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Annals of Laboratory Medicine Jan 2022Immunoassays are powerful qualitative and quantitative analytical techniques. Since the first description of an immunoassay method in 1959, advances have been made in... (Review)
Review
Immunoassays are powerful qualitative and quantitative analytical techniques. Since the first description of an immunoassay method in 1959, advances have been made in assay designs and analytical characteristics, opening the door for their widespread implementation in clinical laboratories. Clinical endocrinology is closely linked to laboratory medicine because hormone quantification is important for the diagnosis, treatment, and prognosis of endocrine disorders. Several interferences in immunoassays have been identified through the years; although some are no longer encountered in daily practice, cross-reaction, heterophile antibodies, biotin, and anti-analyte antibodies still cause problems. Newer interferences are also emerging with the development of new therapies. The interfering substance may be exogenous (e.g., a drug or substance absorbed by the patient) or endogenous (e.g., antibodies produced by the patient), and the bias caused by interference can be positive or negative. The consequences of interference can be deleterious when clinicians consider erroneous results to establish a diagnosis, leading to unnecessary explorations or inappropriate treatments. Clinical laboratories and manufacturers continue to investigate methods for the detection, elimination, and prevention of interferences. However, no system is completely devoid of such incidents. In this review, we focus on the analytical interferences encountered in daily practice and possible solutions for their detection or elimination.
Topics: Antibodies; Biotin; Cross Reactions; Hormones; Humans; Immunoassay
PubMed: 34374345
DOI: 10.3343/alm.2022.42.1.3 -
Nature Chemical Biology Mar 2021Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular...
Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.
Topics: Animals; Betaine-Homocysteine S-Methyltransferase; Biotin; Biotinylation; Blood Proteins; Gene Expression; HEK293 Cells; Hepatocytes; Humans; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Muscle Cells; Myeloid Cells; Organ Specificity; Pericytes; Proteome; Proteomics; Staining and Labeling
PubMed: 33199915
DOI: 10.1038/s41589-020-00698-y -
International Journal of Molecular... Dec 2021Epigenetic changes constitute one of the processes that is involved in the mechanisms of carcinogenicity. They include dysregulation of DNA methylation processes,... (Review)
Review
Epigenetic changes constitute one of the processes that is involved in the mechanisms of carcinogenicity. They include dysregulation of DNA methylation processes, disruption of post-translational patterns of histone modifications, and changes in the composition and/or organization of chromatin. Benzo(a)pyrene (BaP) influences DNA methylation and, depending on its concentrations, as well as the type of cell, tissue and organism it causes hypomethylation or hypermethylation. Moreover, the exposure to polyaromatic hydrocarbons (PAHs), including BaP in tobacco smoke results in an altered methylation status of the offsprings. Researches have indicated a potential relationship between toxicity of BaP and deregulation of the biotin homeostasis pathway that plays an important role in the process of carcinogenesis. Animal studies have shown that parental-induced BaP toxicity can be passed on to the F1 generation as studied on marine medaka (), and the underlying mechanism is likely related to a disturbance in the circadian rhythm. In addition, ancestral exposure of fish to BaP may cause intergenerational osteotoxicity in non-exposed F3 offsprings. Epidemiological studies of lung cancer have indicated that exposure to BaP is associated with changes in methylation levels at 15 CpG; therefore, changes in DNA methylation may be considered as potential mediators of BaP-induced lung cancer. The mechanism of epigenetic changes induced by BaP are mainly due to the formation of CpG-BPDE adducts, between metabolite of BaP-BPDE and CpG, which leads to changes in the level of 5-methylcytosine. BaP also acts through inhibition of DNA methyltransferases activity, as well as by increasing histone deacetylases HDACs, i.e., HDAC2 and HDAC3 activity. The aim of this review is to discuss the mechanism of the epigenetic action of BaP on the basis of the latest publications.
