bluuebunny/crossref_metadata_2025_split huggingface dataset:
Networking for the biologist
Authors: Harper R A 
Published: November 2023

Abstract
Every research worker would like to have the tools on hand to make his job quicker and more efficient, and with the advent of the World Wide Web many of the tasks associated with molecular biology have become freely available online. In the past when a scientist wanted to know something about a particular subject then the first option was to talk to colleagues in the laboratory and ask for their advice. If that was not sufficient then it was off to the library to scan abstracts or the latest journals for the relevant information.
Three-dimensional culture
Authors: Kunz-Schughart L A , Mueller-Kliesert W 
Published: November 2023

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.
Regulation of the cell cycle
Authors: Gitig Diana M , Koff Andrew 
Published: November 2023

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.