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Cold Spring Harbor Protocols Jul 2020This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific...
This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies. Greater care must be taken when labeling monoclonal antibodies to ensure a quality conjugate. The methods outlined here can be adapted for a variety of labels including multiple labels on the same immunoglobulin. The most important consideration when undertaking an antibody labeling experiment is to maintain the activity of the antibody. This is an empirical process and will often require additional experiments to optimize the label of a particular antibody. When successful, these reagents are very useful and adaptable biomolecules. This introduction provides the reader with methods and options for producing a variety of labeled immunological tools.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Antibodies; Biotin; Carbohydrates; Cross-Linking Reagents; Cysteine; Gold Colloid; Humans; Immunoconjugates; Isotope Labeling; Luminescent Proteins; Staining and Labeling; Sulfhydryl Compounds
PubMed: 32611784
DOI: 10.1101/pdb.top099242 -
Nature Methods Mar 2020Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM)...
Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, Kras and Trp53 cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.
Topics: Animals; Biomimetics; Cell Differentiation; Coculture Techniques; Colorectal Neoplasms; Cytophotometry; Enterocytes; Enteroendocrine Cells; Female; Fibroblasts; Gene Expression Regulation; Goblet Cells; Humans; Intestine, Small; Macrophages; Mice; Mice, Inbred C57BL; Organ Culture Techniques; Organoids; Paneth Cells; Signal Transduction; Single-Cell Analysis; Sulfhydryl Compounds; Tumor Suppressor Protein p53
PubMed: 32066960
DOI: 10.1038/s41592-020-0737-8 -
Cellular & Molecular Immunology Jan 2023Boosting tumor immunosurveillance with vaccines has been proven to be a feasible and cost-effective strategy to fight cancer. Although major breakthroughs have been...
Boosting tumor immunosurveillance with vaccines has been proven to be a feasible and cost-effective strategy to fight cancer. Although major breakthroughs have been achieved in preventative tumor vaccines targeting oncogenic viruses, limited advances have been made in curative vaccines for virus-irrelevant malignancies. Accumulating evidence suggests that preconditioning tumor cells with certain cytotoxic drugs can generate whole-cell tumor vaccines with strong prophylactic activities. However, the immunogenicity of these vaccines is not sufficient to restrain the outgrowth of existing tumors. In this study, we identified arsenic trioxide (ATO) as a wide-spectrum cytotoxic and highly immunogenic drug through multiparameter screening. ATO preconditioning could generate whole-cell tumor vaccines with potent antineoplastic effects in both prophylactic and therapeutic settings. The tumor-preventive or tumor-suppressive benefits of these vaccines relied on CD8 T cells and type I and II interferon signaling and could be linked to the release of immunostimulatory danger molecules. Unexpectedly, following ATO-induced oxidative stress, multiple cell death pathways were activated, including autophagy, apoptosis, necroptosis, and ferroptosis. CRISPR‒Cas9-mediated knockout of cell death executors revealed that the absence of Rip3, Mlkl, or Acsl4 largely abolished the efficacy of ATO-based prophylactic and therapeutic cancer vaccines. This therapeutic failure could be rescued by coadministration of danger molecule analogs. In addition, PD-1 blockade synergistically improved the therapeutic efficacy of ATO-based cancer vaccines by augmenting local IFN-γ production.
Topics: Humans; Arsenic Trioxide; Cancer Vaccines; Ferroptosis; CD8-Positive T-Lymphocytes; Necroptosis; Arsenicals; Oxides; Antineoplastic Agents; Apoptosis; Neoplasms; Immunity; Cell Line, Tumor
PubMed: 36447031
DOI: 10.1038/s41423-022-00956-0 -
Organic & Biomolecular Chemistry Apr 2022Heteroaryl sulfonamides are important structural motifs in the medicinal and agrochemical industries. However, their synthesis often relies on the use of heteroaryl...
Heteroaryl sulfonamides are important structural motifs in the medicinal and agrochemical industries. However, their synthesis often relies on the use of heteroaryl sulfonyl chlorides, which are unstable and toxic reagents. Herein, we report a protocol that allows direct oxidative coupling of heteroaryl thiols and primary amines, readily available and inexpensive commodity chemicals. The transformation proceeds under mild reaction conditions and yields the desired -alkylated sulfonamides in good yields. -alkyl heteroaryl sulfonamides can be further transformed using a microwave-promoted Fukuyama-Mitsunobu reaction to ,-dialkyl heteroaryl sulfonamides. The developed protocols thus enable the preparation of previously difficult to prepare sulfonamides (toxic reagents, harsh conditions, and low yields) under mild conditions.
