bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene targeting vectors -considerations and use of loxP
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

In general, the experimental outline of Cre/loxP mediated gene targeting is accomplished in two steps. The first is the incorporation of loxP sites into the genome of ES cells via a corresponding targeting vector and the identification of these homologous recombinant clones. The final genetic modification is obtained by a Cre-mediated recombination event, either stably in cultured ES cells (and the germline of mice derived from these ES cells) or in a conditional manner, in mice by the expression of Cre as a transgene. Because the potential of each targeting construct is greatly expanded with the use of this system, loxP sites should be generally considered for all targeting vectors even if the initial goal is simply to inactivate gene function.
ES cell transfection overview
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

Thinking about addressing a biological question by gene targeting is an exciting aspect of the experiment and one that requires relatively little knowledge about ES cells and the practicalities of gene targeting. Similarly, constructing a targeting vector can also be accomplished with little knowledge of ES cells. The time when ES cells are often first considered, at least in a practical sense, is when the targeting construct is completed and you are ready to introduce the DNA into the ES cells in attempts at homologous recombination. Without any ES cell experience it is at this point that a slight trepidation is often experienced. This feeling is enhanced for the newcomer by the apparent confusion which surrounds the juggling of EF cells for each ES cell manipulation. This section attempts to provide an overview of these manipulations, including the transfection of ES cells, followed by detailed protocols which we have used in generating homologous recombinant ES cell clones and the transfer of the altered allele to the germline. Of course, once your mutation has been transmitted to the germline, the breeding, maintenance, and analysis of the mouse strain is necessary. The topic of mouse genetics is not addressed here but we refer you to an excellent book on this topic (134).
Identification of homologous recombinants
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

One of the biggest strategical questions you must ask about your targeting experiment (preferably before vector construction) is whether or not to screen by PCR. A major advantage to screening by PCR is that the number of colonies which can feasibly be analyzed is much larger than that for screening by Southern analysis. This will of course be important if the locus you wish to target has a low efficiency of undergoing homologous recombination (for unknown reason(s)) and may make the difference between finding a homologous recombinant or not. Another advantage to PCR screening is being able to very quickly discard clones which have not homologously recombined, greatly reducing the amount of tissue culture required when screening by Southern analysis.
Generation of mice -preparation
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

There are several methods in the literature which result in ES cell-directed development and germ.line colonization of a developing mouse blastocyst (16, 140, 147, 168-170). We describe here two methods: the physical injection of ES cells into the blastocoel of blastocysts and the aggregation of ES cells with eight-cell stage/morulas. Although perhaps not as useful for most purposes, ES cells have also been shown to be able to direct the development of a fetus when aggregated together with tetraploid embryos, possibly resulting in viable offspring (147). In our view, the decision as to which of the two general methods to use depends on personal preference but may also be determined by the equipment available, the number of chimeras that need to be produced, the ES cell line used, and the individual’s skill in micromanipulation. In contrast to blastocyst injection, aggregation methods require no expensive equipment (microscope, micromanipulators) and allow the processing of larger numbers of embryos per day. For the production of low numbers of chimeras for transmission of a targeted mutation through the germline, however, both methods are equally useful.
Blastocyst transfer
Authors: Torres Raul M , Kühn Ralf
Published: November 2023

After a days work of injecting blastocysts you still must transfer these embryos to foster mothers. Initially, this can be a source of anxiety but with time can also become quite enjoyable.
Characterization of macrophage antigens and receptors by immunochemistry and fluorescent analysis: expression, endocytosis, and phagocytosis
Authors: Peiser Leanne , Gough Peter J , Darley Elizabeth , Gordon Siamon
Published: November 2023

This chapter describes techniques to identify macrophages and measure their endocytic and phagocytic capabilities. These methods can assist in the analysis of the physiological and pathological situations in which macrophages play a central role including homeostasis, wound repair, and immunity. The expression of cell surface proteins and other molecules varies greatly among macrophage populations as a result of heterogeneity in their differentiation, activation state, or response to external influences in their microenvironment or cell culture conditions (1-3). Inununochemical and fluorescent techniques can beused to investigate the basal expression of antigens, the changes induced in response to a particular stimulus, and to detect antigens in tissues.
Standards and immunogens
Authors: Chaudhry M Q
Published: November 2023

Standards are preparations of analyte that are used for calibration, that is, in an assay ‘unknown’ samples are compared with them to estimate concentrations of analyte in the samples. While for hapten immunoassays standards are chem­ically quite different from the corresponding immunogens, for immunoassays that measure naturally immunogenic antigens the same purified preparation may be used as both standard and immunogen. However, the criteria for a good standard may be quite different from those for a good immunogen. Standards are considered in Section 6.
Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting
Authors: Leger* Olivier J P , Saldanhat Jose w.
Published: November 2023

