: November 2023

The smooth running of the mitotic cell cycle depends on the activity of a family of serine-threonine kinases, the cyclin-dependent kinases (cdks). Agents that regulate the cell cycle ultimately impinge upon the activity of these proteins, either directly (by promoting their degradation or inducing the expression of an inhibitor) or indirectly (by arresting the cell cycle at a point before a particular cdk is active). The cdks are under very strict regulation, as might be expected for enzymes responsible for such a fundamental cellular process. These kinases are activated and inactivated in a sequential manner over the course of the mitotic cycle and their phosphorylation of various substrates allows cells to progress through the mitotic cycle (1). To date, the most extensively characterized of these substrates is the retinoblastoma protein, pRb, whose phosphorylation first by cyclin D/cdk4,6 in early G1 phase and then by cyclin E/cdk2 later in G1 precedes the commencement of DNA synthesis (2, 3) (Figure 1). Other substrates that have been identified are listed in Table 1.
Targeting of nuclear-encoded proteins into and across the thylakoid membrane: isolation and analysis of intact chloroplasts and thylakoids from plants
Authors: Robinson Colin , Mant Alexandra
Published: November 2023

The biogenesis of the chloroplast is a complex process, involving a great deal of protein traffic. The majority of chloroplast proteins (around 80%) are imported from the cytosol, because they are encoded by nuclear genes (reviewed in refs 1 and 2). These proteins must be both specifically targeted into the chloroplast and accurately sorted to their final sites of function within the organelle. With the exception of a subset of outer envelope proteins, which have no cleavable targeting peptides (3), all imported proteins analysed to date are initially synthesized as larger precursors containing amino terminal presequences. The presequences have been shown to contain information which specifies targeting into the chloroplast, and in some cases, localization within the organelle.
Transport into and out of the nucleus
Authors: Fanara Patrizia , Berger Adam C , Green Deanna M , Hagan Henry , Harreman Michelle T , Marfatia Kavita A , Quimby B Booth , Powers Maureen A , Corbett Anita H
Published: November 2023

How macromolecules are transported between the cell nucleus and the cytoplasm is a general question of great importance to cell biology. Diverse macromolecules, including proteins, mRNAs, tRNAs, and ribosomal subunits, for example, are specifically transported between the cytoplasm and the nucleus and many different techniques have been developed to study these processes. This chapter focuses on those techniques that are specific to the transport process rather than the more general methods that have been used to understand the molecular mechanisms that underlie nucleocytoplasmic trafficking. We present:
Receptor biology
Authors: Dosil Mercedes , Mentesana Pamela , Konopka James B
Published: November 2023

Cell surface receptors for hormones, neurotransmitters, and other ligands have long been recognized to play a key role in cellular regulation. Thus, it is with great interest that genome sequencing projects are revealing the existence of new receptors that have not been studied previously. Therefore, we will describe protocols in this chapter that will be helpful for the fundamental characterization of new receptors. The methods will emphasize G protein-coupled receptors since this receptor family is expected to grow by hundreds of new members. However, the techniques will be generally applicable to a wide range of receptors and will also be useful for investigators wishing to add new approaches to previously characterized receptors.
Measurement of signal transduction machinery
Authors: Hodgkin Matthew , Wakelam Michael , Armour Carolyn
Published: November 2023

We describe some of the common methods used to assay signal transduction events in cells. The methods presented quantify both signal transduction enzyme activity and the production of second messenger molecules. In view of the wide variety and complexity of signal transduction pathways that are currently understood, some pathways and enzymes may not be covered in great detail or indeed at all. Therefore, where possible, we have attempted to illustrate how the presented protocols can be adapted to other systems.
Bio-, chemi-, and electrochemiluminescence for fiber-optic biosensors
Authors: Blum Loïc J , Coulet Pierre R
Published: November 2023