Topics: 5-Methylcytosine; Animals; Benzo(a)pyrene; Biotin; Carcinogenesis; DNA Methylation; Epigenesis, Genetic; Epigenomics; Female; Histone Deacetylases; Humans; Pregnancy; Prenatal Exposure Delayed Effects
PubMed: 34948252
DOI: 10.3390/ijms222413453 -
Thyroid : Official Journal of the... Aug 2021Biotin has been reported to interfere with several commonly used laboratory assays resulting in misleading values and possible erroneous diagnosis and treatment. This...
Biotin has been reported to interfere with several commonly used laboratory assays resulting in misleading values and possible erroneous diagnosis and treatment. This report describes a prospective study of possible biotin interference in thyroid-related laboratory assays, with a comparison of different commonly used assay platforms. Thirteen adult subjects (mean age 45 ± 13 years old) were administered biotin 10 mg/day for eight days. Blood specimens were collected at three time points on day 1 and on day 8 (baseline, two, and five hours after biotin ingestion). Thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroxine binding globulin (TBG), and thyroglobulin (Tg) levels were analyzed with four different platforms: Abbott Architect, Roche Cobas 6000, Siemens IMMULITE 2000, and liquid chromatography with tandem mass spectrometry (LC-MS/MS). TSH, fT3, fT4, TT3, and TT4 were measured with Abbott Architect and Roche Cobas 6000. fT3, fT4, TT3, and TT4 were also measured by LC-MS/MS. Tg was measured by Siemens IMMULITE 2000. TBG was assessed with Siemens IMMULITE 2000. Significant changes in TSH, fT4, and TT3 measurements were observed after biotin exposure when the Roche Cobas 6000 platform was used. Biotin intake resulted in a falsely lower Tg level when measurements were performed with Siemens IMMULITE 2000. At the time points examined, maximal biotin interference was observed two hours after biotin exposure both on day 1 and day 8. A daily dose of 10 mg was shown to interfere with specific assays for TSH, fT4, TT3, and Tg. Physicians must be aware of the potential risk of erroneous test results in subjects taking biotin supplements. Altered test results for TSH and Tg can be particularly problematic in patients requiring careful titration of levothyroxine therapy such as those with thyroid cancer.
Topics: Adult; Aged; Biotin; Chromatography, High Pressure Liquid; False Negative Reactions; Female; Humans; Male; Mass Spectrometry; Middle Aged; Prospective Studies; Thyroglobulin; Thyroid Function Tests; Thyroid Hormones; Thyrotropin
PubMed: 34042535
DOI: 10.1089/thy.2020.0866 -
Nutrients Nov 2020Gut microbiota are suspected to affect brain functions and behavior as well as lowering inflammation status. Therefore, an effect on depression has already been... (Randomized Controlled Trial)
Randomized Controlled Trial
Gut microbiota are suspected to affect brain functions and behavior as well as lowering inflammation status. Therefore, an effect on depression has already been suggested by recent research. The aim of this randomized double-blind controlled trial was to evaluate the effect of probiotic treatment in depressed individuals. Within inpatient care, 82 currently depressed individuals were randomly assigned to either receive a multistrain probiotic plus biotin treatment or biotin plus placebo for 28 days. Clinical symptoms as well as gut microbiome were analyzed at the begin of the study, after one and after four weeks. After 16S rRNA analysis, microbiome samples were bioinformatically explored using QIIME, SPSS, R and Piphillin. Both groups improved significantly regarding psychiatric symptoms. and were more abundant and β-diversity was higher in the probiotics group after 28 days. KEGG-analysis showed elevated inflammation-regulatory and metabolic pathways in the intervention group. The elevated abundance of potentially beneficial bacteria after probiotic treatment allows speculations on the functionality of probiotic treatment in depressed individuals. Furthermore, the finding of upregulated vitamin B6 and B7 synthesis underlines the connection between the quality of diet, gut microbiota and mental health through the regulation of metabolic functions, anti-inflammatory and anti-apoptotic properties. Concluding, four-week probiotic plus biotin supplementation, in inpatient individuals with a major depressive disorder diagnosis, showed an overall beneficial effect of clinical treatment. However, probiotic intervention compared to placebo only differed in microbial diversity profile, not in clinical outcome measures.
