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Accounts of Chemical Research Sep 2021Over the past decade, we have contributed to the chemistry of microbial natural products and synthetic ligands, related to riboflavin and uracils, that modulate immune...
Over the past decade, we have contributed to the chemistry of microbial natural products and synthetic ligands, related to riboflavin and uracils, that modulate immune cells called mucosal associated invariant T cells (MAIT cells). These highly abundant T lymphocytes were only discovered in 2003 and have become recognized for their importance in mammalian immunology. Unlike other T cells, MAIT cells are not activated by peptide or lipid antigens. In collaboration with immunology and structural biology research groups, we discovered that they are instead activated by unstable nitrogen-containing heterocycles synthesized by bacteria. The most potent naturally occurring activating compound (antigen) is -(2-oxopropylideneamino)-d-ribitylaminouracil (5-OP-RU). This compound is an imine (Schiff base) formed through condensation between an intermediate in the biosynthesis of riboflavin (vitamin B2) and a metabolic byproduct of mammalian and microbial glycolysis. Although it is very unstable in water due to intramolecular ring closure or hydrolysis, we were able to develop a non-enzymatic synthesis that yields a pure kinetically stable compound in a nonaqueous solvent. This compound has revolutionized the study of MAIT cell immunology due to its potent activation (EC = 2 pM) of MAIT cells and its development into immunological reagents for detecting and characterizing MAIT cells in tissues. MAIT cells are now linked to key physiological processes and disease, including antibacterial defense, tissue repair, regulation of graft--host disease, gastritis, inflammatory bowel diseases, and cancer. 5-OP-RU activates MAIT cells and, like a vaccine, has been shown to protect mice from bacterial infections and cancers. Mechanistic studies on the binding of 5-OP-RU to its dual protein targets, the major histocompatibility complex class I related protein (MR1) and the MAIT cell receptor (MAIT TCR), have involved synthetic chemistry, 2D H NMR spectroscopy, mass spectrometry, computer modeling and molecular dynamics simulations, biochemical, cellular, and immunological assays, and protein structural biology. These combined studies have revealed structural influences for 5-OP-RU in solution on protein binding and antigen presentation and potency; informed the development of potent (EC = 2 nM) and water stable analogues; led to fluorescent analogues for detecting and tracking binding proteins in and on cells; and enabled discovery of drugs and drug-like molecules that bind MR1 and modulate MAIT cell function. MAIT cells offer new opportunities for chemical synthesis to enhance the stability, potency, selectivity, and bioavailability of small molecule ligands for MR1 or MAIT TCR proteins, and to contribute to the understanding of T cell immunity and the development of prospective new immunomodulating medicines.
Topics: Animals; Antigens; Folic Acid; Humans; Molecular Structure; Mucosal-Associated Invariant T Cells; Protein Folding; Riboflavin; Structure-Activity Relationship
PubMed: 34415738
DOI: 10.1021/acs.accounts.1c00359 -
Molecular Medicine (Cambridge, Mass.) Oct 2023High doses of oral thiamine improve clinical fatigue scores in patients with quiescent inflammatory bowel disease (IBD) and chronic fatigue. In this study we analysed... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
High doses of oral thiamine improve clinical fatigue scores in patients with quiescent inflammatory bowel disease (IBD) and chronic fatigue. In this study we analysed plasma samples obtained in a randomised clinical trial and aimed compare levels of vitamins B1, B2, B3 and B6, and their related vitamers and metabolites in patients with IBD, with or without chronic fatigue and with or without effect of high dose oral thiamine for chronic fatigue.
METHODS
Blood samples from patients with fatigue were drawn prior and after thiamine exposure and only once for patients without fatigue. A wide panel of analysis were done at Bevital AS Lab.
RESULTS
Concentration of flavin mononucleotide (FMN) was lower in patients with chronic fatigue compared to patients without fatigue (p = 0.02). Patients with chronic fatigue who reported a positive effect on fatigue after 4 weeks of high dose thiamine treatment had a statistically significantly lower level of riboflavin after thiamine treatment (p = 0.01).
CONCLUSION
FMN and Riboflavin were associated with chronic fatigue in patients with quiescent IBD. Levels of other B vitamins and metabolites were not significantly different between the investigated groups or related to effect of the thiamine intervention.