For many years, our laboratory has been involved in the development of biosensors first based on electrochemical transduction (1). One of the key components in the design of a biosensor is the sensing layer associated with the transducer and involving a biological element which confers molecular recognition properties to the analytical system. Among the several types of transducing modes which have been proposed (2), optical systems, namely those including fiber-optics, appear particularly promising (3). In the present chapter, we will focus on optical biosensors involving bio-, chemi-, and electro-chemiluminescence (ECL).
Determination of metal ions by luorescence anisotropy: A practical biosensing approach
Authors: Thompson Richard , Maliwal Badri , Zeng Hui Hui , Cramer Michele Loetz
Published: November 2023

Fluorescence anisotropy is a well-known experimental technique which has hitherto been little used for chemical analysis or sensing, the major exception to this being fluorescence polarization immunoassay (1, 2). Yet it has important advantages among photoluminescence techniques (fluorescence, phosphores-cence) that argue for its use in this regard. Note that polarization and anisotropy measure the same phenomenon, the polarization of fluorescence emission; the two measurements are normalized differently, but are algebraically interconvertible with ease.
The cell culture laboratory
Authors: Wigley Caroline B
Published: November 2023

The history of animal cell culture dates from the turn of the 20th century (Table 1), with Ross Harrison’s demonstration that explants of neural tissue from frog embryos, sealed into a chamber containing frog lymph, generated fibres which extended into the lymph clot. He thus showed, for the first time, that nerve axons are formed by the outgrowth of processes from the neuronal cell body. These processes lengthen as a result of the amoeboid movement of their distal tip, now called the growth cone. This observation finally resolved one of the long-standing controversies in neurobiology, showing clearly that each nerve fibre is the outgrowth of a single nerve cell.
Microscopy of living cells
Authors: Dobbie Ian , Zicha Daniel
Published: November 2023

The ability to observe living cells has always been dependent on the availability of appropriate technology. Despite the fact that the principle of how to make powerful compound microscopes was known as far back as the 16th century, the quality of actual devices from this period was very poor due to optical aberrations. Indeed, the first observation of individual bacteria and protozoa – by Anton von Leeuwenhoek – was not reported until 1674 and even this was achieved using a single lens microscope.
Good Laboratory Practice in the cell culture laboratory
Authors: Clitherow Barbara
Published: November 2023

Suppose that you are about to subculture a cell line: an activity common to all cell culture scientists, so familiar as to be completed almost without thinking. You clean your laminar flow cabinet, warm your bottle of fresh medium, locate your culture, and ensure that all your other equipment and materials are to hand. Then you disinfect your gloves, transfer one-fifth of the culture volume into a new flask, aseptically add four volumes of fresh medium, similarly add any other separate but essential medium components, label the new culture, and return this to the incubator. Finally you clean your cabinet, discard your contaminated materials, and return the medium and components to the fridge or freezer. Comfortable?
Cloning and expression of cytokine genes
Authors: Declercq W , Logghe M , Fiers W , Contreras R
Published: November 2023

Cytokines are proteins secreted into the extracellular fluid or are membrane exposed by a cell. There they exert their effects on the same cells (autocrine activity) or on neighbouring cells (paracrine activity) by interacting with specific receptors. Most cytokine proteins have, on a molar basis, very high biological activities (for instance, colony-stimulating factors are active at 10-11 to 10-13 M concentrations) which can usually be assayed in rapid, sensitive, and fairly specific in vitro test systems. These fast and simple detection systems have played a crucial role in cloning the corresponding cytokine genes by providing tools for either purification of the protein, the mRNA, or the direct detection of biologically active clones in cDNA expression libraries. Other cytokines, such as the strongly inducible chemokine family act in much higher concentrations (10-3 to 10-9 M) and have often been cloned by different techniques such as induction-specific differential hybridization or subtracted libraries. The mRNA of these molecules can represent 1% of the total poly(A)+ RNA after stimulation. Recently the phenomenon of in silica cloning has been introduced, due to the fact that nowadays computer technology allows searches of protein and DNA databases to be made for sequence similarities (1, 2). In this way several related cytokines and cytokine receptors have been identified in recent years.
Measurement of prollferatlve, cytostatlc, and cytolytlc activity of cytoklnes
Authors: Burke Frances , Balkwill Frances R
Published: November 2023

