bluuebunny/crossref_metadata_2025 huggingface dataset:
Directed mutagenesis in embryonic stem cells
Authors: Plagge Antonius , Kelsey Gavin , Allen Nicholas D
Published: November 2023

Since the first description of homologous recombination in mouse embryonic stem (ES) cells (1,2), gene targeting has become a routine technology in mouse molecular and developmental genetics. At its simplest it allows the introduction of disabling mutations into a gene of interest in ES cells and passage of the disrupted allele through the mouse germ line in order to assess gene function from the phenotypic consequences. Mutations in some hundreds of genes have now been made by this route (see the transgenic/targeted mutation database and Mouse Knockout and Mutation Database (MKMD);. Over the past few years, approaches have become more sophisticated and the potential for more refined sequence manipulation is being realized (see Table J). Amongst these refinements are, for example, conditional mutations which can be induced at defined times in development, or within specific lineages, to assess aspects of function of a gene for which a constitutively null mutation is lethal. In addition, targeted alleles can be ‘cleaned up’ by removing selectable markers to avoid potentially confounding effects arising from the presence of heterologous sequences. Finally, it is feasible to introduce subtle mutations, even single nucleotide exchanges, into genes. Many of these extra possibilities have come about through the exploitation of site-specific recombinases (reviewed in ref. 3). The potential of such technology should be carefully considered before embarking on a simple targeting experiment. Although generation of a more elaborate construct may be more time-consuming, in comparison with the time involved in targeting ES cells and obtaining germline transmission it is insignificant and the additional time investment in designing a more versatile construct might have significant longer-term benefits. In this chapter, we provide an overview of targeting strategies and the factors to be taken into account in the design of a targeting experiment, from initial construct preparation to the generation and analysis of mice carrying the desired mutation.
Optimization
Authors: Ezan Eric , Grassi Jacques
Published: November 2023

The ultimate goal of immunoassay optimization is to satisfy previously determined criteria of accuracy and precision along with the achievement of adequate sensitivity and practicability. All of these four basic characteristics must be kept in mind at all three stages of assay development:

Choice of assay format and reagents,

Setting assay conditions (optimization),

Validation, including application to biological samples.
lmmunoaffinity chromatography of macromolecules
Authors: Hobbs Steve
Published: November 2023

The epitope specificity of an antibody combining site allows the antibody to bind to its corresponding ligand in the presence of other complex molecules such as whole cell lysates or animal sera. If the antibody is immobilized to a solid surface the complex can easily be separated from the mixture, and the reversible nature of the antibody-antigen interaction allows the ligand to be recovered by dissociation once the unbound molecules have been washed away. This process is called immunoaffinity chromatography, a technique now in widespread use (1, 2). It can be used either to purify an antigen, or to remove a specific antigen from a solution in which case recovery of biologically active molecules is not an issue (3). The advent of monoclonal antibody technology (4) has enabled the preparation of homogeneous populations of antibody molecules with identical structure, epitope specificity, and affinity. These features are ideally suited to solid phase antigen isolation because the supply of antibody is reproducible, and elution of antigen from the immobilized antibody will take place under a narrow range of conditions.
Detection of chemically modified DNA in lymphocytes of patients undergoing chemotherapy
Authors: Tilby Michael J
Published: November 2023

Antibodies that recognize DNA occur spontaneously in people and animals exhibiting certain types of ‘autoimmune’ diseases. In rodents not exhibiting autoimmune symptoms, DNA is a weak immunogen, possibly because B cells exhibiting specificity for DNA are deleted (1). However, it was discovered that alteration of the normal structure of DNA by irradiation with lN light greatly increased its immunogenicity and the antibodies, that were then produced, specifically recognized the lN-induced thymine dimers (2). In the 1950s it was also reported that the immunogenicity of DNA could be enhanced by forming an electrostatic complex with methylated bovine serum albumin (2). The mechanisms by which antibodies are made against DNA are not understood. They show the conventional features of oligoclonality, somatic mutation, gene re arrangements, and class switching, but there is no known way that DNA molecules can be processed and presented to T cells in the context of HIA class II molecules (1, 3). Since the 1960s a large number of antisera and monoclonal antibodies have been produced which recognize various DNA structures or modifications. These can be divided pragmatically into three broad groups:
Monoclonal antibody therapy in rheumatoid arthritis
Authors: Choy Ernest H S , Kingsley, Gabrielle H , Panayi Gabriel S
Published: November 2023

Rheumatoid arthritis (RA) is the commonest inflammatory and destructive arthropathy in the world with a prevalence of 1000 per 100000. It is characterized by a chronic symmetrical polyarthritis that leads to joint damage in most patients. The commonest affected joints are the small joints of the hands and feet but it can involve any synovial joint. Rheumatoid factor is present in over 70% of the patients. The long-term prognosis of RA is poor as most patients develop significant disability despite treatment (1). Hence it is a major socio economical burden. In the UK, the cost of RA was estimated at £1.3 billion/year in 1992 (2).
Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates
Authors: Van Der Geer Peter , Luo Kunxin , Sefton Bartholomew M , Hunter Tony
Published: November 2023

