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This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non-lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single-cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single-cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures., (© 2023. Springer Nature Limited.)

Förster resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) spectroscopy are complementary techniques for quantifying distances in the nanometer range. Both approaches are commonly employed for probing the conformations and conformational changes of biological macromolecules based on site-directed fluorescent or paramagnetic labeling. FRET can be applied in solution at ambient temperature and thus provides direct access to dynamics, especially if used at the single-molecule level, whereas EPR requires immobilization or work at cryogenic temperatures but provides data that can be more reliably used to extract distance distributions. However, a combined analysis of the complementary data from the two techniques has been complicated by the lack of a common modeling framework. Here, we demonstrate a systematic analysis approach based on rotamer libraries for both FRET and EPR labels to predict distance distributions between two labels from a structural model. Dynamics of the fluorophores within these distance distributions are taken into account by diffusional averaging, which improves the agreement with experiment. Benchmarking this methodology with a series of surface-exposed pairs of sites in a structured protein domain reveals that the lowest resolved distance differences can be as small as ∼0.25 nm for both techniques, with quantitative agreement between experimental and simulated transfer efficiencies within a range of ±0.045. Rotamer library analysis thus establishes a coherent way of treating experimental data from EPR and FRET and provides a basis for integrative structural modeling, including studies of conformational distributions and dynamics of biological macromolecules using both techniques., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The relaxation-induced dipolar modulation enhancement (RIDME) technique allows the determination of distances and distance distributions in pairs containing two paramagnetic metal centers, a paramagnetic metal center and an organic radical, and, under some conditions, also in pairs of organic radicals. The strengths of the RIDME technique are its simple setup requirements, and the absence of bandwidth limitations for spin inversion which occurs through relaxation. A strong limitation of the RIDME technique is the background decay, which is often steeper than that in the double electron electron resonance experiment, and the absence of an appropriate description of the intermolecular background signal. Here we address the latter problem and present an analytical calculation of the RIDME background decay in the simple case of two types of randomly distributed spin centers each with total spin S = 1/2. The obtained equations allow the explaination of the key trends in RIDME experiments on frozen chelated metal ion solutions, and singly spin-labeled proteins. At low spin label concentrations, the RIDME background shape is determined by nuclear-driven spectral diffusion processes. This fact opens up a new path for structural characterization of soft matter and biomacromolecules through the determination of the local distribution of protons in the vicinity of the spin-labeled site.

Morphological and molecular characteristics determine the function of biological tissues. Attempts to combine immunofluorescence and electron microscopy invariably compromise the quality of the ultrastructure of tissue sections. We developed NATIVE, a correlated light and electron microscopy approach that preserves ultrastructure while showing the locations of multiple molecular moieties, even deep within tissues. This technique allowed the large-scale 3D reconstruction of a volume of mouse hippocampal CA3 tissue at nanometer resolution.

A combined method, employing NMR and EPR spectroscopies, has demonstrated its strength in solving structures of protein/RNA and other types of biomolecular complexes. This method works particularly well when the large biomolecular complex consists of a limited number of rigid building blocks, such as RNA-binding protein domains (RBDs). A variety of spin labels is available for such studies, allowing for conventional as well as spectroscopically orthogonal double electron-electron resonance (DEER) measurements in EPR. In this work, we compare different types of nitroxide-based and Gd(iii)-based spin labels attached to isolated RBDs of the polypyrimidine-tract binding protein 1 (PTBP1) and to short RNA fragments. In particular, we demonstrate experiments on spectroscopically orthogonal labelled RBD/RNA complexes. For all experiments we analyse spin labelling, DEER method performance, resulting distance distributions, and their consistency with the predictions from the spin label rotamers analysis. This work provides a set of intra-domain calibration DEER data, which can serve as a basis to start structure determination of the full length PTBP1 complex with an RNA derived from encephalomycarditis virus (EMCV) internal ribosomal entry site (IRES). For a series of tested labelling sites, we discuss their particular advantages and drawbacks in such a structure determination approach.

Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron-electron resonance (DEER) EPR measurements were performed for both the nitroxide-nitroxide and Gd(III)-nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