Most cytokines have proliferative, cytostatic and/or cytolytic activity depending on the context in which they are acting. IFN-α, -β. and -γ, TNF-α and -β. IL-1, and IL-4, have all been shown to have proliferative, cytostatic, and cytolytic activity depending on the conditions (1). Synergistic interactions between cytokines can often produce powerful antiproliferative and cytotoxic effects (2), as can a combination of cytokines and inhibitors of transcription. For some cytokines, such as IFN-γ, the cytokine has to be continually present in the tissue culture medium for two or more days to have an irreversible effect on cell growth (3). Other cytokines such as Fas ligand and TRAIL can induce apoptosis within hours. Some of the methods used to assess the above parameters will be described in this chapter. Other assays for measuring the action of cytokines on cell growth are described in Chapter 13, this volume.
Human B-cell responses to cytoklnes
Authors: Gallard Robin E , Katowicz Karolena T
Published: November 2023

B-lyrnphocyte responses are determined by complex interactions between antigen, cell-surface ligands, and soluble growth and differentiation factors (cytokines) binding to specific receptors on the cell surface. To date, at least 17 out of more than 40 recombinant cytokines are known to stimulate B-cell activation, proliferation, and/or differentiation (Table 1 ). Many of these cytokines are derived from T cells and are involved in T-cell/B-cell collaboration, but some are made by B cells, monocytes, and a variety of non-lymphoid cells such as endothelial cells and :fibroblasts.
The generation and quantltatlon of cell-mediated cytotoxlclty
Authors: Grimm Elizabeth
Published: November 2023

Cell-mediated cytoxicity (CMC) is defined as the process by which a cell of the immune system directly kills another cell. CMC is composed of a specific series of events occurring between an effector cell (lymphocyte or monocyte) and a susceptible target cell, which ultimately results in the delivery of a ‘lethal hit’ by the effector to the target cell. An obligate event in CMC, which distinguishes it from other modes of immunologically mediated target-cell killing (e.g. com­plement-mediated cytolysis) is intimate cell membrane contact between the effector and the target, referred to as conjugate formation. This brief chapter will be restricted solely to the description of lymphocyte-mediated CMC systems. Many excellent reviews cover the complicated processes which encompass effector cell-mediated recognition, conjugate formation, triggering, and delivery of the lethal hit ultimately resulting in the death of the specific target (1, 2).
Assays for leucocyte migration
Authors: Van Damme Jo , Lenaerts Jean-Pierre , Struyf Sofie
Published: November 2023

In view of the recent developments in cytokine research, there has been a revived interest in chemotaxis assays. Indeed, within a short time a number of novel chemotactic cytokines (chemokines) have been identified. These low molecular weight proteins are different from other cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF), previously reported to be chemotactic for leukocytes. Chemokines belong to a large family of small proteins that exert their chemotactic activity different leucocytic cell types (1-4). Like several other chemotactic substances, such as formylmethionyl peptides (e.g. fMLP), the anaphylatoxin C5a, and leukotriene B4 (LTB4), chemokines bind to distinct serpentine-like receptors. Some chemokine receptors are essential for HN-entry into monocytes or lymphocytes. Several chemokines, such as IL-8, have now been studied in detail with respect to leucocyte migration in vitro and in vivo. More recent members of the chemokine family have so far only been biochemically characterized and their role in cell migration has not yet been studied in great detail.
Assays for macrophage activation by cytokines
Authors: Doyle Anthony , Stein Michael , Keshav Satish , Gordon Siamon
Published: November 2023

The circulating monocyte may undergo a wide spectrum of functional changes during its further differentiation. In the absence of inflammatory stimuli monocytes enter the tissues constitutively to become resident macrophages (1). These cells are relatively quiescent with respect to secretory or microbicidal activity and may have as yet uncharacterized trophic functions. In contrast, inflammatory recruitment causes monocytes to rapidly induce their secretory activity. These cells produce a large variety of enzymes, growth factors, and cytokines when appropriately triggered. Lymphokines produced at the site of inflammation regulate the fate of elicited macrophages determining whether they are further stimulated to a heightened state of activation with maximal host-defence func­tion, e.g. by IFN-γ, or whether their secretory and defence activity is held in check (e.g. by IL-4 and IL-10).
Flow cytometrlc detection of Intracellular cytoklnes
Authors: Pennycook Alasdair M J , Pala Pietro , Matthews Steve
Published: November 2023