Phosphopeptide mapping enables the investigator to study the phosphorylation of a protein of interest in detail. The technique can be used to estimate the number of sites of phosphorylation in a protein, to determine the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for the phosphorylation of specific sites. Additionally, comparative phosphopeptide mapping is an invaluable tool for the determination of the identity, or lack of identity, of phosphoproteins obtainable only in trace amounts.
Assay of protein kinases and phosphatases using specific peptide substrates
Authors: Ruzzene Maria , Pinna Lorenzo A
Published: November 2023

The successful and widespread use of peptide substrates for the assay of protein kinases is grounded on the observation that the great majority of these enzymes are able to recognize phosphoacceptor sites defined by local structural features within their target proteins. Although this local sequence specificity is not the only tool ensuring the selectivity of protein kinases (indeed, in some cases, notably protein-tyrosine kinases, it may not even be a major one) many members of the protein kinase family phosphorylate short peptides reproducing their natural phosphoacceptor sites. The advantage of synthetic peptides, compared with protein substrates, for kinase assays and specificity studies include: they can be readily obtained in large amounts.
Interaction cloning of protein kinase partners
Authors: Stone Julie M , Walker John C
Published: November 2023

Historically, protein kinase partner proteins have been isolated by biochemical means, which requires purification of the interacting protein for antibody production or microsequencing before a cDNA clone can be obtained. However, two molecular biological approaches, the yeast two-hybrid system (Chapter 14) and interaction (or expression) cloning (1), directly yield a cDNA clone encoding the interacting protein. In this chapter we describe some of the recent advances in interaction cloning as a simple, rapid, and powerful alternative to biochemical approaches to identify protein kinase partners. The recombinant protein kinase of interest is enzymatically labelled with 32P by virtue of a five amino recognition site for cAMP-dependent protein kinase (PKA), to facilitate the identification of cDNA clones encoding interacting partners. Briefly, expression of polypeptides encoded by cDNA inserts from a bacteriophage X. expression library is induced, and the polypeptides adsorbed on to nitrocellulose filters. The library is then screened with the radiolabelled protein probe to identify cDNA clones expressing interacting protein kinase partners (see Figure 1).
Identifying protein kinase substrates by expression screening with solid-phase phosphorylation
Authors: Fukunaga Rikiro , Hunter Tony
Published: November 2023

The biological activities of protein kinases are evoked through phosphorylation of their substrate proteins. Phosphorylation of target proteins causes changes in their structure, stability, enzymatic activity, ability to interact with other molecules, or subcellular localization, leading to regulation of a wide variety of cellular processes. Indeed, the identification of physiological targets has been a high priority ever since the first protein kinase was purified. However, the conventional approach of purifying substrate proteins by biochemical techniques is laborious and time consuming, and is especially difficult in the case of scarce proteins. Among the approaches developed to identify protein kinase substrates in a systematic manner are various techniques for determining consensus phosphorylation site sequences using oriented peptide libraries (1) (see Chapter 16), interaction screening for protein substrates by the far-Western method (2), or the yeast two-hybrid system (3, 4) (see Chapter 14).
Analysis of protein kinase interactions using the two-hybrid method
Authors: Jiang Rong
Published: November 2023

The yeast two-hybrid system is a genetic approach that detects proteinprotein interactions (1, 2). This method relies on the functional reconstitution of transcriptional activators, such as Gal4, in the nucleus of yeast cells. The interaction of two-hybrid proteins, one bearing a DNA-binding domain and the other bearing a transactivation domain, results in transcriptional activation of a reporter gene. Certain features of the two-hybrid system make it particularly suitable to analyse protein kinase interactions. First, it is highly sensitive and can detect transient, as well as stable, interactions. This probably results from the fact that the two-hybrid system uses gene expression as a readout to amplify the signal generated by protein-protein interactions. The two-hybrid method has uncovered interactions not easily revealed with other methods. For example, the interaction between the Raf kinase and Ras is readily detectable by the two-hybrid method, but not by co-immunoprecipitation (3, 4). Secondly, as the two-hybrid system analyses interactions within the environment of a eukaryotic cell, many types of post-translational modifications critical for interaction are preserved. While yeast is the popular host for this method, a mammalian version of the two-hybrid system has also been developed (5, 6). Thirdly, the two-hybrid method detects bridged as well as direct interactions, which facilitates the dissection of protein interactions within kinase complexes. Such analyses have revealed important roles for scaffold proteins, bridging proteins, and adaptor proteins in kinase signalling. Finally, many kinases contain separable catalytic and regulatory domains, making protein kinases themselves amenable to the structure/function analysis offered by the two-hybrid system.
Simultaneous in situ detection of DNA and proteins
Authors: Ersfeld Klaus , Stone Elisa M
Published: November 2023

