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Nature Protocols Aug 2019Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol...
Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA. Total RNA isolated from a tissue of interest is then sequenced with SLAMseq, which introduces thymine to cytosine (T>C) conversions at the sites of the incorporated 4-thiouracil. The resulting sequencing reads are then mapped with the T>C-aware alignment software, SLAM-DUNK, which allows mapping of reads containing T>C mismatches. The number of T>C conversions per transcript is further analyzed to identify which transcripts are synthesized in the UPRT-expressing cells. Thus, our method, SLAM-ITseq (SLAMseq in tissue), enables cell-specific transcriptomics without laborious FACS-based cell sorting or biochemical isolation of the labeled transcripts used in TU tagging. In the murine tissues we assessed previously, this method identified ~5,000 genes that are expressed in a cell type of interest from the total RNA pool from the tissue. Any laboratory with access to a high-throughput sequencer and high-power computing can adapt this protocol with ease, and the entire pipeline can be completed in <5 d.
Topics: Animals; Female; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Organ Specificity; Pentosyltransferases; Sequence Analysis, RNA; Thiouracil; Transcriptome
PubMed: 31243395
DOI: 10.1038/s41596-019-0179-x -
Different Oxidation Pathways of 2-Selenouracil and 2-Thiouracil, Natural Components of Transfer RNA.International Journal of Molecular... Aug 2020Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and...
Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and tuning of protein biosynthesis in all three domains of life. Both sulfur and selenium chalcogens functionally operate as key elements of biological molecules involved in the protection of cells against oxidative damage. In this work, 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura) were treated with hydrogen peroxide at 1:0.5, 1:1, and 1:10 molar ratios and at selected pH values ranging from 5 to 8. It was found that Se2Ura was more prone to oxidation than its sulfur analog, and if reacted with HO at a 1:1 or lower molar ratio, it predominantly produced diselenide Ura-Se-Se-Ura, which spontaneously transformed to a previously unknown Se-containing two-ring compound. Its deselenation furnished the major reaction product, a structure not related to any known biological species. Under the same conditions, only a small amount of S2Ura was oxidized to form Ura-SOH and uracil (Ura). In contrast, 10-fold excess hydrogen peroxide converted Se2Ura and S2Ura into corresponding Ura-SeOH and Ura-SOH intermediates, which decomposed with the release of selenium and sulfur oxide(s) to yield Ura as either a predominant or exclusive product, respectively. Our results confirmed significantly different oxidation pathways of 2-selenouracil and 2-thiouracil.
Topics: Hydrogen Peroxide; Oxidation-Reduction; Thiouracil; Uracil
PubMed: 32825053
DOI: 10.3390/ijms21175956 -
Physiology & Behavior Mar 2022Past research has identified distinct phenotypic differences in responses to sweet taste, although the origins of these differences remain unclear. One possibility is...
Past research has identified distinct phenotypic differences in responses to sweet taste, although the origins of these differences remain unclear. One possibility is that these individual differences in sweet-liking are a manifestation of the more widely known differences in sensitivity to the bitter tastant 6-n-propylthiouracil (PROP), which has been related to wider differences in food liking and preference. However, previous studies exploring the relationship between sweet-liking and PROP-tasting have had mixed outcomes. This is possibly due to older studies using a more simplistic dichotic characterisation of sweet likers, whereas recent research suggests three sweet-liking phenotypes (extreme sweet likers, ESL; moderate sweet likers, MSL; and sweet dislikers, SD). To re-assess how sweet-liking and PROP tasting are inter-related, 236 volunteers evaluated their liking for 1.0 M sucrose and the intensity of three concentrations of each NaCl and PROP. Using three different methods for classifying PROP taster status, our analysis confirmed that all three sweet-liking phenotypes were represented in all three PROP taster groups (super-tasters, ST; medium tasters, MT; and non-tasters, NT), but relatively few ESL were classified as ST, or SD as NT. Overall, these data suggest that while PROP tasting and sweet-liking are not causally related, the SD phenotype may partly be explained by a broader tendency for anhedonia.
Topics: Emotions; Food Preferences; Humans; Propylthiouracil; Sodium Chloride; Sucrose; Taste
PubMed: 35016967
DOI: 10.1016/j.physbeh.2022.113702 -
Journal of the National Medical... Jan 1991The case of a 43-year-old female with propylthiouracil-induced hepatitis is reported. The case is unique because the patient's liver function deteriorated 2 weeks after...
The case of a 43-year-old female with propylthiouracil-induced hepatitis is reported. The case is unique because the patient's liver function deteriorated 2 weeks after medication was discontinued.