Recent technological advances in the availability and capability of flow cytometric analysis, coupled with advances in knowledge over the last 10 years, have made intracellular staining for cytokine expression an increasingly ubiquitous technique. It began with the pioneering work of the Anderssons at Stockholm University in the 1980s, who developed a system of cell fixation and permeabilization that left target antigens intact (1). Modifications since then have optim­ized the technique to allow the detection and further characterization of very few cytokine secreting cells within a heterogeneous population (2, 3).
Development of antibodies to cytoklnes
Authors: Poole S
Published: November 2023

Antibodies to cytokines can greatly facilitate research on many aspects of these mediators, including: the development of specific and sensitive immunoassays (see Chapter 12); the detection of cytokines in biological fluids, tissues, and cells using immunochemical and immunocytochemical techniques; immmunoneutralization of biological activities; and delineation of external regions (epitopes) of the cytokines.
Cadherin adhesion regulation in keratinocytes
Authors: Braga Vania
Published: November 2023

This chapter will describe the keratinocyte system as a model to study the regulation of cadherin-mediated cell–cell adhesion. However, some of the principles discussed here can be applied to the analyses of different adhesive systems in other cell types. The chapter describes the potentials and pitfalls of the keratinocyte culture system and the various techniques used to investigate the regulation of cell–cell adhesiveness. Among these, a detailed description is given of microinjection in epithelial cells as a technique to investigate regulation of cadherin function by members of the small GTPase family. The analysis of the microinjection results by immunostaining and confocal microscopy is also presented. In addition, this chapter illustrates biochemical methods to determine the composition of cadherin complexes and their phosphorylation status (orthophosphate labelling and phosphopeptide mapping). Finally, two functional assays are discussed: the use of inhibitory antibodies to block cadherin-dependent adhesion and a technique to cluster cadherin receptors artificially.
Analysis of intercellular adhesion molecules in endothelial cells
Authors: Bazzoni Gianfranco , Lampugnani Maria G , Martinez-Estrada Ofelia M
Published: November 2023

Intercellular adhesion between endothelial cells (ECs) is mediated by members of the cadherin, immunoglobulin, integrin, and proteoglycan families of cell adhesion molecules. Some of these molecules are concentrated at intercellular structures known as adherens junctions and tight junctions. Other molecules, such as platelet–endothelial cell adhesion molecule-1 (PECAM-1), are localized at cell–cell contacts, but outside of the junctional complexes. Although the morphological and molecular organization of these complexes are similar in ECs and in other cell types, some components are more specific for the endothelium. For instance, vascular endothelial-cadherin (VE-cadherin) is expressed exclusively in ECs at the adherens junctions, while claudin-5 and the alpha minus isoform of zonula occludens-1 (ZO-1), two tight junction components, are expressed preferentially in the endothelium (1, 2). By interacting (either homophilically or heterophilically) with their receptors, junctional adhesive molecules provide physical links that hold ECs together and regulate the paracellular passage of ions, solutes, and transmigrating leukocytes. Also, by interacting with intracellular partners, the adhesive receptors may strengthen their own linkage with the cytoskeleton. Finally, besides providing attachment for neighbouring cells and the cytoskeleton, junctional molecules may also impinge upon cell signalling and trigger responses that are translated into changes in cell morphology and eventually in the organization of three-dimensional networks of patent tubes (3).
Studying cell interactions during development of the nervous system in Drosophila
Authors: Shepherd David , Block Louise , Folwell James , Williams Darren
Published: November 2023