The development of fluorescent in situ hybridization (FISH) in combination with digital imaging technology has contributed greatly to our current view on how the nucleus of eukaryotic cells is organized. It has been shown that chromatin is not randomly distributed but is confined to defined sub-regions of the nucleus (1–4). There is mounting evidence that the non-random chromatin distribution is caused by anchoring of chromosomal sub-regions to elements of the nucleoskeleton, such as the nuclear lamina. The compartmentalization of genetic material and its interaction with specific proteins has important functional implications for various processes such as replication (5), transcription (6) or gene silencing (7–10) and chromosome partitioning (11-15) (Figure JA-G).
Evolutionary biology of the nuclear receptor superfamily
Authors: Escriva H , Robinson M , Laudet V
Published: November 2023

The study of the interplay between gene regulation, embryonic development, and the evolution of animal forms is receiving a growing interest. Numerous authors have studied the Hox gene complexes which control in all metazoan phyla. the establishment of antero-posterior polarity of organisms. By deciphering the phylogenetic relationships between individual genes of the Hox complex, as well a through isolation of homologues of Hox genes in a wide variety of organisms, from Hydrato various insects and vertebrates. the major role played by these genes in the evolutionary process is under intense scrutiny (reviewed in ref.1).
Analysis of steroid/nuclear receptor phosphorylation
Authors: Rowan H G , Weigel N L
Published: November 2023

As with other transcription factors. phosphorylation of nuclear receptors has been shown to be important in regulating receptor function. Studies in this area have utilized a variety of approaches and methodology to analyse receptor phosphorylation and the relation of phosphorylation to receptor function. This chapter will outline these different approaches and the current methodologies available lo identify phosphorylation sites. identify candidate kinases. and assay the functional significance of nuclear receptor phosphorylation. The advantages and disadvantages of each methodology are presented as well as a discussion of general considerations for examining receptor phosphorylation. This information should enable: the beginning investigator to choose the mos1 appropriate method to study receptor phosphorylation in different model systems.
Ligand- and cofactor-regulated transcription with chromatin templates
Authors: Kraus W L , Kadonaga J T
Published: November 2023

Nuclear hormone receptors function as DNA binding, ligand-, and cofactor-regulated transcription factors in the chromatin environment of the cell nucleus (1). Experiments aimed at understanding the detailed mechanisms of transcriptional activation by nuclear hormone receptors should use a bio-chemical approach and should include both the receptor and non-receptor (i.e. co-activators, co-repressors, chromatin, ligands) components of the nuclear hormone receptor signalling pathway. This chapter describes biochemical approaches for the analysis of lignnd- and cofactor-regulated transcription by nuclear hormone receptors. Methods are presented for the synthesis and purification of nuclear hormone receptors and co-activators (Protocols 1and 2). as5embly of chromatin templates i11 virro (Protocols 3and 4).and ligand- and co-activator-regulated transcription of the assembled chromatin template in vitro (Protocols 5—7).
Rho
Authors: Ridley Anne J
Published: November 2023

Rho was first identified in 1985 as a cDNA encoding a small GTP-binding protein related to Ras. Later, Rho was identified as the target of the Clostridium botulinum exoenzyme C3 transferase, which had been shown previously to induce changes to the morphology of mammalian tissue-culture cells. Subsequently, Rho and then a closely related protein, Rae, were shown to be major regulators of the actin cytoskeleton in all eukaryotic cells. A major surprise at that time was that Rae was also required for activation of the NADPH oxidase enzyme complex in phagocytic cells, a process seemingly independent of the actin cytoskeleton. Since then, additional members of the Rho family have been identified and many other cellular responses have been linked to them, including activation of MAPK cascades and regulation of transcription factors, secretion, endocytosis, cell polarity, and the cell cycle. Members of the Rho family have emerged as important coordinators of signal transduction processes activated by a wide variety of both extracellular and intrinsic cell cycle linked signals. Accordingly, they play crucial roles in the development and behaviour of multicellular organisms.
Rab
Authors: Collins Ruth N , Brennwald Patrick
Published: November 2023

A hallmark of eukaryotic cells is their possession of a set of membrane-enclosed organelles. The traffic of proteins between different organelles of the secretory and endocytic pathways is mediated by membrane-bound containers, termed vesicles. Figure 1 illustrates the basic stages of transport. Vesicles form by budding off from a donor compartment. The vesicle is transported to the acceptor compartment where it docks onto the membrane. This is followed by the final stage of fusion which releases the vesicle contents into the acceptor compartment. A variety of experimental strategies have begun to identify components of the transport machinery, which appear to be conserved in all eukaryotic cells. Every transport event is thought to require representatives from a set of conserved protein families. One of these families is that of the Rab proteins, a group of small, monomeric GTP-binding proteins related to Ras. The Rab GTPases will be the focus of this chapter.
An introduction to the nerve tertninal
Authors: Kelly Regis b.
Published: November 2023