Topics: Amines; Indicators and Reagents; Microwaves; Sulfhydryl Compounds; Sulfonamides
PubMed: 35343994
DOI: 10.1039/d2ob00345g -
Current Opinion in Chemical Biology Oct 2020Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes, fulfilling specific functions with unmatched precision.... (Review)
Review
Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes, fulfilling specific functions with unmatched precision. This unique specificity makes proteins an ideal scaffold to generate tools for the exploration of natural systems or for the construction of modern therapeutics. Thus, the chemoselective modification of proteins with functionalities that are not defined by the genetic code has become an indispensable approach for life science research and the development of therapeutics. Amongst site-selective strategies for protein modification, cysteine-selective approaches have long been used for the generation of functional protein conjugates and new reactions continue to emerge, offering solutions for diverse research questions. In this review, we are highlighting new strategies for the chemoselective modification of cysteine residues in peptides, proteins and antibodies with a particular focus on the most recent years. We lay special focus on new reagents for efficient cysteine conjugation that produce stable conjugation products with significant pharmaceutical application.
Topics: Antibodies; Drug Discovery; Humans; Peptides; Proteins; Sulfhydryl Compounds
PubMed: 32645576
DOI: 10.1016/j.cbpa.2020.04.017 -
Annals of Hematology Dec 2022The advent of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the outcomes of acute promyelocytic leukemia (APL); nevertheless, a... (Review)
Review
The advent of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the outcomes of acute promyelocytic leukemia (APL); nevertheless, a small fraction of patients still experience relapse. Due to the infrequency of APL relapse coupled with the rapid change in the therapeutic landscape, there are limited available data regarding the treatment of relapsed APL. In this situation, however, ATO-based therapy has been shown to result in high rates of hematological and molecular complete remission (CR). Autologous hematopoietic cell transplantation (HCT) is considered the postremission therapy of choice when patients achieve molecular CR, whereas recent studies have suggested that molecular CR is not prerequisite for the success of autologous HCT. Allogeneic HCT can be reserved for selected patients, i.e., those who cannot achieve CR and those who relapse after autologous HCT, because of high toxicities and the expectation of highly favorable outcomes with autologous HCT during CR. For patients who are ineligible for HCT, prolonged administration of ATRA + ATO would be a viable option. To further refine the therapy for patients with relapsed APL, it is imperative to aggregate clinical data of patients who relapse after the ATRA + ATO frontline therapy within the framework of national and international collaboration.
Topics: Humans; Leukemia, Promyelocytic, Acute; Arsenicals; Oxides; Treatment Outcome; Arsenic Trioxide; Tretinoin; Antineoplastic Combined Chemotherapy Protocols; Recurrence
PubMed: 35972562
DOI: 10.1007/s00277-022-04954-0 -
Methods in Molecular Biology (Clifton,... 2022Arsenic is either notorious toxicant or miracle cure for acute promyelocytic leukemia and several other diseases. It interacts with mitochondria directly or indirectly,...
Arsenic is either notorious toxicant or miracle cure for acute promyelocytic leukemia and several other diseases. It interacts with mitochondria directly or indirectly, by interacting with mitochondrial enzymes, such as respiratory chain complexes and tricarboxylic acid cycle proteins, or affecting mitochondrial homeostasis via ROS or mitochondrial outer membrane permeabilization. Given the ubiquitous presence of mitochondria and indispensable role in cellular metabolism, arsenical-mitochondrial interactions may manifest clinical importance by revealing mechanism of disease curation, preventing severe side effects, and foreseeing potential health issues. Here, we described the interaction between isolated mitochondria and arsenicals.