The fruit fly Drosophila melanogaster is one of the key model organisms for studying both the genetic and molecular mechanisms of cell interactions. The versatility of the fly as a model experimental organism means that by working with Drosophila it is possible to combine molecular, genetic, and micromanipulation techniques to address fundamental questions in embryology, cell biology, and neurobiology. This versatility has attracted many research workers to Drosophila and has generated a vast literature. The volume of work done and the high degree of evolutionary conservation have meant that Drosophila has had a huge impact on many areas of biological research. The completed sequencing of the Drosophila genome further adds to its power as a research organism. The combination of the genome database and the available experimental tools mean that the humble fruit fly will play a key role in the post-genomic era in establishing the functions of the gene sequences held in the databases. This will ensure that Drosophila will remain one of the key model organisms. Within the scope of this chapter it would be impossible to provide a comprehensive review of the techniques and strategies used to study Drosophila. This chapter, therefore, focuses specifically on techniques used to study cell interactions during nervous system development and function. Many of these techniques can be applied to other areas of Drosophila research. It is our aim that the chapter will be useful to newcomers and experienced research workers alike. The chapter is divided into two sections. The first describes techniques and strategies for visualizing cells in the nervous system. The second details strategies for experimentally manipulating cells. Each describes the methodologies used in our laboratory and gives full details of the techniques.
Reflective Theories
Authors: Walsh Joseph
Published: November 2023

In this chapter, and the four chapters that follow, we consider how endings can be addressed from the perspectives of a variety of prominent clinical practice theories. All these theoretical perspectives have much in common with regard to our topic, but they also contain significant differences. It is not my intention to endorse one or several of these theories as inherently preferable for clinical practice but rather to provide an assortment so that you can make comparisons and get a sense of the breadth of current thinking about appropriate ways to end intervention. Those of you who utilize a theoretical framework described in the next five chapters may recognize some of the ending principles from that perspective, but I hope that you may get ideas for using ending procedures associated with other theories as well.
Aida Luz Maisonet Giachello: Improving Health in the Latino Community
Authors: Sherraden Margaret S
Published: November 2023

On a frigid and windy Chicago morning, Aida Luz Maisonet Giachello pulled over in her old black Mercedes to pick me up for our breakfast interview. After driving back and forth on North Avenue several times looking for Nellie’s, a popular Puerto Rican restaurant, Aida turned to me and smiled. ‘‘I’m a little absent-minded,’’ she admitted. Her mind was not on details. Like most other mornings, her mind was on work.
Professor Armaity Desai: Interpreting Social Work in a Developing Country
Authors: Banerjee Mahasweta
Published: November 2023

I am honored to be able to write about a person I loved and admired in the initial years of my professional life. Dr. Armaity Desai has been a role model, mentor, and someone I have known since 1984. At that time, I was catapulted, through a set of unforeseen circumstances, into the positions of acting prin- cipal of the Calcutta School of Social Work and deputy director of the Jaya Prakash Institute of Social Change. I had yet to go back to school for my doctorate, and it was Dr. Desai who helped me to see the necessity of this move.
Building Research into Practice
Authors: Fischer Joel
Published: November 2023

The purpose of this chapter is to focus on the process of technique building and validation in practice with individuals. It is hoped that this will encourage explicitness in the kind of outcome-oriented approach that can serve as the foundation for clinical experimentation and innovation. A general term for such practice-oriented research has been proposed by Chassan (1967) who distinguishes between the “extensive” and “intensive” models of research. The extensive design refers primarily to the testing of hypotheses with respect to groups of subjects, e.g., controlled group experimental designs (it also has been called “nomothetic research,” i.e., research concerned with statements regarding general classes of events; Marx, 1963). Such a design might profitably, in fact, should, be used when there is substantial evidence from practice that its hypotheses can be validated. This is not to negate the importance of hypothesis-testing with the potential for discarding untenable positions such as already has occurred in the field of casework.
Research and Knowledge-building: The Case for Research Reviews
Authors: Fischer Joel
Published: November 2023