Synaptic transmission between nerve cells can usefully be viewed as a specialized example of cell-to-cell communication. A cell in a metazoan organism communicates most simply by releasing a signal which diffuses until it is detected by receptors on a neighbouring cell, a process called paracrine communication. If the signalling cell is very far away from the target cell simple diffusion will not work. In such cases the signalling cells can release the signal into the bloodstream, which carries it by the blood flow into the vicinity of the target cell. This is called endocrine communication. We can immediately see the problem facing an endocrine signalling cell. If enough signal is to reach the target, the endocrine cell must release into the body vast quantities of signal compared with what is needed for paracrine signalling. This requirement is reflected in the morphology of endocrine cells. Their cytoplasm is packed with secretory vesicles that contain the signalling molecule at concentrations approaching those that cause crystallization.
Neurotransmitter transporters
Authors: Krantz Davide , Chaudhry farrukh A , Edwards Robert H
Published: November 2023

Synaptic transmission involves two distinct types of neurotransmitter transport activity. Plasma membrane reuptake is generally considered to terminate the action of neurotransmitter in the extracellular space. Transport from the cytoplasm into secretory vesicles packages transmitter for regulated release by exocytosis. Although the two activities thus have important if not essential roles in synaptic transmission, the mechanisms of active transport, their regulation and roles in signalling remain poorly understood. Molecular analysis has also suggested previously unanticipated functions for plasma membrane and vesicular neurotransmitter transporters.
Studying mutants that affect neurotranstnitter release in C.elegans
Authors: Nonet Michael L
Published: November 2023

Sydney Brenner initiated the study of the nematode Caenorhabditis elegans in the early 1960s with the specific intent of developing the organism as a model to study the nervous system. Primarily through the isolation and characterization of mutants that disrupt the functioning of the nervous system at the behavioural level, studies of the nematode have contributed to our understanding of the molecular mechanisms underlying a wide variety of neuronal processes including axonal outgrowth (1), sensory reception (2, 3), and synaptic transmission (4). The strength of the nematode as a model organism is undoubtedly the ability to apply powerful genetics to a biological problem. However, in the long-term, the dissection of the role of molecules in a complex process such as synaptic transmission requires the synthesis of results obtained using a variety of experimental approaches. Here, I first review the approaches available in C. elegans which have been utilized to characterize the molecules underlying the regulated release of neurotransmitter. Subsequently, I examine the complexity of the synaptic transmission apparatus in C. elegans through a discussion of mutants in a variety of genes which perturb the process. Finally, I discuss the avenues of research where the study of C. elegans is most likely to provide future insight that is applicable to understanding the machinery operating at synapses in all organisms.
Laboratory computing in medical microbiology
Authors: Lovering Andrew M
Published: November 2023

There can be few, if any, clinical microbiology laboratories in the UK that are without some form of computer system. Indeed. many laboratories will be running their second, or even, third generation of system and most will be acutely aware of the relative advantages and disadvantages of a laboratory computer system. Consequently this chapter will not address the basic principles and methods of operation of microbiology laboratory computer systems, as these will be familiar to most readers, but highlight some of the developing issues in Information Technology (IT) that are impacting on microbiology laboratories. For those readers unfamiliar with such basic principals the chapter by MacGowan et al. (1) and the paper by Kenny (2) are strongly recommended.
The nature and significance of protein folding
Authors: Dobson Christopher M , Kiefhaber Thomas
Published: November 2023

Proteins are involved in virtually every biological process in a living system. They are synthesized on ribosomes as linear chains of typically several hundred amino acid residues in a specific order from information encoded within the cellular DNA. In order to function, it is necessary for these chains to fold into the unique, native, three-dimensional structures that are characteristic of the individual proteins. It is clear that this folding process can take place rapidly.
Kinetic models in protein folding
Authors: Bieri Oliver , Kiefhaber Thomas
Published: November 2023

Experimental work on the mechanism of protein folding has been greatly influenced by Levinthal’s famous paper of 1969 (1), in which he pointed out that a polypeptide chain would require an astronomical time to explore at random all possible con formations in order to finally reach the native state. This motivated the search for partially folded intermediates guiding the protein to the native state. Although many proteins have been found to fold through transient intermediates (for reviews see refs 2-10), more recent theoretical studies have sparked off a discussion on their role in the folding process.
The molten globule state: the physical picture and biological significance
Authors: Kuwajima Kunjhjro , Arai Munehito
Published: November 2023

According to a classic view of protein folding, there is a specific folding pathway on which a protein has to go through a sequence of conformational intermediates in order to fold into the native structure. For this reason, studies on protein folding have been focused on the detection and characterization of folding intermediates (1) and, in particular, the molten globule state, which has been regarded as a general inter mediate of protein folding (2–4).
Protein folding and import into organelles
Authors: Rassow Joachim
Published: November 2023