The biotechnological generation of high affinity monoclonal antibodies has traditionally involved the production of hybridomas from spleen cells of immunized animals. Recent advances in recombinant technology, using phage dis play technology, have opened a new route for the generation of antibodies. Instead of immortalizing B cells for production of monoclonal antibodies, the antibody heavy (H) and light (L) chain V genes are immortalized by gene technology. The IgG mRNA of B cells of immunized animals can be used as a source of variable region (V) gene libraries. Murine VH and VL genes are amplified using PCR and used to construct libraries of millions of recombinant single chain Fvs (scFvs). ScFv antibodies consist of the variable regions of the light and heavy chains of antibodies linked via a short peptide spacer (1). Repertoires of scFv are cloned into the gene encoding the minor coat protein (gene III) of filamentous bacteriophage, thus creating a large library of phage, each displaying an individual heavy and light chain combination. The fusion protein created, consisting of the antibody fragment at the N-terminus of the coat protein, is incorporated into the phage particle (a phage antibody). Each recombinant phage genome contains the DNA encoding the specific antibody displayed on its surface, allowing the phage particle carrying antibody gene to be selected directly using the binding properties of the expressed protein.
Laboratory based methods for small scale production of monoclonal antibodies
Authors: Griffiths Bryan
Published: November 2023

Although hybridoma cells are spherical and well adapted to suspension culture they do grow well in stationary culture resting on the substrate, sometimes with very light attachment. This means that a wide selection of culture vessels and systems are available for their culture ranging from a simple tissue culture plate or flask to highly sophisticated bioreactors with full instrumentation to control the physiological environment (1-3). Commercially hybridoma cell lines are grown at scales up to 2000 litres and beyond in culture units scaled-up from laboratory size vessels. Laboratory, pilot, and production scale is ill-defined so in this chapter laboratory scale will be taken as 10 litre volume cultures, and below.
Non-radioactive antibody probes
Authors: Wisdom G Brian
Published: November 2023

The high specificity of monoclonal antibodies makes them valuable reagents, however, to exploit them fully requires a means of detecting them at low con centration. This is usually done by labelling the antibody in one of various ways. Radioisotopes are widely used (Chapter 9) as are non-isotopic labels such as enzymes, fluorogenic and luminogenic molecules, as well as various low molecular weight groups that can themselves be easily detected by a secondary reagent. The non-isotopic labels provide low detection limits in a wide range of techniques without the complications of handling radioisotopes, although it should be noted that all the non-isotopic labelling procedures have the hazards associated with protein-modifying reagents.
Monoclonal antibody therapy in organ transplantation
Authors: Wise Matt , Zelenika Diana
Published: November 2023

In the two decades since they were first described by Kohler and Milstein mono clonal antibodies have proved to be invaluable tools for research, not only in immunology, but also in many aspects of biomedical research. Monoclonal anti bodies revolutionized lymphocyte biology by permitting the precise characterization of cell surface molecules which until that time were ill-defined by polyclonal sera. To date numerous mAbs have been used in animal models as either immunosuppressants or agents to induce tolerance. It may therefore come as some surprise to the reader that since their inception only two mAbs have been licensed for this purpose in humans.
Analysis of protein kinase interactions using biomolecular interaction analysis
Authors: Herberg Friedrich W , Zimmermann Bastian
Published: November 2023

Novel technologies employing surface plasmon resonance (Biacore AB) or resonance mirrors (lAsys, Lab Systems) can be used for the characterization of protein-protein, protein-DNA and ligand-receptor interactions (for review see refs 1-3). These techniques directly measure the binding of a molecule in the soluble phase (the ‘analyte’, also called the ‘ligate’) to a ‘ligand’ molecule immobilized on a sensor surface. Thereby the association (k  a) and dissociation rate constants (k  d) are determined separately. With a known concentration of the analyte, apparent equilibrium binding constants (K  D or K  A) can be calculated. The chemistries for coupling molecules to the surface via amine, sulfhydryl, carboxyl, and other groups are well defined and reproducible. Non-covalent, site-directed immobilization can be achieved by avidin-biotin interaction, by the use of antibodies, or by binding fusion proteins to specific surfaces. Surfaces containing lipid monolayers are also available. This technology can be used for monitoring interactions, in real time without the use of labels, between two or more molecules, e.g. between proteins, peptides, nucleic acids, or pharmaceuticals.
Basic immunofluorescence
Authors: Allan Victoria J
Published: November 2023

Immunofluorescence on fixed samples forms a vital part of most investigations into protein function, since it provides an idea of the whereabouts of a protein within the cell, which in turn can give important clues as to the protein’s function. In addition, immunofluorescence can tell us whether this location varies through the cell cycle or development, and whether this is consistent in cells from different tissues or species. All of this information is very valuable, particularly now that genome sequencing data are available. However, in order to obtain meaningful results, and to interpret them correctly, it is important that the methods and limitations of immunofluorescence are understood.
Regulation of heterologous proteins by fusion to a hormone binding domain
Authors: Picard D
Published: November 2023