Topics: Adult; Chemical and Drug Induced Liver Injury; Female; Graves Disease; Humans; Liver Function Tests; Propylthiouracil
PubMed: 1994070
DOI: No ID Found -
Annals of Human Biology 2001The ability to taste the bitter compound phenylthiocarbamide (PTC) and related chemicals is bimodal, and all human populations tested to date contain some people who can... (Review)
Review
The ability to taste the bitter compound phenylthiocarbamide (PTC) and related chemicals is bimodal, and all human populations tested to date contain some people who can and some people who cannot taste PTC. Why this trait has been maintained in the population is uncertain but this polymorphism may influence food selection, nutritional status or thyroid metabolism. The gene product that gives rise to this phenotype is unknown, and its characterization would provide insight into the mechanism of bitter taste perception. Although this trait is often considered a simple Mendelian trait, i.e. one gene two alleles, a recent linkage study found a major locus on chromosome 5p15 and evidence for an additional locus on chromosome 7. The development of methods to identify these genes will provide a good stepping-stone between single-gene disorders and polygenic traits.
Topics: Antimetabolites; Genetics, Population; Humans; Phenotype; Phenylcarbamates; Propylthiouracil; Structure-Activity Relationship; Taste; Thiocarbamates
PubMed: 11293722
DOI: 10.1080/03014460151056310 -
International Journal of Paediatric... Mar 2022PROP test (6-n-propylthiouracil) for the identification of genetic sensitivity to caries in young individuals has emerged as a useful tool for caries risk assessment. (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
PROP test (6-n-propylthiouracil) for the identification of genetic sensitivity to caries in young individuals has emerged as a useful tool for caries risk assessment.
AIM
To systematically appraise available evidence on the association between genetic taste sensitivity, as detected by (PROP), and caries.
DESIGN
Seven databases, as of March 2020, were searched. Search terms included 'caries', 'taste predisposition', 'PROP'. Risk of bias assessment was performed using ROBINS-I tool, and the quality of evidence was assessed with GRADE. Random-effects meta-analyses were conducted to synthesize data, and pooled effects were estimated through standardized mean differences (SMDs) and associated confidence Intervals (95% CIs).
RESULTS
Of 92 articles initially retrieved, 12 were eligible for inclusion. Seven contributed to the meta-analyses. All were cross-sectional studies, with moderate-to-serious risk of bias. The non-tasters of PROP exhibited a significantly higher value for the DMFT compared with tasters (SMD: 1.23; 95% CI: 0.90, 1.56; P < .001), whereas the association for the DMFS was SMD: 1.34; 95% CI: 0.66, 2.01; P < .001 (non-tasters versus super-tasters). The quality of evidence was very low overall.
CONCLUSIONS
Within the limitations of this study, non-tasters to PROP exhibited higher caries experience, with subsequent clinical implications for follow-up and management of the 'high-susceptibility' individuals.
Topics: Adolescent; Child; Dental Caries; Humans; Propylthiouracil; Taste
PubMed: 34080244
DOI: 10.1111/ipd.12845 -
Methods in Molecular Biology (Clifton,... 2022RNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to...
RNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to environmental changes. Accordingly, analyzing RNA expression remains an important step to understand the molecular basis controlling the formation of cellular phenotypes, cellular homeostasis or disease progression. Steady-state RNA levels in the cells are controlled by the sum of highly dynamic molecular processes contributing to RNA expression and can be classified in transcription, maturation and degradation. The main goal of analyzing RNA dynamics is to disentangle the individual contribution of these molecular processes to the life cycle of a given RNA under different physiological conditions. In the recent years, the use of nonradioactive nucleotide/nucleoside analogs and improved chemistry, in combination with time-dependent and high-throughput analysis, have greatly expanded our understanding of RNA metabolism across various cell types, organisms, and growth conditions.In this chapter, we describe a step-by-step protocol allowing pulse labeling of RNA with the nonradioactive nucleotide analog, 4-thiouracil , in the eukaryotic model organism Saccharomyces cerevisiae and the model archaeon Haloferax volcanii .
Topics: Haloferax volcanii; Nucleotides; RNA; Saccharomyces cerevisiae; Thiouracil
PubMed: 35796990
DOI: 10.1007/978-1-0716-2501-9_12 -
Physiology & Behavior Dec 1994Taste worlds of humans vary because of taste blindness to phenylthiocarbamide (PTC) and its chemical relative, 6-n-propylthiouracil (PROP). We review early PTC studies... (Review)
Review
Taste worlds of humans vary because of taste blindness to phenylthiocarbamide (PTC) and its chemical relative, 6-n-propylthiouracil (PROP). We review early PTC studies and apply modern statistical analyses to show that a higher frequency of women tasted PTC crystals, and were tasters (threshold classification). In our laboratory, scaling of PROP bitterness led to the identification of a subset of tasters (supertasters) who rate PROP as intensely bitter. Supertasters also perceive stronger tastes from a variety of bitter and sweet substances, and perceive more burn from oral irritants (alcohol and capsaicin). The density of taste receptors on the anterior tongue (fungiform papillae, taste buds) correlate significantly with perceived bitterness of PROP and support the supertaster concept. Psychophysical data from studies in our laboratory also show a sex effect; women are supertasters more frequently. The anatomical data also support the sex difference; women have more fungiform papillae and more taste buds. Future investigations of PTC/PROP tasting and food behaviors should include scaling to identify supertasters and separate sex effects.