How do proteins find their distinct functional location within the cell after they have been synthesized? In the last 15 years this basic biological question has opened a new field of molecular cell biology. The sites of protein synthesis and function are often separated by membranes. Therefore, the structures and mechanisms which drive proteins across membranes are of fundamental importance in the correct distribution of newly synthesized proteins in the cell (1). In this chapter, several of the techniques will be described which are applied to characterize intracellular protein traffic. All the protocols involved are easy to perform and do not require special equipment so they can be readily adopted by non-specialists. More sophisticated protocols can be obtained from the literature cited in the references.
Functional significance of O-GlcNAc glycoproteins in the nucleus and cytoplasm
Authors: Kreppel Lisa K , Hart Gerald W
Published: November 2023

O-GlcNAcylation is a form of intracellular protein glycoslyation consisting of a single N-acetylglucosamine residue [3-O-linked (O-GlcNAc) to the hydroxyl group of serine and threonine. Unlike other carbohydrate modifications, O-GlcNAc is not elongated to more complex structures and is found almost exclusively in the nucleus and cytoplasm (1, 2). O-GlcNAc-modified proteins are found in virtually all eukaryotic organisms, but not in bacteria (3, 4). Studies have demonstrated that the O-GlcNAc modification is dynamic and abundant, exhibiting characteristics more like phosphorylation than typical N-and O-linked glycosylation (5-8). Increasing evidence suggests that O-GlcNAc is a regulatory modification that may modulate numerous biological processes.
Molecular differentiation of bacterial strains
Authors: Brown Eric W
Published: November 2023

Understanding the epidemiological principles that govern bacterial populations has become paramount in the fields of medical, environmental, and agricultural microbiology (1). The introduction of bacterial strains which exhibit more aggressive and deadly pathological phenotypes, development of multi-drug antibiotic resistance, and acquisition of novel modes of transmission between host organisms has placed a renewed pressure on researchers to be able to identify and monitor the origins and dissemination of destructive genetic variants (strains) within a species. It has become apparent that the best approaches to characterizing individual strains are founded in the practical application of molecular biology. Molecular genetic techniques such as polymerase chain reaction (PCR), nucleic acid fingerprinting, and DNA sequence analysis of the bacterial chromo-some have allowed for remarkable advances in bacterial identification as in many other areas of microbiology (2). Whether the need for molecular typing involves the tracking of nosocomial (hospital-borne) outbreaks, identifying the reservoirs of food-borne infections, or the discrimination of plant pathogenic strains in the field, the isolation of a specific genotype in conjunction with a specific bacteria often allows for a larger understanding of the epidemiological principles that govern the evolution and spread of many bacterial diseases.
Evolutionary analysis of molecular sequence data
Authors: Hughes Austin L , Silva Jack da , Verra Federica
Published: November 2023

In the 140 years since Darwin’s Origin of Species was published, evolutionary ideas have formed an important part of the theoretical framework of biological research, but their practical consequences have been few for most of that period. The situation has changed since the advent of techniques for rapidly sequencing nucleic acids. Because evolution is nothing other than change in nucleotide sequences, the availability of sequence data enables us to study evolution far more directly than was previously possible. Molecular evolutionary analysis has now become an important part of the conceptual arsenal used by biologists in a wide variety of disciplines, including epidemiology.
HLA-peptide tetrameric complexes
Authors: Ogg Grahams.
Published: November 2023

HLA-peptide tetrameric complexes allow the direct visualization of antigen specific T cells by flow cytometry (1-3). Therefore, unlike other techniques used to study antigen-specific T cells, the method is independent of a functional activity such as cytokine production or lysis of target cells. Furthermore, the complexes are sufficiently sensitive (down to 0.02% of CDS+ T cells) to be used directly ex vivo, in contrast to assays of cytolytic function, which usually require a period of expansion in vitro before lysis becomes detectable. HLA-peptide tetrameric complexes are synthesized by the addition of biotin to the inferior aspect of refolded HLA molecules. Streptavidin has four biotin-binding sites and so will induce the formation of tetrameric structures of biotinylated HLA molecules. HLA heavy chain and β2microglobulin ( 2m) are generated in Escherichia coli as inclusion bodies. Only the extracellular portion of the heavy chain is included, and this is modified by the addition of a C-terminal signal sequence containing a site for biotinylation by the bacterial biotin ligase enzyme BirA. The inclusion bodies are solubilized in urea and then added to a refolding reaction along with an epitope peptide. The process of diluting the denaturant urea in the refolding buffer allows a proportion of the HLA heavy chain to refold with β2 and peptide into a conformationally correct form. The complex is then biotinylated at room temperature by the enzyme BirA in the presence of biotin, ATP, and Mg2+. Purification by gel filtration and ion exchange is followed by the addition of fluorescent streptavidin in a 1:4 molar ratio with the refolded complex to generate the tetramers. A tetrameric structure overcomes the low affinity of T-cell receptors (TCRs) for peptide/HLA, which for single complex interactions has an off-rate half-life of 12-30 sec at 25°C (reviewed in ref. 4). In fact, a trimeric structure may be sufficient-trimers and tetramers of a cytochrome c/I-Ek complex were shown to induce intracellular pH changes in specific T cells, whereas monomers and dimers were inactive (4).
Further applications to cell biology
Authors: Ormerod Michael G
Published: November 2023

