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Radiolabeling is a highly sensitive method for protein detection, which is easily performed by the incorporation of radioactive amino acids into proteins. This makes radiolabeling a method of choice for visualizing proteins separated on two-dimensional (2-D) gels. This chapter presents protocols to determine in vivo labeling conditions and to label proteins for the comparison of protein samples by means of 2-D gel electrophoresis.

International audience; Genetic diversity can be introduced into polyploid crop species through meiotic recombination by exchanges between homologous or homoeologous chromosomes. Fluorescent in situ hybridization (FISH) enables the characterization of these homologous and homoeologous chromosome pairs during meiosis and identification of structural rearrangements during mitosis in metaphase I. In this chapter, we describe a protocol for the multicolored fluorescent labeling of chromosome spreads. This protocol allows the characterization of each A and C homoeologous subgenomes in a polyploid species using a genome-specific BAC combined with specific chromosome labeling BAC sequences.

Cryopreservation has been successfully used in the banking and maintenance of cultures of microorganisms, from bacteria to yeasts, since the onset of cryobiology. Biobanking of marine biological resources is crucial for development of scientific knowledge as researchers rely on guaranteed access to reliable, stable resources. Culture collections play a key role in the provision of marine biological resources as they ensure long-term ex situ storage of biological resources that are made available for public and private sector research and education. In this chapter, we provide protocols for cryopreservation of different types of algae cultures. European Marine Biological Resource Centre Biobank | Ref. EAPA_501/2016 INTERREG-IV-Atlantic project EBB (European Blue Biobank)

Marine invertebrates represent the vast majority of marine biodiversity; they are extremely diverse playing a key role in marine ecosystems, thus playing an important role at the socioeconomic level. Some invertebrates such as sea urchins, ascidians, and horse-shoe crabs are very well-known model organisms for research and biocompound discovery. In this chapter we revisit the importance of cryopreservation for the conservation and rational use in research, fisheries management, or aquaculture and provide comprehensive protocols for the cryopreservation of sperm, embryos, and larvae. Xunta de Galicia Fundação para a Ciência e a Tecnologia

International audience; At the dawn of the renaissance of bacteriophage therapy, the full acceptation of bacteriophages as anti-bacterial agents requires the determination of their basic pharmacokinetic parameters. Such data, known for all conventional drugs used in human and veterinary medicine, allow optimizing dose regimens, efficacy, and help to limit toxicity. Here, we describe basic methods to experimentally obtain pharmacokinetic data and give also examples of data calculation to determine key parameters related to the biodistribution and elimination of bacteriophages in vivo.

Anther culture is a biotechnological method that allows to obtain, in one step, homozygous plants, very important to plant breeding, due to their numerous applications in mutation research, selection, genome sequencing, genetic analysis, and transformation. To induce the microspores, i.e., the immature male gametes, to switch from the normal gametophytic pathway to the sporophytic one, it is necessary to submit them to a type of stress, such as high or low temperature, starvation, or magnetic field. Stress can be applied to the donor plants and/or the fl oral buds or the anthers or the isolated microspores, before or during the culture. In this chapter, the protocol to induce gametic embryogenesis from anther culture of several cultivars of Citrus clementina Hort. ex Tan. is reported.

International audience; Arginine (Arg) has long been known to be a major regulator of immunity via its metabolic and physiological functions. In the 1950s, it was classified as a non-essential amino acid by Rose since Arg could be synthesized at the whole body level, mainly in the kidneys, after the conversion of intestinal citrulline (Cit) via argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). But things changed when Barbul et al. (Surg Forum 28:101–103, 1977) observed, surprisingly, that Arg was an immunomodulator via a thymic effect. At the same time, they observed that Arg could be essential in several situations, like growth or sepsis. Finally in the 1990s, Albina et al. (J Immunol 147:144–148, 1991) demonstrated that macrophages were able to produce nitric oxide (NO). These pioneering observations opened a new field of research focused on the regulation of macrophage functions by Arg.

Prostheses can release some metallic elements to the surrounding tissues, particularly when they are not covered with a biomaterial layer and when an unsealing process happens. We try to measure major and trace elements in these tissues with an experimentally sensitive method. Proton-induced X-ray emission is used to detect about 10 elements in tissue. Tissues are calcinated and deposited in a thin layer before irradiation. Results are obtained in a standard and samples from three patients. We observe contamination by Ti, Cr, Ni, and Zn in the tissues. Correlations are to be studied between these atomic transfers and prosthesis in the patient.

Better defining the dynamics of biomolecular interactions is an important step in understanding molecular biology and cellular processes. DNA-protein interactions, and specifically hormone-triggered DNA-nuclear receptor interactions, are key events which are still poorly understood. To date, the most commonly used approach in studying chromatin interactions is the immunoprecipitation of chemically cross-linked chromatin (ChIP) coupled with single gene or global genomic analyses. Currently, establishing a stable interplay between nucleic acids and proteins (DNA-protein cross-link) is mainly obtained through conventional, diffusion-triggered, chemical methods using formaldehyde. Here we describe an alternative method, called Laser-ChIP (LChIP), for the specific analysis of interactions between chromatin and nuclear receptors driven by a UV laser energy source. Photo-induced cross-linking in LChIP is achieved very rapidly, allowing the study of transient interactions, depending on laser source parameters.

International audience; The NMDA subtypes of glutamatergic receptors (NMDARs) are unusual in that their activation requires the binding of both glutamate and a co-agonist glycine or D-serine. Whereas glycine was first suggested to play such a role, it was later established that D-serine could serve as an endogenous co-agonist at different central synapses. We still do not know the exact nature of the endogenous co-agonist(s) of NMDARs and the function of the so-called glycine B site in many brain structures. We introduced few years ago the use of enzymes that specifically degrade either D-serine or glycine to decipher the influence of these amino acids on NMDA receptors function. The use of these enzymatic scavengers represents an invaluable technique for neurophysiologists investigating the neuromodulation of the glycine B site in the CNS. Here, we describe the proper way to manipulate these enzymes during electrophysiological recordings in acute brain slices and highlight the experimental tricks.

An implanted biomaterial can be transformed into young bone after some months, but it has not necessary reached full biofunctionality. Mineral concentration kinetics and crystal-structure studies, still being carried out in our group, are completed here by biofunctionality determinations. A natural coral is implanted in vivo at the cortical level of the femoral diaphyoff++ in rabbits. Diphosphonates molecules labeled with radioactive 99mTc are then injected in rabbits and the fixation of the radioactivity is analyzed in several sites for 8 mo after the implantation. Nuclear instruments and methods are used for the measurements. Four successive cycles of osseous remodeling are determined before reaching a biofunctional phase.