It is often helpful or even a necessity in biological studies to be able to regulate.: the expression or the activity of a protein of interest. Intracellular receptors by virtue of being ligand-regulated transcription factors have naturally been examined as regulatory tools. The application that is the focus of this chapter exploits the normal regulatory activity of the hormone binding domain (HBD) of vertebrate steroid receptors for the regulation of heterologous proteins in cis.Upon fusion to a heterologous protein. (he ITRD can function as an autonomous regulatory cassette and subject various protein functions to hormonal control (1. 2). Thus. one or several functional activities of the heterologous moiety may be turned offin the absence of steroid ligand. While the chimeric protein can he expressed constitutively, it only become-, active upon addition of hormone. Within at most a few minutes the hormonally-induced conformational change in the HBD results in the relieve of its protein inactivation function and activation of the chimeric protein.
Construction and screening of a subtractive cDNA library
Authors: Akopian Armen N , Wood John N
Published: November 2023

Subtractive library production and screening are powerful methods to isolate transcripts that are exclusively or selectively expressed either in certain tissues, or during development or in disease states. This chapter describes procedures for the isolation of specific transcripts using construction and screening of sub tractive libraries, and illustrates examples of typical problems arising during the experiments. The original method was developed by Davis and colleagues in classical work on the isolation of T-lymphocyte-specific transcripts, including a third type of murine T-cell receptor gene (1, 2). Over the years this approach has been modified. However, the principle behind the method is essentially the same, and only a few modifications have improved on the remarkable sensitivity and efficiency of the original method. Construction and screening of a sub tractive cDNA library can be divided into several steps Figure 1).
SADE: a microassay for serial analysis of gene expression
Authors: Cheval Lydie , Virion Bérangère , Elalouf Jean-Marc
Published: November 2023

SAGE has been shown to provide rapid and detailed information on transcript abundance and diversity (7-10). It involves several steps for mRNA purification, cDNA tags generation and isolation, and polymerase chain reaction (PCR) ampli fication. We reasoned that increasing the yield of the various extraction pro cedures, together with slight modifications in the number of PCR cycles could enlarge SAGE potentiality. Here we present a mkroadaptation of SAGE, referred to as SADE (11) as, in contrast to the original method, it allows to provide quantitative gene expression data on a small number (30000-50000) of cells.
Protein folding and human disease
Authors: Raso Stephen W , King Jonathan
Published: November 2023

The earliest studies on the refolding of purified proteins in the test tube identified the failure of such processes, in the form of inactive and insoluble chains (1, 2). For decades these aggregated states were thought to be unrelated to physiological processes taking place within cells. However, the recognition that the failure of the expression of cloned genes in bacteria was often due to aggregation of intracellular folding intermediates focused attention on these processes (3-5). The discovery that heat-shock proteins in almost all organisms include chaperonins that aid protein folding, established that failure of protein folding is a very general phenomenon, at least at elevated temperatures and under other stressful circumstances (6). In parallel, the discovery that amyloid deposits recovered from human tissues represent fibrillar forms of particular protein fragments revealed that protein aggregation was associated with a number of human pathologies (7, 8).
Analysis of protein phosphorylation
Authors: Walaas S Ivar , ∅stvold Anne Carine
Published: November 2023

Extensive studies have demonstrated that extracellular signals, e.g. hormones, neurotransmitters, growth factors, as well as intracellular signals and effectors, e.g. cyclic nucleotides, Ca2+ ions, and phospholipid derivatives, mediate many of their effects by regulating the activities of protein phosphorylation systems (1-3). The role of reversible protein phosphorylation in regulating and integrating the activity of eukaryotic cells has thus become widely appreciated during the last decades (4). Cellular protein phosphorylation systems consist of a minimum of three components: (a) The phosphorylated proteins themselves. (b) A large number of protein kinases which catalyse the incorporation of phosphate from the terminal phosphate of ATP into specific serine, threonine, tyrosine, and possibly histidine (5) residues in the substrate phosphoproteins.
Carbohydrate recognition in leukocyte-endothelial cell interactions
Authors: Homeister Jonathon W , Lowe John B
Published: November 2023

Mammalian cell surfaces are decorated with complex carbohydrate molecules, also known as oligosaccharides or glycoconjugates. These molecules display a remarkable degree of structural diversity, which is in turn a function of the cell type or tissue, or the particular stage of development of the cell or tissue. Cell surface oligosaccharide structure varies with the differentiation state or lineage of a cell. These observations suggest that these molecules play important roles in mediating information transfer into and out of the cell. During mammalian development, for example, dynamic changes are observed in cell surface glycoconjugate expression. This work suggests that complex mechanisms have evolved to regulate these processes, and that they have important functional correlates. The existence of an ever-expanding group of mammalian carbohydrate binding proteins, or lectins (1), suggests that cell surface carbohydrate molecules may participate as ligands in adhesive processes mediate by mammalian lectins. Indeed, oligosaccharides have been shown to operate in processes whereby lysosomal enzymes are targeted to the lysosome (via the mannose-6-phosphate receptor; ref. 2), in the clearance of desialylated serum glycoproteins (via the hepatic asialoglycoprotein receptor; ref. 3), and in the clearance of circulating pituitary glycoprotein hormones like leutropin (4). Experimental studies during the past several years have implicated members of a family of mammalian lectins, known as the selectins, and a specific group of cell surface oligosaccharide determinants, in adhesive processes that mediate leukocyte adhesion to vascular endothelium. This chapter will summarize experimental approaches that have led to the discovery of these processes, and will discuss the enzymes, selectins and other molecules that participate in carbohydrate dependent leukocyte adhesion.
Polysialic acid in neural cell developtnent: roles, regulation and tnechanistn
Authors: Bruses Juan L , Rutishauser Urs
Published: November 2023