It is particularly important to point out right at the outset of this chapter that knowledge development, as a field, includes more than research (see, e.g., the Sage Publication journal, Knowledge Creation, Diffusion, Utilization [first issue, September 1979]). Perhaps the seminal work in social work in this arena has been Ed Thomas’ attempts to conceptualize the knowledge development process in a series of articles (Thomas, 1978a, 1978b, 1978c). Thomas has proposed a systematic way of looking at a whole range of sources of basic information for social work. These sources include contributions from basic, applied, indigenous research; technology from science, allied technologies, social innovation, and practice; values and ideology; legal policy; and practice experience. Thomas translates each of these into five generation or transfer processes (the processes by which information from the basic source is converted for use by social workers) and spells out operational steps for the phases of the knowledge development process (the phases are analysis, development, evaluation, diffusion, and adaptation).
Increasing the Relevance of Research Education: The University of Hawaii Research Program
Authors: Fischer Joel
Published: November 2023

There is substantial evidence that research training of graduate social work students has been less than successful. Research courses often are viewed by the students as the least helpful courses in the curriculum (1). After graduation, few practitioners either conduct research (2) or read, consult, utilize, or share research findings, especially when faced with difficult practice situations (3). And even when they do read research, it appears that social workers’ judgments of the quality of the research and its implications for practice are strongly influenced by whether the findings support their own biases (4).
Uniformity Myths in Eclectic and Integrative Psychotherapy
Authors: Fischer Joel
Published: November 2023

Almost thirty years ago, Kiesler (1966) described a number of “uniformity assumption myths” that he believed were impeding progress in psychotherapy research. His argument was that nearly all domains of variables in psychotherapy and psychotherapy research are considerably more heterogeneous than the then-current descriptive labels implied. Thus, lack of progress in psychotherapy research was based partly on assuming that those variables (e.g., clients, problems, psychotherapists) were homogeneous and thereby asking the wrong questions. While it is not clear that Kiesler’s concerns have been adequately addressed, even today, it does appear as though a new class of uniformity myths may be affecting the newest developments in the field of psychotherapy. These myths are assumptions underlying some of the key foundations of the fields of integrative and eclectic psychotherapy. In sum, these myths are: (1) that all psychotherapies have equivalent outcomes; (2) that all theories (or systems) of psychotherapy are equally sound; and (3) that cultural homogeneity exists for all eclectic and integrative clients and psychotherapists, encouraging the uncritical dissemination of Western approaches to psychotherapists from other traditions and cultures.
Mobilizing Communities for Social Change: Integrating Mindfulness and Passionate Politics
Authors: Todd Sarah
Published: November 2023

Laura Pulido (2003) suggests that political action is not just about changing the external world but is also an ‘‘exercise in creating and changing ourselves’’ (p. 51). For Pulido, the interior includes ‘‘such things as our emotions, psychological development, souls and passions, as well as our minds’’ (47). In this chapter I explore the ways that mindfulness practices can be useful in mobilizing community members and sustaining community change. In particular, I will explore what these practices offer for communities that are negotiating passionate emotions and are attempting to engage others in their activities, and that want to enhance people’s ability to listen to each other and want to create sustainable change. Through this integration, I will articulate the possibilities for progressive social change that can be enhanced and sustained by an understanding of how our interior lives shape our external worlds and the possibilities that mindfulness practices have for increasing the transformative potential of this interaction.
Complete Loss of RelA and SpoT Homologs in Arabidopsis Reveals the Importance of the Plastidial Stringent Response in the Interplay between Chloroplast Metabolism and Plant Defense Response
Authors: Inazu Masataka , Nemoto Takanari , Omata Yuto , Suzuki Sae , Ono Sumire , Kanno Yuri , Seo Mitsunori , Oikawa Akira , Masuda Shinji
Published: November 2023

The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases and characterized its phenotype. The mutant showed over 20-fold less ppGpp levels than the wild type under normal growth conditions and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense.
Effects of glucocorticoid use on survival of advanced non-small-cell lung cancer patients treated with immune checkpoint inhibitors
Authors: Li Nijiao , Zheng Xuliang , Gan Jinyan , Zhuo Ting , Li Xiaohong , Yang Chuyi , Wu Yanbin , Qin Shouming
Published: November 2023

Background:

Lung cancer is the second most common cancer worldwide, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. Patients with NSCLC have achieved great survival benefits from immunotherapies targeting immune checkpoints. Glucocorticoids (GCs) are frequently used for palliation of cancer-associated symptoms, as supportive care for non-cancer-associated symptoms, and for management of immune-related adverse events (irAEs). The aim of this study was to clarify the safety and prognostic significance of glucocorticoid use in advanced patients with NSCLC treated with immune checkpoint inhibitors (ICIs).