CLINICAL TRIAL REGISTRATION
ClinicalTrials.gov study identifier NCT036347359. Registered 15 August 2018, https://clinicaltrials.gov/study/NCT03634735?cond=Inflammatory%20Bowel%20Diseases&intr=Thiamine&rank=1.
Topics: Humans; Vitamin B Complex; Thiamine; Fatigue Syndrome, Chronic; Riboflavin; Inflammatory Bowel Diseases
PubMed: 37880581
DOI: 10.1186/s10020-023-00741-3 -
Genes Jul 2023Riboflavin transporter 1 (RFVT1) deficiency is an ultrarare metabolic disorder due to autosomal dominant pathogenic variants in . The RFVT1 protein is mainly expressed...
Riboflavin transporter 1 (RFVT1) deficiency is an ultrarare metabolic disorder due to autosomal dominant pathogenic variants in . The RFVT1 protein is mainly expressed in the placenta and intestine. To our knowledge, only five cases of RFVT1 deficiency from three families have been reported so far. While newborns and infants with variants mainly showed a multiple acyl-CoA dehydrogenase deficiency-like presentation, individuals identified in adulthood were usually clinically asymptomatic. We report two patients with novel heterozygous variants. Patient 1 presented at the age of 62 with mild hyperammonemia following gastroenteritis. An acylcarnitine analysis in dried blood spots was abnormal with a multiple acyl-CoA dehydrogenase deficiency-like pattern, and genetic analysis confirmed a heterozygous variant, c.68C > A, p. Ser23Tyr. Patient 2 presented with recurrent seizures and hypsarrhythmia at the age of 7 months. Metabolic investigations yielded unremarkable results. However, whole exome sequencing revealed a heterozygous start loss variant, c.3G > A, p. Met1Ile in These two cases expand the clinical spectrum of riboflavin transporter 1 deficiency and demonstrate that symptomatic presentation in adulthood is possible.
Topics: Female; Humans; Infant; Infant, Newborn; Pregnancy; Heterozygote; Multiple Acyl Coenzyme A Dehydrogenase Deficiency; Receptors, G-Protein-Coupled; Riboflavin; Membrane Transport Proteins
PubMed: 37510312
DOI: 10.3390/genes14071408 -
Bioorganic & Medicinal Chemistry Letters Sep 2021The riboflavin biosynthetic pathway is a promising target for the development of novel antimycobacterial drugs given the lack of riboflavin transporter in M....
The riboflavin biosynthetic pathway is a promising target for the development of novel antimycobacterial drugs given the lack of riboflavin transporter in M. tuberculosis. Herein, a series of riboflavin derivatives was designed, synthesized and screened for their antimycobacterial and antibacterial activity. The compounds 1a, 1b, 2a, 3a and 5a displayed noticeable antitubercular activity against M. tuberculosis with minimum inhibitory concentration (MIC) in the range of 6.25 to 25 μM. The lead compound 5a had a selectivity index of 10.7 in the present study. The compounds 2a, 2b, 2c, 4c and 4d showed relatively low to moderate antibacterial activity (MIC = 100-200 μM) against gram-positive strains. Notably, the compounds do not show any inhibition against gram-negative strains even at 200 μM concentration. Further, molecular docking and binding experiments with representative flavin mononucleotide (FMN) riboswitch suggested that the riboflavin analogs exhibited antimycobacterial activity plausibly through FMN riboswitch-mediated repression of riboflavin biosynthesis. In addition to FMN riboswitch, flavoproteins involved in the flavin biosynthesis could also be target of riboflavin derivatives. In conclusion, the potency and low toxicity of riboflavin analogs particularly 5a (MIC = 6.25) make it a lead compound for the synthesis of new analogs for antimycobacterial therapy.
Topics: Antitubercular Agents; Dose-Response Relationship, Drug; Microbial Sensitivity Tests; Molecular Structure; Mycobacterium tuberculosis; Riboflavin; Structure-Activity Relationship
PubMed: 34242760
DOI: 10.1016/j.bmcl.2021.128236 -
International Urogynecology Journal Sep 2022To determine if administration of a standard 400 mg oral dose of riboflavin (vitamin B2) was comparable to phenazopyridine (pyridium) for evaluating ease of... (Randomized Controlled Trial)
Randomized Controlled Trial
INTRODUCTION AND HYPOTHESIS
To determine if administration of a standard 400 mg oral dose of riboflavin (vitamin B2) was comparable to phenazopyridine (pyridium) for evaluating ease of visualization of ureteral jets at the time of cystoscopy.