There are a multitude of applications for flow cytometry, the most important of which have been covered in earlier chapters. In this chapter, five further applications to cell biology are described. The first is a method for estimating cell viability; the second, a way of monitoring the effect of high electric fields on cells; the third, a method for measuring the production of oxidative species in cells; and the last section discusses two methods for characterizing drug resistance in cancer cells, including a method for measuring intracellular glutathione.
Lmmunocytochemistry for light microscopy
Authors: Satiat-Jeunemaitre Beatrice
Published: November 2023

With the near completion of many of the genome sequencing programmes, the discipline of proteomics (the quantitative and physical mapping of cellular proteins and their function) will become one of the dominant topics in biology for the foreseeable future. As part of this, the unravelling of the spatial pattern of protein distribution and other molecular components throughout the cell is essential if we are to fully understand cell function. To provide such information, immunohistochemistry and immunocytochemistry have been increasingly valuable research techniques since they were first introduced in the late 60s.
The roles of the major cytoplasmic chaperones in normal eukaryotic growth: Hsc70 and its cofactors
Authors: Pfund Christine , Yan Wei , Craig Elizabeth A
Published: November 2023

The role of chaperones in the prevention of protein aggregation in cells experiencing stress is well established (see Chapters 1 and 9); however, problems of protein misfolding and aggregation are not confined to the stressed cell. Even under optimal growth conditions, there is a need for chaperone action. In the past few years, this aspect of chaperone function has become increasingly obvious as researchers focus their attention on the processes of translation, translocation across membranes, and the assembly/ disassembly of protein complexes. Within the crowded environment of the cell, each protein must be protected from misfolding and aggregation. In this chapter, we discuss the processes in the normal cell that require chaperone assistance and the evidence from studies using eukaryotes that support a role for both Hsp70s and their cohort proteins in these processes.
Laying Down The Vertebrate Body Plan
Authors: Tickle C , Davey M
Published: November 2023

How do different parts of the body arise in their proper positions in vertebrate embryos? In the first chapter, the principles of pattern formation were reviewed. This chapter outlines how the body plan is laid down, and provides the embryological background for the following chapters that deal in detail with patterning of mesoderm (Chapters 3 and 4), nervous system (Chapters 5, 6, 7, and 8), and limbs (Chapter 9) and the molecules involved. Laying down the body plan is essentially a matter of defining the main body axes: the anteroposterior axis (head to tail axis, sometimes known as rostral-caudal axis) and dorsoventral axis (back to front). Once these two axes have been set up, this also defines right and left (Fig. 1). In addition, the three main body layers—ectoderm, mesoderm, and endoderm—are formed and arranged so that endoderm comes to lie on the inside, ectoderm on the outside, and mesoderm in between. Establishment of these layers in their proper positions requires considerable cell rearrangements, which must be precisely choreographed.
Constructing phage display libraries by oligonucleotide- directed mutagenesis
Authors: Sidhu Sachdev S , Weiss Gregory A
Published: November 2023

As with any selection technology, the success of phage display depends on library diversity and quality. This chapter details robust methods that can be used to rapidly construct highly diverse phage-displayed libraries. Our protocols optimize previously described methods, allowing for the facile construction of libraries almost 100-fold greater than previously thought practical (1). Libraries with diversities greater than 10 can be readily constructed with a single reaction, and multiple reactions can be combined to generate libraries with diversities on the order of 10.
Murine chromosome preparation
Authors: Mai Sabine , Wiener Francis
Published: November 2023

Cytogenetic analysis of mouse chromosomes demands high quality metaphase plates suitable for various banding procedures and in situ hybridization protocols.
Multicolour FISH and spectral karyotyping
Authors: Bayani Jane , Squire Jeremy A
Published: November 2023

It is often difficult to determine the identity of a small structural chromosomal aberration with certainty using conventional cytogenetic banding methods alone. The problems that can typically arise in both clinical and cancer cytogenetics are the presence of structural chromosome aberrations with unidentifiable chromosomal regions, or very complex chromosomes (sometimes called ‘marker chromosomes’) in which no recognizable region appears to be present. Confirmation of the cytogenetic origins of such chromosomal aberrations can sometimes be obtained by the judicious application of locus-specific FISH analysis (see Chapter 9) if the investigator has some general impression regarding a possible identity. However, such an approach is very subjective, risky, and requires some knowledge of the specific loci and available probes likely to be involved in the aberration.
Popular Influence and State Power
Authors: Ginsberg Benjamin
Published: November 2023