Topics: Female; Humans; Male; Phenylthiourea; Propylthiouracil; Psychophysics; Sex Factors; Taste; Taste Buds; Taste Threshold
PubMed: 7878086
DOI: 10.1016/0031-9384(94)90361-1 -
International Journal of Molecular... Jul 2019The molecular structure of 2-thiouracil, 4-thiouracil and 2,4-dithiouracil was analyzed under the effect of the first and second hydration shell by using the B3LYP...
The molecular structure of 2-thiouracil, 4-thiouracil and 2,4-dithiouracil was analyzed under the effect of the first and second hydration shell by using the B3LYP density functional (DFT) method, and the results were compared to those obtained for the uracil molecule. A slight difference in the water distribution appears in these molecules. On the hydration of these molecules several trends in bond lengths and atomic charges were established. The ring in uracil molecule appears easier to be deformed and adapted to different environments as compared to that when it is thio-substituted. Molecular docking calculations of 2-thiouracil against three different pathogens: , and were carried out. Docking calculations of 2,4-dithiouracil ligand with various targeted proteins were also performed. Different DNA: RNA hybrid microhelixes with uridine, 2-thiouridine, 4-thiouridine and 2,4-dithiouridine nucleosides were optimized in a simple model with three nucleotide base pairs. Two main types of microhelixes were analyzed in detail depending on the intramolecular H-bond of the 2'-OH group. The weaker Watson-Crick (WC) base pair formed with thio-substituted uracil than with unsubstituted ones slightly deforms the helical and backbone parameters, especially with 2,4-dithiouridine. However, the thio-substitution significantly increases the dipole moment of the A-type microhelixes, as well as the rise and propeller twist parameters.
Topics: Animals; Anti-Infective Agents; Bacillus subtilis; Candida albicans; DNA; Escherichia coli; Humans; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Protein Binding; RNA; Thiouracil
PubMed: 31311161
DOI: 10.3390/ijms20143477 -
Pediatric Research Oct 2022Although less frequent than in adults, taste loss also occurs in childhood. "Taste Strips" are frequently used for diagnosing taste dysfunction; however, normative...
BACKGROUND
Although less frequent than in adults, taste loss also occurs in childhood. "Taste Strips" are frequently used for diagnosing taste dysfunction; however, normative values are lacking for children. In this study, we will create normative values for the "Taste Strips" in children.
METHODS
This cross-sectional study included 609 children aged 6-15 years. "Taste Strips" were used to determine sweet, sour, salty, and bitter taste scores by a non-forced procedure. The 10th percentile was used to distinguish normal taste function from a reduced sense of taste. Multivariable generalized linear models (GLM) were estimated to study the effect of age (group), sex, and 6-n-propylthiouracil (PROP) status on taste function.
RESULTS
Taste function changed with age, allowing for a distinction of three age groups: (I) 6-7 years, (II) 8-9 years, and (III) 10-15 years. Normative values were created for the age groups and boys and girls separately. Additionally, GLM showed a significant effect of (1) age (group) on sweet, salty, bitter, and total taste scores; (2) sex on sweet, sour, and total taste scores; and (3) PROP status on total taste scores.
CONCLUSIONS
This study provided normative values for the "Taste Strips" in children, highlighting age- and sex-related differences.
IMPACT
Taste dysfunction can be harmful and impacts quality of life, a topic that became increasingly important since the COVID-19 pandemic. Although taste dysfunction is thought to be rare in childhood, the detrimental impact of such dysfunction might be large, as children's eating habits are strongly influenced by input from the chemical senses. Measuring taste function may elucidate the relationship between taste dysfunction and disease, fostering the development of more appropriate supportive strategies. However, adequate tools are lacking for children. Normative values of the "Taste Strips" are now available for children, which bolster the clinical utility of this test.
Topics: Adult; Male; Child; Female; Humans; Taste; Propylthiouracil; Cross-Sectional Studies; Quality of Life; Pandemics; COVID-19; Taste Disorders
PubMed: 34963699
DOI: 10.1038/s41390-021-01920-w