Polysialic acid (PSA) was discovered as a major component of vertebrate brains, largely on the basis of its unusual composition and size (1). It is a linear homopolymer of a-2,8-linked sialic acid (from about eight to over 100 residues) attached to an otherwise typical N-linked carbohydrate core (for review, see ref. 2, see also Fig. 10 in chapter 1). However, unlike most carbohydrates found on the cell surface, PSA is attached exclusively to the polypeptide of the neural cell adhesion molecule (NCAM) (3-5). PSA’s function was first recognized through its ability to decrease NCAM mediated membrane-membrane adhesion in vitro (6, 7). It was later observed that PSA could also affect other cell interactions that do not appear to depend directly on NCAM’s intrinsic binding function (5, 8). For example, PSA can attenuate the function of the Ll class of axon-associated adhesion molecules, apparently without any direct association of L1 with NCAM or PSA.
Single-sperm typing: a rapid alternative to family-based linkage analysis
Authors: Cullen Michael , Carrington Mary
Published: November 2023

Single-sperm typing provides an efficient means of measuring recombination frequencies relative to the traditional methods of genetic cross and pedigree analysis. Using this method, it is possible to rapidly determine the haplotype of each meiotic product directly using PCR to identify alleles at multiple poly morphic markers along the chromosome. Traditional pedigree analysis, on the other hand, requires genotypes collected from children of parents informative (heterozygous) for markers of interest. For example, mapping resolutions of 1–2 cM were reported for the Genethon human linkage map based on 5264 microsatellites genotyped for eight CEPH (Centre d’Etudes du Polymorphisme Humaine) families, to talling 134 individuals (186 meioses) (1). However, markers used to construct a family-based linkage map must have high joint polymorphic information content (PIC), i.e. a high proportion and number of informative meioses per marker pair (2). In regions of low marker density this is problematic since the total number of markers with high PIC values is decreased. This issue becomes less relevant with the use of single-sperm typing because donors who are heterozygous at the appropriate markers can be selected regardless of the PIC value of those markers. Furthermore, unlimited numbers of meiotic events provide a means of analysing tightly linked markers at resolutions of 0.1–0.2 cM, presently not possible by pedigree analysis (3–5).
HLA typing methods
Authors: Krausa Pete , Browning Michael
Published: November 2023

The human major histocompatibility complex or HLA system is a set of highly polymorphic genes clustered on chromosome 6. There are three main groups of HLA genes, namely: HLA class I, class II, and class III. HLA class I and class II genes code for molecules that present antigen in the form of peptides at the cell surface. HLA class I molecules are expressed on most nucleated cells and platelets, and present antigen to CD8+ T cells. HLA class II genes are constitutively expressed on professional antigen-presenting cells (B lymphocytes, macrophages, monocytes, and dendritic cells). In addition, HLA class II expression can be induced on a range of other cell types, including activated T cells, activated endothelium, and many epithelial cells. HLA class II molecules present antigen to CD4+ T cells. HLA class III genes code for certain complement components, cytokines (TNF-a and -13), and a variety of non-immunological products, but their identification will not be covered in this chapter.
Preparing suspensions of single cells
Authors: Ormerod Collated by Michael G
Published: November 2023

A suspension of single cells is the essential element of flow cytometry. The sample should contain as little debris and as few dead cells and clumps as pos sible. A set of protocols for the preparation of cell suspensions has been collected in this chapter. It is by no means comprehensive but should form a good starting point for any new investigation. Extensive sets of methods have also been described by Cheetham et al. (1) and Pallavicini (2).

The first six protocols have been taken from N. P. Carter’s Chapter 3 in the first edition of this book.
Flow sorting
Authors: Carter Nigel P , Ormerodt Michael G
Published: November 2023

Flow sorting enables subpopulations of cells or particles to be separated from the sample suspension with a high degree of purity. The purified fractions (usually95% pure) are thus made available for morphological examination or use in functional or other assays, so greatly extending the research and diagnostic potential of the flow cytometer. Any combination of parameters can be applied as criteria to decide whether a cell is or is not sorted, thereby harnessing the full analytical potential of the instrument in the separation process. One of the main original applications of flow sorting was the purification of morphologically similar, but functionally distinct, lymphocyte subpopulations using fluorescent staining with polyclonal antibodies. However, the ability to utilize any analytical parameter or combinations of parameters has led to flow sorting being used in diverse applications, ranging from cloning rare cell transfectants directly into multi-well plates to the bulk sorting of human chromosomes for the production of DNA libraries.
The application of fluorescence in situ hybridization to chromosome analysis
Authors: Kearney L , Buckle V J
Published: November 2023