Topics: Apoptosis; Arsenic Trioxide; Arsenicals; Membrane Potential, Mitochondrial; Mitochondria; Oxides; Reactive Oxygen Species
PubMed: 35771442
DOI: 10.1007/978-1-0716-2309-1_11 -
Marine Drugs Jul 2023Microalgae are abundant components of the biosphere rich in low molecular weight carbohydrate-containing natural products (glycoconjugates). Glycoconjugates take part in... (Review)
Review
Microalgae are abundant components of the biosphere rich in low molecular weight carbohydrate-containing natural products (glycoconjugates). Glycoconjugates take part in the processes of photosynthesis, provide producers with important biological molecules, influence other organisms and are known by their biological activities. Some of them, for example, glycosylated toxins and arsenicals, are detrimental and can be transferred via food chains into higher organisms, including humans. So far, the studies on a series of particular groups of microalgal glycoconjugates were not comprehensively discussed in special reviews. In this review, a special focus is given to glycoconjugates' isolation, structure determination, properties and approaches to search for new bioactive metabolites. Analysis of literature data concerning structures, functions and biological activities of ribosylated arsenicals, galactosylated and sulfoquinovosylated lipids, phosphoglycolipids, glycoside derivatives of toxins, and other groups of glycoconjugates was carried out and discussed. Recent studies were fundamental in the discovery of a great variety of new carbohydrate-containing metabolites and their biological activities in defining the role of microalgal viral infections in regulating microalgal blooms as well as in the detection of glycoconjugates with potent immunomodulatory properties. Those discoveries support growing interest in these molecules.
Topics: Humans; Microalgae; Molecular Weight; Glycosides; Photosynthesis; Arsenicals
PubMed: 37623708
DOI: 10.3390/md21080427 -
Journal of Exposure Science &... Nov 2023Exposure assessment of inorganic arsenic is challenging due to the existence of multiple species, complexity of arsenic metabolism, and variety of exposure sources.... (Review)
Review
Exposure assessment of inorganic arsenic is challenging due to the existence of multiple species, complexity of arsenic metabolism, and variety of exposure sources. Exposure assessment of arsenic during pregnancy is further complicated by the physiological changes that occur to support fetal growth. Given the well-established toxicity of inorganic arsenic at high concentrations, continued research into the potential health effects of low-level exposure on maternal and fetal health is necessary. Our objectives were to review the value of and challenges inherent in measuring inorganic arsenic species in pregnancy and highlight related research priorities. We discussed how the physiological changes of pregnancy influence arsenic metabolism and necessitate the need for pregnancy-specific data. We reviewed the biomonitoring challenges according to common and novel biological matrices and discussed how each matrix differs according to half-life, bioavailability, availability of laboratory methods, and interpretation within pregnancy. Exposure assessment in both established and novel matrices that accounts for the physiological changes of pregnancy and complexity of speciation is a research priority. Standardization of laboratory method for novel matrices will help address these data gaps. Research is particularly lacking in contemporary populations of pregnant women without naturally elevated arsenic drinking water concentrations (i.e. <10 µg/l).
Topics: Pregnancy; Humans; Female; Arsenic; Biological Monitoring; Arsenicals; Drinking Water; Fetus
PubMed: 35948664
DOI: 10.1038/s41370-022-00457-2 -
Journal of the American Chemical Society Jan 2024A new bioconjugation reagent containing silicon has been developed for the selective reaction with thiols. The inclusion of silicon significantly improves...
A new bioconjugation reagent containing silicon has been developed for the selective reaction with thiols. The inclusion of silicon significantly improves chemoselectivity and suppresses retro processes, thereby exceeding the capabilities of traditional reagents. The method is versatile and compatible with a broad range of thiols and unsaturated carbonyl compounds and yields moderate to high results. These reactions can be conducted under biocompatible conditions, thereby making them suitable for protein bioconjugation. The resulting conjugates display good stability in the presence of various biomolecules, which suggests their potential application for the synthesis of antibody-drug conjugates. Furthermore, the presence of a silicon moiety within the conjugated products opens up new avenues for drug release and bridging inorganics with other disciplines. This new class of silicon-containing thiol-specific bioconjugation reagents has significant implications for researchers working in bioanalytical science and medicinal chemistry and leads to innovative opportunities for advancing the field of bioconjugation research and medicinal chemistry.
Topics: Silicon; Sulfhydryl Compounds; Indicators and Reagents; Proteins; Immunoconjugates
PubMed: 38198597
DOI: 10.1021/jacs.3c12050