Critics of the American political process seldom hesitate to propose remedies for the nation’s supposed political ills. Proponents of new campaign finance rules, lobbying regulations, voting reforms, and the like all seem confident that their nostrums will promote civic virtue and enhance popular influence. Few reformers, however, are sufficiently radical to confront the fundamental problem of citizen influence in political life. To put the matter succinctly, popular influence is inversely related to the state’s power. Weak governments are vulnerable to popular pressure. The more powerful the government and the greater the scope of its activities, the less the chance that it will concern itself with citizens’ views.
Gender And Genome
Authors: Hamilton W D
Published: November 2023

IN his The Genetical Theory of Natural Selection R. A Fisher included a certain passage about sex,ratio selection that after his death became famous among evolutionary biologists. It is a good example of what may be called his slit-trench style-short and deep. Many have fallen into this trench and been slow either to escape or to feel sheltered there, and I was one. Nowadays the trench catches fewer because there are many other good accounts of how selection works on the sex ratio; indeed, I believe that, in a purely qualitative way, the opening passage of my 1967 paper presents Fisher’s idea more clearly than he did. However, by the time I was able to write such a stepwise account, my struggles had revealed to me that there was a lot that Fisher’s argument had not covered. I saw that there were at least two kinds of influence that might affect selection of tendency to produce one sex or the other. First, there is the question of what genetic element is imagined to be controlling the sex ratio, and in which sex and at what stage the element acts. Second, there is the question of whether the population might be grouped such that the all with all (panmictic) competition assumed in Fisher’s argument would not apply.
Funeral Feasts
Authors: Hamilton W D
Published: November 2023

MY interest in the odd sex ratios of arthropods had tangled to some degree with my older interest in the potential role of haplodiploidy in shaping the ‘eusocial’ pattern of insect social ltfe. Most of the animals with inbreeding and male-deficient sex ratios were small.
Discordant Insects
Authors: Hamilton W D
Published: November 2023

Reading about them was not enough, they were too fantastic. Only in 1963 in Brazil did I first thoroughly believe in fig wasps, the tiny black insects that both breed in and pollinate all fig trees. Worldwide this means 600 species, not just the one cultivated species that is most familiar to Northern Europeans for its dried fruits in the brown, oblong packets. The 600 mainly inhabit tropical countries where many are giants of the forest. To this great assemblage the almost shrubby European fig stands an outlier in several ways. For example, besides its geographic placement as the world’s most northerly fig, and its small stature, it is dioecious-that is, has male and female trees separate like holly or yew. Again, generally each fig species has one kind of wasp adapted to pollinate it, but the European can sometimes have none; for the most practical and novel pollination feature of the European fig is that although it has a wasp not all its varieties need it, or at least not for their crops.
Prognostic Significance of Signs of Life in Out-of-Hospital Cardiac Arrest Patients Undergoing Extracorporeal Cardiopulmonary Resuscitation*
Authors: Bunya Naofumi , Ohnishi Hirofumi , Kasai Takehiko , Katayama Yoichi , Kakizaki Ryuichiro , Nara Satoshi , Ijuin Shinichi , Inoue Akihiko , Hifumi Toru , Sakamoto Tetsuya , Kuroda Yasuhiro , Narimatsu Eichi ,   Study of Advanced life support for Ventricular fibrillation with Extracorporeal circulation in Japan II (SAVE-J II) Study Group
Published: November 2023

OBJECTIVES:

Signs of life (SOLs) during cardiac arrest (gasping, pupillary light reaction, or any form of body movement) are suggested to be associated with favorable neurologic outcomes in out-of-hospital cardiac arrest (OHCA). While data has demonstrated that extracorporeal cardiopulmonary resuscitation (ECPR) can improve outcomes in cases of refractory cardiac arrest, it is expected that other contributing factors lead to positive outcomes. This study aimed to investigate whether SOL on arrival is associated with neurologic outcomes in patients with OHCA who have undergone ECPR.

DESIGN:

Retrospective multicenter registry study.

SETTING:

Thirty-six facilities participating in the Study of Advanced life support for Ventricular fibrillation with Extracorporeal circulation in Japan II (SAVE-J II).

PATIENTS:

Consecutive patients older than 18 years old who were admitted to the Emergency Department with OHCA between January 1, 2013, and December 31, 2018, and received ECPR.

INTERVENTIONS:

None.

MEASUREMENTS AND MAIN RESULTS:

Patients were classified into two groups according to the presence or absence of SOL on arrival. The primary outcome was a favorable neurologic outcome (Cerebral Performance Category 1 or 2) at discharge. Of the 2157 patients registered in the SAVE-J II database, 1395 met the inclusion criteria, and 250 (17.9%) had SOL upon arrival. Patients with SOL had more favorable neurologic outcomes than those without SOL (38.0% vs. 8.1%; p < 0.001). Multivariate analysis showed that SOL on arrival was independently associated with favorable neurologic outcomes (odds ratio, 5.65 [95% CI, 3.97–8.03]; p < 0.001).