For over two decades banding has remained the ‘gold standard’ of cytogenetic analysis, providing the first genome-wide screen for abnormalities. However, conventional cytogenetic banding techniques are limited to the detection of rearrangements involving more than 2 Mb of DNA. In addition, the identification of de nova unbalanced chromosome rearrangements provides a particular challenge for chromosome banding to decipher. In recent years a number of techniques based around FISH have evolved, all of which complement the conventional banding approaches and which have steadily increased the accuracy of cytogenetic diagnosis.
The lymphomas and chronic lymphoid leukaemias
Authors: Harrison C J
Published: November 2023

Malignant lymphomas and chronic lymphoid leukacmias rcpresc not two highly heterogeneous groups of malignant disease. Both arise as a result of neoplastic proliferation of lymphoid cells. A number of chromosomal abnormalities have been associated with histopathology. disease staging, cmd prognosis. Although these are fewer than any of the other haematological disorders, cytogenetic information is increasing in importance as an aid to diagnosis and patient management. This chapter presents those techniques that have been the most successful in producing cytogenetic results and a SL1mma1y of the impo11ant cytogenetic findings.
The role of cytogenetics in the diagnosis and classification of haematological neoplasms
Authors: Bain B J
Published: November 2023

Cytogenetic analysis is undertaken in patients with haemalological disorders for two main reasons: firstly, to aid in the management of the patient and secondly, for the advancement of scientific knowledge. Often, both purposes are served simultaneously. The patient may benefit from cor1firmation of a provisional diagnosis and from the availability of prognostic information that can influence m;inagcmcnt. Cytogc-111:tic analysis can be very useful during follow up. firstly for monitoring the response to therapy and secondly for detecting rebpse and distinguishing relapse from a new therapy-related leukaemia. Cytogenetic analysis may also be required before patients are entered into therapeutic trials and may influence stratification within these trials.
Chromosome instability syndromes
Authors: Howell R T
Published: November 2023

A number of clinically diverse inherited human diseases, unified by the abnormal behaviour of their chromosomes. are somelimes grouped together under the heading of chromosome instability syndromes. Depending on the syndrome, the abnormal chromosome behaviour may take the form of:
Introduction to optical microscopy for plant cell biology
Authors: Shaw P J
Published: November 2023

The development of the optical microscope in the seventeenth Century opened up new areas of study in many fields of science, In particular, the observations of plant and animal tissues and micro-organisms gave rise to cell biology, although our modern idea of what a ‘cell’ actually is arose somewhat later. Some of the data recorded by the early microscopists, using only primitive microscopes and drawing freehand what they saw, are quite remarkable. Hooke, Malpighi, and Leuwenhoek are the best known seventeenth century microscopists, but Nehemiah Grew was the first true specialist in plant microscopy. He produced detailed descriptions of plant microanatomy which have proved remarkably accurate. An example is shown, along with Grew’s original legend (1), in Figure 1. Although there is only space to show one such picture, the original volumes contain many pages of such detailed, painstaking hand-engravings. With today’s access to photography, video cameras, and computer image processing, this should serve as a reminder of how much can be achieved with careful observation and a very simple microscope. Many biologists overlook the useful information that can be rapidly obtained by even a very simple laboratory microscope.
Transient expression, a tool to address questions in plant cell biology
Authors: Hadlington Jane L , Denecke Jurgen
Published: November 2023

Transient expression in plant systems is a rapidly expanding technique, which has opened up new strategies to approach biological problems without the necessary delay and environmental risks that are associated with the production of stable transformants. DNA is introduced into cells via naked DNA transfer or biological vectors such as bacteria or viruses, and gene expression is assayed for a limited time after the initial transfer. Some methods allow the introduction of RNA or proteins as well. Results can be obtained much faster without extensive resources and time, and in case of cell suspensions or protoplasts, experiments are more reproducible due to the high number of cells and the homogeneous nature of the suspensions. In some cases, i.e. the analysis of cytotoxins or dominant mutant approaches disrupting essential pathways or mechanisms, transient expression is the only option available, because transgenic plants would not be viable. Finally, with the current debate on the safety and necessity of genetically modified organisms, it is important to introduce alternative ways of addressing biological questions in plants. In many cases, transient expression has been shown to provide ultimately the same information as approaches using transgenic plants, thus justifying a more consequent and widespread use of the technique.
Electrophysiological methods: monitoring exo- and endocytosis in real time
Authors: Thiel* Gerhard , Sutter, Jens-Uwe , Homann* Ulrike
Published: November 2023

About two decades ago Neher and Marty (1) showed that the patch-clamp technique in combination with some impedance analysis could be used for monitoring the membrane capacitance as a quasi real time assay for exocytosis and endocytosis. This method allows recording of exo- and endocytosis in single animal cells in vivo with such a high resolution, that single exo- and endocytotic events from vesicles with a diameter under 100 nm can be recorded with a temporal resolution in the order of some 10 msec. This method has been adopted with success in plant physiology laboratories to examine these processes in plant protoplasts (reviewed in ref. 2). Here we aim to present the range of problems which can be addressed with this methodology and provide guidance to the methodological approach.
Organelle isolation
Authors: Robinson David G , Hinz Giselbert
Published: November 2023