Cancer is a major cause of death. Common chemo- and radiation-therapies damage healthy tissue and cause painful side effects. The enzyme horseradish peroxidase (HRP) has been shown to activate the plant hormone indole-3-acetic acid (IAA) to a powerful anticancer agent in in vitro studies, but gene directed enzyme prodrug therapy (GDEPT) studies showed ambivalent results. Thus, HRP/IAA in antibody directed enzyme prodrug therapy (ADEPT) was investigated as an alternative. However, this approach has not been intensively studied, since the enzyme preparation from plant describes an undefined mixture of isoenzymes with a heterogenic glycosylation pattern incompatible with the human system. Here, we describe the recombinant production of the two HRP isoenzymes C1A and A2A in a Pichia pastoris benchmark strain and a glyco-engineered strain with a knockout of the α-1,6-mannosyltransferase (OCH1) responsible for hypermannosylation. We biochemically characterized the enzyme variants, tested them with IAA and applied them on cancer cells. In the absence of H2 O2 , HRP C1A turned out to be highly active with IAA, independent of its surface glycosylation. Subsequent in vitro cytotoxicity studies with human T24 bladder carcinoma and MDA-MB-231 breast carcinoma cells underlined the applicability of recombinant HRP C1A with reduced surface glycoslyation for targeted cancer treatment. Summarizing, this is the first study describing the successful use of recombinantly produced HRP for targeted cancer treatment. Our findings might pave the way for an increased use of the powerful isoenzyme HRP C1A in cancer research in the future., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 Mut(S) strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.

Recombinant protein production in microorganisms is one of the most studied areas of research in biotechnology today. In this respect the yeast Pichia pastoris is an important microbial production host due to its capability of secreting the target protein and performing posttranslational modifications. In a recent study, we described the development of a robust bioprocess for a glyco-engineered recombinant P. pastoris strain where the native α-1,6-mannosyltransfrease OCH1 was knocked out (Δoch1 strain). This strain produced the glycosylated enzyme horseradish peroxidase (HRP) with more homogeneous and shorter surface glycans than the respective benchmark strain. However, the recombinant Δoch1 strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and consequent intensive foam formation. Thus, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on (1) cell physiology, (2) cell morphology, (3) cell lysis, (4) productivity and (5) product purity in a multivariate manner. However, not only process parameters might influence these characteristics, but also media supplements might have an impact. Here, we describe the effects of different heme-precursors as well as of a protease-inhibitor cocktail on the production of active HRP in therecombinant P. pastoris Δoch1strain.

The methylotrophic yeast Pichia pastoris is a widely used host organism for recombinant protein production in biotechnology and pharmaceutical industry. However, if the target product describes a glycoprotein, an α-1,6-mannosyltransferase located in the Golgi apparatus of P. pastoris, called OCH1, triggers hypermannosylation of the recombinant protein which significantly impedes following unit operations and hampers biopharmaceutical product applications. A knockout of the och1 gene allows the production of less-glycosylated proteins-however, morphology and physiology of P. pastoris also change, complicating the upstream process. Here, we describe a controlled and efficient bioprocess based on the specific substrate uptake rate (q s) for a recombinant P. pastoris OCH1 knockout strain expressing a peroxidase as model protein.

The yeast Pichia pastoris is a common host for the recombinant production of biopharmaceuticals, capable of performing posttranslational modifications like glycosylation of secreted proteins. However, the activity of the OCH1 encoded α-1,6-mannosyltransferase triggers hypermannosylation of secreted proteins at great heterogeneity, considerably hampering downstream processing and reproducibility. Horseradish peroxidases are versatile enzymes with applications in diagnostics, bioremediation and cancer treatment. Despite the importance of these enzymes, they are still isolated from plant at low yields with different biochemical properties. Here we show the production of homogeneous glycoprotein species of recombinant horseradish peroxidase by using a P. pastoris platform strain in which OCH1 was deleted. This och1 knockout strain showed a growth impaired phenotype and considerable rearrangements of cell wall components, but nevertheless secreted more homogeneously glycosylated protein carrying mainly Man8 instead of Man10 N-glycans as a dominant core glycan structure at a volumetric productivity of 70% of the wildtype strain.

The female members of two different gym squads who where subject to hard training (12 to 15 hours a week), were investigated in regard to their growth parameters shortly after the beginning of intensive training and at yearly intervals thereafter. 12 of the initial 25 girls gave up training before reaching their 14th year. Only data compiled during the training period were included in the study. At the beginning of training, the gymnasts were comparable to the standard population (Zurich Longitudinal Study of Growth and Development) in regard to height and bone age: their weight was slightly subaverage. Height, growth rate, bone age, weight development and skinfold thickness remained clearly below standard values during the years of training. Due to a prolonged growth spurt during puberty, the final height and body weight were again comparable to standard values. Thelarche, pubarche and menarche were also significantly delayed; thelarche and menarche did occur, though, when the same body measurements (height, weight) as compared to the standard population were reached. FH, FSH and estradiol showed the puberty rise only after the 14th year, i.e. with considerable delay. When separately analyzing the data of the girls who gave up training before completing their 14th year, preexisting greater body measurements (including bone age) as well as more rapid development during the first years of training were found. Thus, the observed physical growth retardation is partly due to predisposition in the form of a constitutional late puberty, and is partly caused by the intensive training. A permanent physical developmental loss was not observed.