Topics: Adult; Biodiversity; Biotin; Cohort Studies; Depression; Dietary Supplements; Female; Gastrointestinal Microbiome; Haptoglobins; Humans; Male; Placebos; Principal Component Analysis; Probiotics; Protein Precursors
PubMed: 33171595
DOI: 10.3390/nu12113422 -
Gut Dec 2022Gut microbiota is a key component in obesity and type 2 diabetes, yet mechanisms and metabolites central to this interaction remain unclear. We examined the human gut...
OBJECTIVES
Gut microbiota is a key component in obesity and type 2 diabetes, yet mechanisms and metabolites central to this interaction remain unclear. We examined the human gut microbiome's functional composition in healthy metabolic state and the most severe states of obesity and type 2 diabetes within the MetaCardis cohort. We focused on the role of B vitamins and B7/B8 biotin for regulation of host metabolic state, as these vitamins influence both microbial function and host metabolism and inflammation.
DESIGN
We performed metagenomic analyses in 1545 subjects from the MetaCardis cohorts and different murine experiments, including germ-free and antibiotic treated animals, faecal microbiota transfer, bariatric surgery and supplementation with biotin and prebiotics in mice.
RESULTS
Severe obesity is associated with an absolute deficiency in bacterial biotin producers and transporters, whose abundances correlate with host metabolic and inflammatory phenotypes. We found suboptimal circulating biotin levels in severe obesity and altered expression of biotin-associated genes in human adipose tissue. In mice, the absence or depletion of gut microbiota by antibiotics confirmed the microbial contribution to host biotin levels. Bariatric surgery, which improves metabolism and inflammation, associates with increased bacterial biotin producers and improved host systemic biotin in humans and mice. Finally, supplementing high-fat diet-fed mice with fructo-oligosaccharides and biotin improves not only the microbiome diversity, but also the potential of bacterial production of biotin and B vitamins, while limiting weight gain and glycaemic deterioration.
CONCLUSION
Strategies combining biotin and prebiotic supplementation could help prevent the deterioration of metabolic states in severe obesity.
TRIAL REGISTRATION NUMBER
NCT02059538.
Topics: Humans; Mice; Animals; Prebiotics; Gastrointestinal Microbiome; Obesity, Morbid; Biotin; Diabetes Mellitus, Type 2; Vitamin B Complex; Mice, Inbred C57BL; Obesity; Inflammation
PubMed: 35017197
DOI: 10.1136/gutjnl-2021-325753 -
The Plant Cell Mar 2023Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically...
Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.
Topics: Arabidopsis; Arabidopsis Proteins; Biotin; Biotinylation; Brassinosteroids; Phosphorylation; Protein Kinases; Proteomics; Signal Transduction
PubMed: 36660928
DOI: 10.1093/plcell/koad013 -
The Plant Cell Nov 2020Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase...
Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.
Topics: Arabidopsis; Biotin; Biotinylation; Carbon-Nitrogen Ligases; Cell Membrane; Escherichia coli Proteins; Green Fluorescent Proteins; Lotus; Solanum lycopersicum; Plant Proteins; Plants, Genetically Modified; Protein Interaction Maps; Protein Subunits; Recombinant Proteins; Repressor Proteins; Temperature; Nicotiana
PubMed: 32843435
DOI: 10.1105/tpc.20.00235 -
Nature Communications May 2022Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We...
Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states.
Topics: Animals; Astrocytes; Biotin; Biotinylation; Brain; Mice; Neurons; Proteome; Proteomics
PubMed: 35614064
DOI: 10.1038/s41467-022-30623-x -
ELife May 2020Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a...
Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4 complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.
Topics: Biotin; Biotinylation; Carbon-Nitrogen Ligases; Cell Survival; Escherichia coli; Escherichia coli Proteins; HEK293 Cells; Humans; Mutation; Protein Engineering; Protein Interaction Mapping; Protein Interaction Maps; Recombinant Fusion Proteins; Repressor Proteins
PubMed: 32391793
DOI: 10.7554/eLife.54983