Subcellular fractionation has, as its general principle, the application of three consecutive procedures. The first, homogenization, results in the abolition of cellular integrity. Depending on the forces involved, individual compartments or organelles may also no longer remain intact. The second step entails imposing a new system of order among the randomly dispersed organelles in the homogenate. This is usually achieved through separations based on size (gel filtration), mass (differential or rate zonal centrifugations), density (isopycnic centrifugation), or surface charge (electrophoresis, phase-partitioning). Finally, the subcellular units or fractions obtained have to be checked for their homogeneity. For this, electron microscopical (negatively stained or thin sectioned samples), chemical (enzyme tests), and immunological (Western blotting) methods can be employed.
FISH probes and labeling techniques
Authors: Bray-Ward Patricia
Published: November 2023

A Fluor is a chemical moiety that, in response to absorption of a photon of energy provided by a light source with a suitable emission wavelength, develops an excited electron singlet state. This creates a transient conformational change and partial dissipation of the excitation energy. This is followed by emission of a photon of lower energy and therefore longer wavelength. Toe difference in energy or wavelength of excitation and emission is called the Stokes shift. This is a critical element in fluorescence imaging since detection systems must exclude wavelengths of light from the excitation source. Stokes shift can be affected by the physical characteristics of the fluor, for example covalent linkage to a protein, the polarity and hydrophobicity of the solvent, and the pH of the solution. Generally fluors with larger Stoke shifts are preferred since they allow filter sets to be used that have wider bandwidths to permit maximum capture sensitivity.
The WY Domain of an RxLr Effector Drives Interactions with a Host Target Phosphatase to Mimic Host Regulatory Proteins and Promote Phytophthora infestans Infection
Authors: Bentham Adam R. , Wang Wei , Trusch Franziska , Varden Freya A. , Birch Paul R. J. , Banfield Mark J.
Published: November 2023

Plant pathogens manipulate the cellular environment of the host to facilitate infection and colonization that often lead to plant diseases. To accomplish this, many specialized pathogens secrete virulence proteins called effectors into the host cell, which subvert processes such as immune signaling, gene transcription, and host metabolism. Phytophthora infestans, the causative agent of potato late blight, employs an expanded repertoire of RxLR effectors with WY domains to manipulate the host through direct interaction with protein targets. However, our understanding of the molecular mechanisms underlying the interactions between WY effectors and their host targets remains limited. In this study, we performed a structural and biophysical characterization of the P. infestans WY effector Pi04314 in complex with the potato Protein Phosphatase 1-c (PP1c). We elucidate how Pi04314 uses a WY domain and a specialized C-terminal loop carrying a KVxF motif that interact with conserved surfaces on PP1c, known to be used by host regulatory proteins for guiding function. Through biophysical and in planta analyses, we demonstrate that Pi04314 WY or KVxF mutants lose their ability to bind PP1c. The loss of PP1c binding correlates with changes in PP1c nucleolar localization and a decrease in lesion size in plant infection assays. This study provides insights into the manipulation of plant hosts by pathogens, revealing how effectors exploit key regulatory interfaces in host proteins to modify their function and facilitate disease.

[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
Early Prognostication of Critical Patients With Spinal Cord Injury
Authors: Fan Guoxin , Liu Huaqing , Yang Sheng , Luo Libo , Pang Mao , Liu Bin , Zhang Liangming , Han Lanqing , Rong Limin , Liao Xiang
Published: November 2023

Study Design.

A retrospective case-series.

Objective.

The study aims to use machine learning to predict the discharge destination of spinal cord injury (SCI) patients in the intensive care unit.

Summary of Background Data.

Prognostication following SCI is vital, especially for critical patients who need intensive care.

Patients and Methods.

Clinical data of patients diagnosed with SCI were extracted from a publicly available intensive care unit database. The first recorded data of the included patients were used to develop a total of 98 machine learning classifiers, seeking to predict discharge destination (eg, death, further medical care, home, etc.). The microaverage area under the curve (AUC) was the main indicator to assess discrimination. The best average-AUC classifier and the best death-sensitivity classifier were integrated into an ensemble classifier. The discrimination of the ensemble classifier was compared with top death-sensitivity classifiers and top average-AUC classifiers. In addition, prediction consistency and clinical utility were also assessed.

Results.

A total of 1485 SCI patients were included. The ensemble classifier had a microaverage AUC of 0.851, which was only slightly inferior to the best average-AUC classifier (P=0.10). The best average-AUC classifier death sensitivity was much lower than that of the ensemble classifier. The ensemble classifier had a death sensitivity of 0.452, which was inferior to the top 8 death-sensitivity classifiers, whose microaverage AUC were inferior to the ensemble classifier (P<0.05). In addition, the ensemble classifier demonstrated a comparable Brier score and superior net benefit in the DCA when compared with the performance of the origin classifiers.