METHODS
A three-arm double-blinded, randomized controlled study was performed consisting of thiamine as placebo, phenazopyridine, and riboflavin. Agents were administered the morning of surgery prior to surgical procedure. The primary outcome was the ease of visualization of the ureteral jets based on a grading of urine stain intensity on a 7-point color scale, where 1-2 were minimal yellow staining, 3-4 were moderate yellow staining, and 5-7 defined as intense yellow staining. Analysis of covariance (ANCOVA) was used with pairwise comparison to characterize urine stain intensity as a continuous variable among the three groups controlling for age, BMI, creatinine, and time from ingestion of medication to first cystoscopy.
RESULTS
Eighty-four subjects were randomized with a mean ± SD age of 46.25 + 11.36 and BMI of 32.46 + 6.59. Riboflavin did have moderate or intense staining in 57% of cases; however, there was no significant difference between urine staining intensity compared to placebo (p = 0.21). There was a statistically significant increased urine staining intensity for phenazopyridine compared to placebo (p = 0.001) and for phenazopyridine compared to riboflavin (p = 0.001).
CONCLUSIONS
Phenazopyridine provided statistically significantly greater urine staining compared to both riboflavin and placebo and should be considered primarily for ease of ureteral jet visualization.
Topics: Administration, Oral; Coloring Agents; Humans; Phenazopyridine; Riboflavin; Staining and Labeling; Ureter
PubMed: 34057543
DOI: 10.1007/s00192-021-04867-y -
Current Eye Research Apr 2022The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts.
PURPOSE/ AIM
The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts.
MATERIALS AND METHODS
The tenon tissue was harvested from a patient undergoing strabismus surgery. The human tenon fibroblast cell culture and isolation were performed according to the standard laboratory cell culturing protocol. The cells were divided into three groups: control, treatment with irradiated and non-irradiated riboflavin. There were five different concentrations (0.00156%, 0.003125%, 0.00625%, 0.0125%, 0.025%) in each group of riboflavin. The fibroblasts were treated with riboflavin and the cellular viability was assessed at 24-hour and 48-hour post treatment with MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide colorimetric assay. The absorbance values were analysed using Magellan microplate reader data analysis. A triplicate of readings was taken. The data were presented as mean ± standard deviation of the triplicates. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) analysis version 23.
RESULTS
Irradiated riboflavin caused a concentration-dependent cell death in human tenon fibroblast cell culture ( < .05). The antiproliferative difference between irradiated and non-irradiated riboflavin was significant up to 48 hours ( < .05). Post hoc multiple comparisons showed higher concentrations of irradiated riboflavin (0.0125% and 0.025%) caused more reduction in cellular viability in human tenon fibroblast cells ( < .05). The duration of treatment is not a causative factor in this study.
CONCLUSIONS
This pilot experiment demonstrated that irradiated riboflavin induced cell death in human tenon fibroblast culture in a concentration-dependent manner, but is not time-dependent. Further exploratory investigations should be performed to determine the mechanism of cell death. We postulate that apoptosis occurred in these irradiated riboflavin-treated cells.
Topics: Cell Survival; Cells, Cultured; Fibroblasts; Humans; Riboflavin; Tenon Capsule
PubMed: 34963422
DOI: 10.1080/02713683.2021.2011326 -
Microbial Cell Factories May 2023Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed...
BACKGROUND
Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria.
RESULTS
In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain.
CONCLUSION
The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.
Topics: Riboflavin; Acetolactate Synthase; Eremothecium; Flavoproteins; Mutation; Mitochondrial Proteins; Mitochondria; Reactive Oxygen Species; Amino Acids, Branched-Chain
PubMed: 37217979
DOI: 10.1186/s12934-023-02114-1 -
Proceedings of the National Academy of... Sep 2022Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon...
Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon (e.g., sugars) and nitrogen (e.g., ammonia) inputs are required. Gas-fixing microorganisms that can grow on H from renewable water splitting and gaseous CO and N offer a renewable path to overcoming these limitations but confront challenges owing to the scarcity of genetic engineering in such organisms. Here, we demonstrate that the hydrogen-oxidizing carbon- and nitrogen-fixing microorganism grown on a CO/N/H gas mixture can overproduce the vitamin riboflavin (vitamin B). We identify plasmids and promoters for use in this bacterium and employ a constitutive promoter to overexpress riboflavin pathway enzymes. Riboflavin production is quantified at 15 times that of the wild-type organism. We demonstrate that riboflavin overproduction is maintained when the bacterium is grown under hybrid inorganic-biological conditions, in which H from water splitting, along with CO and N, is fed to the bacterium, establishing the viability of the approach to sustainably produce food and nutrients.
Topics: Carbon Dioxide; Nitrogen; Riboflavin; Water; Xanthobacter
PubMed: 36067303
DOI: 10.1073/pnas.2210538119 -
Methods in Molecular Biology (Clifton,... 2021Riboflavin (vitamin B) is a vitamin of the B group involved in essential biological pathways, including redox reactions and the electron transport chain. Some lactic...
Riboflavin (vitamin B) is a vitamin of the B group involved in essential biological pathways, including redox reactions and the electron transport chain. Some lactic acid bacteria (LAB) can synthesize riboflavin and this capability is strain-dependent. In the last years, a growing interest has focused on the selection of riboflavin-overproducing food-grade LAB for the vitamin biofortification of fermented foods, as well as for the formulation of innovative functional products.In this chapter we report fast and inexpensive techniques in order to (1) screen LAB isolates able to produce riboflavin from different matrices, (2) select spontaneous roseoflavin-resistant riboflavin overproducing strains, and (3) quantify vitamin B in culture media by fluorescence detection.These protocols could be useful to select new overproducing strains and/or species from different ecological niches, as well as to optimize the conditions for vitamin bioproduction.
Topics: Bacteriological Techniques; Culture Media; Drug Resistance, Bacterial; Fermented Foods; Fluorescence; Lactobacillales; Riboflavin
PubMed: 33751425
DOI: 10.1007/978-1-0716-1286-6_1 -
Cornea Jul 2023The aim of this study was to assess changes in visual acuity after epithelium-on ("epi-on") corneal crosslinking after a diagnosis of keratoconus. (Randomized Controlled Trial)
Randomized Controlled Trial
PURPOSE
The aim of this study was to assess changes in visual acuity after epithelium-on ("epi-on") corneal crosslinking after a diagnosis of keratoconus.
METHODS
Subjects with corneal ectatic diseases were enrolled in a prospective, randomized, controlled, open-label, multicenter trial. Subjects were randomized to 1 of 3 treatment groups and treated with an epi-on crosslinking system including riboflavin/sodium iodide and pulsed UVA exposure (EpiSmart, CXL Ophthalmics, Encinitas, CA). The UVA treatment groups were 2.4 J/cm 2 over 20 minutes, 3.6 J/cm 2 over 20 minutes, and 3.6 J/cm 2 over 30 minutes. The primary end point was logarithm of the minimum angle of resolution corrected distance visual acuity (CDVA). Secondary end points were logarithm of the minimum angle of resolution uncorrected distance visual acuity (UCVA), maximum corneal curvature (Kmax), and minimum corneal thickness. Data were assessed 6 and 12 months post-operatively, using t -tests for differences from baseline.
RESULTS
Two thousand two hundred twenty-eight subjects were treated with epi-on crosslinking. One thousand nine hundred twenty-two subjects had a diagnosis of keratoconus; other treated eyes had postsurgical and other ectasias. At 6 and 12 months, the subjects with keratoconus demonstrated significant improvements in CDVA, UCVA, and Kmax; minimum corneal thickness was unchanged. One hundred ninety-five subjects (8.7%) reported at least 1 adverse event (AE). A mild corneal epithelial defect was reported in 31 cases (1.4%) and was the only AE reported in >1% of subjects. There were no serious AEs related to the treatment.
CONCLUSIONS
EpiSmart epi-on crosslinking resulted in mean improvements in CDVA, UCVA, and Kmax at both 6 and 12 months and an excellent safety and efficacy profile in subjects with keratoconus, with few significant side effects. Differences between UVA treatment groups were not significant.
Topics: Humans; Keratoconus; Photosensitizing Agents; Photochemotherapy; Prospective Studies; Ultraviolet Rays; Corneal Topography; Cross-Linking Reagents; Collagen; Riboflavin; Dilatation, Pathologic
PubMed: 36173242
DOI: 10.1097/ICO.0000000000003136