CONCLUSIONS:

SOL on arrival was associated with favorable neurologic outcomes in patients with OHCA undergoing ECPR. In patients considered for ECPR, the presence of SOL on arrival can assist the decision to perform ECPR.
Beyond the Operating Room: Exploring Gender Bias in Leadership Positions in German Plastic Surgery
Authors: Saadoun Rakan , Soqia Jameel , Ataya Jamal , Fischer Katharina S. , De La Cruz Carolyn , Kamal Abdallah , Knoedler Leonard , Risse Eva-Maria
Published: November 2023

Background:

Despite efforts to promote gender equity in medicine, gender disparities persist in various medical specialties, including plastic surgery. This study aimed to investigate the representation of female physicians in leadership positions in German plastic surgery departments.

Methods:

This cross-sectional study collected data about the physician workforce in the German plastic surgery field. The primary outcome was the proportion of female physicians in plastic surgery departments. Data were collected from 94 departments. The physician workforce was stratified based on gender and leadership.

Results:

We included 812 physicians working in different German plastic surgery departments. Of those, 76.8% were in leadership positions, and 35.1% were women. There was a significant association between being male sex and holding a leadership position (n = 158/188, 84% versus n = 30/188, 16%, P < 0.0001). This association persisted even after accounting for the academic grade of each physician in a multivariable regression model (OR 2.565; 95% confidence interval, 1.628–4.041)

Conclusions:

Women are significantly underrepresented in leadership positions in German plastic surgery, with only 16% of female physicians holding such positions. Furthermore, being male sex was significantly associated with holding a leadership position, even after adjusting for the academic grade. These findings emphasize the existence of gender bias in the selection process for leadership positions in plastic surgery.
Cardiovascular disease prevention in women – the current state in 2023
Authors: Purohit Aarti , Kim Yoo Jin , Michos Erin D.
Published: November 2023

Purpose of review

Cardiovascular disease (CVD) remains a leading cause of morbidity and mortality for women globally. The purpose of this review is to provide an updated overview of CVD prevention in women, focusing on what is currently understood about female-specific or female-predominant CVD risk factors and the importance of tailored strategies for risk assessment and medical interventions.

Recent findings

Recent studies have demonstrated the need to account for risk factors specific to women in current risk assessment models for CVD, including early menarche, polycystic ovary syndrome, adverse pregnancy outcomes, early menopause, and chronic inflammatory conditions. Incorporation of these findings has led to advancements in sex-specific guidelines, diagnostic tools, and treatment approaches that have led to improvement in the precision of CVD prevention strategies. At-risk women benefit similarly to lipid-lowering and other preventive therapies as men but are less likely to be treated.

CVD prevention in women has made substantial progress over the past decade, marked by increasing awareness among clinicians, improved understanding of sex-specific risk-enhancing factors, and incorporation of sex-specific guidelines for management. However, there remain knowledge gaps that warrant ongoing efforts to optimize CVD prevention strategies in women, which will ultimately lead to improved cardiovascular health outcomes.
Baseline Liver Ultrasound Findings in Preschool Children From the Praziquantel in Preschoolers Trial in Lake Albert, Uganda
Authors: Pach Sophie , Webb Emily L. , Edielu Andrew , Nagawa Roy , Anguajibi Victor , Mpooya Simon , Wu Hannah , Colt Susannah , Mawa Patrice , Richter Joachim , Friedman Jennifer F. , Bustinduy Amaya L.
Published: November 2023

Background:

Periportal fibrosis is a late-stage manifestation of chronic infection withSchistosoma mansoni. Praziquantel (PZQ), the only drug available for the treatment of schistosomiasis, has limited effect in treating established morbidity. Preschool-age children (PSAC) are not considered to be an at-risk population for severe morbidity. However, the prevalence of periportal fibrosis in PSAC inS. mansoniendemic settings is unknown.

Methods:

As part of a phase II clinical trial comparing different dosing regimens of PZQ in children age 12–47 months infected withS. mansoniin Uganda (“praziquantel in preschoolers” trial), we present baseline results assessing liver ultrasound (US) findings as per the WHO Niamey Protocol.

Results:

A total of 7/347 (2%) PSAC had Image Pattern C with pipe stems and echogenic rings suggestive of periportal fibrosis, 29/347 (8%) had Image Pattern B and 58 (17%) had evidence of periportal thickening There were higher adjusted odds of periportal thickening with older age [odds ratio (OR): 1.04; 95% confidence interval (CI): 1.00–1.07], primary maternal education (OR: 1.04; 95% CI: 1.00–1.07) and being taken to the lake weekly (OR: 3.02; 95% CI: 1.19–7.63). A further 44/347 children (13%) had a rounded caudal liver edge which was associated with highS. mansoniinfection intensity (adjusted OR: 3.31; 95% CI: 1.46–7.51).

Conclusions:

Incipient schistosomiasis-related liver morbidity was detected in young children enrolled in the praziquantel in preschoolers trial. Adequate age-adjusted reference measurements for liver ultrasound findings in very small children are lacking but urgently needed. Schistosomiasis-related fibrosis may be delayed or averted with early and repeated PZQ treatment.