Conclusions.

The ensemble classifier shows an overall superior performance in predicting discharge destination, considering discrimination ability, prediction consistency, and clinical utility. This classifier system may aid in the clinical management of critical SCI patients in the early phase following injury.

Level of Evidence:

Level 3.
Three Cases of Catheter-related Bloodstream Infection Caused by Phialemonium curvata: A Case Report and Literature Review
Authors: Özen Seval , Karabiçak Nilgün , Uzuntaş Sema Turan , Kanik-Yüksek Saliha , Yildiz Selin , Güder Latife , Üçkardeş Fatih , Şahin Seda , Dinç Bedia , Özyörük Derya , Parlakay Asli Nur Özkaya , Bayhan Gülsüm İclal
Published: November 2023

Background:

With recent developments in the field of microbiology, an increasing number of yeasts and molds with the potential to cause infections in humans are identified every year. In addition to the challenges in identifying clinical isolates, there is limited antifungal susceptibility data available for Phialemonium species, leading to uncertainty in optimal treatment recommendations.

Methods:

In this article, catheter-related bloodstream infections caused by Phialemonium curvata (previously Phialemonium curvatum) in 3 immunosuppressed patients are presented. Furthermore, the literature was reviewed to identify the clinical spectrum and treatment approaches for the reported infections.

Results:

The cases presented here were analyzed along with 24 cases reported in the literature. Among all cases, 21 (77.7%) patients had an underlying condition. Nine (33.3%) patients had hematologic/oncologic malignancies and solid organ transplants. Twenty-two (81.4%) patients had a history of device or invasive interventions. Surgical procedures, removal of contaminated devices or tissue were found to reduce the risk of death by 86.7%. Correspondence analysis revealed a significant association between antifungal treatment and outcome (P < 0.001). The correspondence analysis could explain 53.9% of this relationship. Monotherapy and combination therapy were associated with survival. While salvage treatment or no antifungal therapy was associated with mortality, intravitreal injection or topical application of voriconazole was associated with sequelae.

Conclusions:

Surgical intervention and removal of contaminated devices or tissue should be considered at an early stage.
Cefiderocol Red Wine Urine Syndrome in Pediatric Patients: A Multicenter Case Series
Authors: Shapiro Kate , Ungar Stephanie P. , Krugman Jessica , McGarrity Orlagh , Cross Shane J. , Indrakumar Bairavi , Hatcher James , Ratner Adam J. , Wolf Joshua
Published: November 2023

Cefiderocol is a novel cephalosporin antibiotic with activity against multidrug-resistant Gram-negative bacteria and limited pediatric experience. This case series describes 3 immunocompromised children receiving blood transfusion who developed benign red or purple urine with administration of cefiderocol. Interaction with iron from blood products is a possible mechanism. It is important to recognize this phenomenon and distinguish it from hematuria to avoid unnecessary diagnostic testing.
Impact of Respiratory Bacterial Codetection on Outcomes in Ventilated Infants With Bronchiolitis
Authors: Akande Manzilat , Spencer Sandra P. , Moore-Clingenpeel Melissa , Jamieson Nathan , Karsies Todd
Published: November 2023

Background:

Viral bronchiolitis is a common cause of acute respiratory failure requiring intubation for infants. Bacterial respiratory tract infections can occur with bronchiolitis, although their prevalence and impact on outcomes are unclear, especially with increased use of noninvasive respiratory support.

Methods:

This was a single-center retrospective cohort study of children <2 years old requiring intubation in the emergency department for bronchiolitis from 2012 to 2017 who had viral testing plus a lower respiratory culture obtained. We evaluated the impact of bacterial codetection (positive respiratory culture plus moderate or many polymorphonuclear neutrophils on Gram stain) on mechanical ventilation (MV) duration and intensive care unit length of stay using multivariable gamma regression.

Results:

Of 149 patients enrolled, 52% had bacterial codetection. In adjusted analysis, patients with codetection had shorter MV duration [adjusted relative risk (aRR) 0.819, 95% confidence interval (CI): 0.69–0.98; marginal mean duration of 5.31 days (4.71–5.99) compared to 6.48 days (5.72–7.35) without codetection]. Patients with codetection had a shorter intensive care unit stay [aRR 0.806 (0.69–0.94); marginal mean length of stay 6.9 days (6.21–7.68) vs. 8.57 days (7.68–9.56) without codetection]. The association between codetection and duration of ventilation appears confined to those receiving earlier antibiotics (less than the median time) rather than later antibiotics [aRR 0.738 (0.56–0.95) for earlier vs. aRR 0.92 (0.70–1.18) for later].