Methods:

The study searched publications from PubMed, Embase, Cochrane Library, Web of Science, China Biology Medicine disc, Chinese National Knowledge Infrastructure, Wanfang Data, and Chinese Science and Technology Journal Database up to March 1st, 2022, and conducted a meta-analysis to assess the effects of glucocorticoid use on overall survival (OS) and progression-free survival (PFS) in NSCLC patients treated with ICIs through the available data. The study calculated the pooled hazard ratios (HRs) and 95% confidence intervals (CIs).

Results:

This study included data from 25 literatures that were mainly retrospective, with 8713 patients included. Patients taking GCs had a higher risk for tumor progression and death compared with those not taking GCs (PFS: HR = 1.57, 95% CI: 1.33–1.86, P <0.001; OS: HR = 1.63, 95% CI: 1.41–1.88, P <0.001). GCs used for cancer-associated symptoms caused an obviously negative effect on both PFS and OS (PFS: HR = 1.74, 95% CI: 1.32–2.29, P <0.001; OS: HR = 1.76, 95% CI: 1.52–2.04, P <0.001). However, GCs used for irAEs management did not negatively affect prognosis (PFS: HR = 0.68, 95% CI: 0.46–1.00, P = 0.050; OS: HR = 0.53, 95% CI: 0.34–0.83, P = 0.005), and GCs used for non-cancer-associated indications had no effect on prognosis (PFS: HR = 0.92, 95%CI: 0.63–1.32, P = 0.640; OS: HR = 0.91, 95% CI: 0.59–1.41, P = 0.680).

Conclusions:

In advanced NSCLC patients treated with ICIs, the use of GCs for palliation of cancer-associated symptoms may result in a worse PFS and OS, indicating that they increase the risk of tumor progression and death. But, in NSCLC patients treated with ICIs, the use of GCs for the management of irAEs may be safe, and the use of GCs for the treatment of non-cancer-associated symptoms may not affect the ICIs’ survival benefits. Therefore, it is necessary to be careful and evaluate indications rationally before administering GCs in individualized clinical management.
FABP7 Progenitors are a Targetable Metabolic Root in theBRCA1Breast
Authors: McCloskey Curtis W. , Zhang Bowen , Waas Matthew , Abazari Golnaz , Vyas Foram , Aliar Kazeera , Tharmapalan Pirashaanthy , Kuttanamkuzhi Abhijith , Narala Swami , Cruikshank Jennifer , Hofer Stefan O.P. , Jackson Hartland W. , Kislinger Thomas , Berman Hal K. , Khokha Rama
Published: November 2023

It has been nearly 3 decades since the discovery of theBRCA1/2genes and their link to breast cancer risk, with prophylactic mastectomy remaining the primary management option for these high-risk mutation carriers. The current paucity of interception strategies is due to undefined, targetable cancer precursor populations in the high-risk breast. Despite known cellular alterations in theBRCA1breast, epithelial populations at the root of unwarranted cell state transitions remain unresolved. Here, we identify a root progenitor population that is dysregulated inBRCA1carriers stemming from the metabolic role of BRCA1. This fatty-acid binding protein 7 (FABP7) expressing luminal progenitor population is spatially confined to the mammary ducts, has enhanced clonogenic capacity, and is the predicted origin of mixed basal-luminal differentiation in theBRCA1but notBRCA2breast. We show global H3K27 acetylation is reduced within ductal FABP7 cells inBRCA1carriersin situ, linking to a non-canonical metabolic role of BRCA1 in regulating acetyl-CoA pools andde novofatty acid synthesis. We demonstrate FABP7 progenitor capacity is preferentially ablated inBRCA1carriers through inhibition of fatty acid metabolism using an FDA-approved fatty acid synthase (FASN) inhibitor. This study lays the foundation for metabolic control of breast progenitor dynamics to mitigate breast cancer risk in theBRCA1breast.