Conclusions:

Respiratory bacterial codetection is common and associated with shorter MV duration in infants requiring early intubation for bronchiolitis. Early antibiotics may contribute to these outcomes, but further multicenter studies are needed to understand the role of codetection and antibiotics on bronchiolitis outcomes.
Ukrainian War Refugee Children With Particularly Severe Viral Infections: A Case Series Report
Authors: Pluta Magdalena , Karny Katarzyna , Lipińska Marta , Mańdziuk Joanna , Podsiadły Edyta , Kuchar Ernest , Pokorska-Śpiewak Maria , Okarska-Napierała Magdalena
Published: November 2023

The humanitarian crisis in Ukraine in 2022 led to a massive migration of refugees to Poland. Immigrant children, living in overcrowded humanitarian hubs, were exposed to multiple stressful factors likely affecting their immune systems. This case series study aimed to describe a particularly severe course of common viral infections, in Ukrainian refugee children. We present 2 case series of Ukrainian refugee children: 5 hospitalized due to either adenovirus (AdV) and 8 with rotavirus (RV) infection, admitted within 3 months in each case series, recruited retrospectively. Most patients lived in humanitarian hubs and were neglected on admission (dehydrated, with poor hygiene and anxious). All RV infection cases had symptoms of severe gastroenteritis requiring intravenous rehydration. Metabolic acidosis was present in 6 children, and hypoglycemia in 4 participants. None of them were vaccinated against RV. All children with AdV infection had prolonged fever, dyspnea requiring oxygen therapy and hyperinflammation. In 2 AdV infection cases with no clinical improvement and increasing inflammatory markers, intravenous immunoglobulins and glucocorticosteroids were used. The combination of stressful factors and living in overcrowded hubs during the high prevalence of viral infections led to a particularly severe course of viral infections in Ukrainian refugee children.
Improvement in Epiphora Using TEARS Grading Following Surgical Treatment of Meibomian Gland Inversion in Patients With Facial Nerve Palsy
Authors: Yvon Camille , Malhotra Raman
Published: November 2023

Purpose:

To assess epiphora outcomes using the TEARS grading score in patients with concomitant meibomian gland inversion (MGI) and facial nerve palsy (FNP) undergoing correction of MGI.

Methods:

Retrospective, 5-year, noncomparative, single-center study of patients with MGI and FNP, treated with MGI correction, under the supervision of a single surgeon. A validated “TEAR” score was used to assess changes in epiphora.

Results:

Ten patients with FNP, MGI, and epiphora were analyzed from a group of 160 patients with FNP who underwent MGI surgery between 2017 and 2022. The mean age at surgery was 50 years (range, 13–76 years). T, E, and A scores significantly improved (p < 0.05). Eighty percent of patients saw a reduction in tearing frequency (T), with 60% gaining ≥ 2-grade improvement. Improvements in clinical effects (E) and activity limitation (A) were seen in 80% of patients, with 40% and 50% gaining ≥ 2-grade improvement, respectively. R scores (related to reflex tearing) improved by 60%, with 40% seeing ≥ 2-grade improvement. Nine patients (90%) improved symptomatically with an average improvement “S” score of 65% over a mean follow-up period of 30 months. All patients demonstrated restoration of the normal anatomical position of the meibomian glands.

Conclusions:

MGI can cause epiphora in patients with FNP and may explain cases where symptoms persist despite standard surgical intervention. This study provides proof of concept that MGI correction can improve epiphora and that identifying MGI may be considered a critical step in the treatment algorithm for epiphora in patients with FNP.
Flammability of Ocular Surface Lubricants
Authors: Gupta Akash , Mudie Lucy I. , Yen Michael T.
Published: November 2023

Purpose:

Surgical fires pose a substantial risk to patients and can cause significant injury, especially in oculofacial surgery. Ocular surface lubricants can potentially act as fuel for an operating room fire. We present an experimental analysis of the flammability of 9 commonly used ophthalmic lubricants under 4 ignition sources used in oculofacial surgery with and without supplemental oxygen.

Methods:

The flammability of 9 ophthalmic lubricants were tested under various operating room conditions. Each lubricant was exposed to 4 different ignition sources: an open flame lighter, monopolar cautery, bipolar cautery, and hand-held high temperature cautery, and the response of the lubricant was recorded. The testing was conducted both in room air and with 6 L/minute of 100% oxygen directed at the lubricant through a nasal cannula. Any reaction in which there was ignition, sparking, smoking, or a transient or permanent change in appearance of the lubricant was deemed notable.

Results:

Of the 9 lubricants tested, 4 displayed a reaction to the ignition source. Without supplemental oxygen, 100% petrolatum and neomycin-polysporin-bacitracin-hydrocortisone ointment produced some smoke when applied with the high temperature cautery. Notably, under both the conditions of no supplemental oxygen and with the addition of 6 L/minute of 100% oxygen, the carboxymethylcellulose drops and lidocaine jelly both conducted and sparked with the monopolar cautery leaving visible burn marks on the paper.

Conclusions:

The overall fire hazard posed by ocular surface lubricants is low. Some topical lubricants can conduct electricity from monopolar cautery, which could increase the risk of inadvertent electrical burns. Certain lubricants could potentially become a fuel source when used in combination with hand-held high temperature battery cautery. Bipolar cautery was not associated with either increased conductivity or flammability with any of the lubricants tested.