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Microbial Cell Factories Aug 2019Roseoflavin, a promising broad-spectrum antibiotic, is naturally produced by the bacteria Streptomyces davaonensis and Streptomyces cinnabarinus. The key enzymes...
BACKGROUND
Roseoflavin, a promising broad-spectrum antibiotic, is naturally produced by the bacteria Streptomyces davaonensis and Streptomyces cinnabarinus. The key enzymes responsible for roseoflavin biosynthesis and the corresponding genes were recently identified. In this study we aimed to enhance roseoflavin production in S. davaonensis and to synthesize roseoflavin in the heterologous hosts Bacillus subtilis and Corynebacterium glutamicum by (over)expression of the roseoflavin biosynthesis genes.
RESULTS
While expression of the roseoflavin biosynthesis genes from S. davaonensis was not observed in recombinant strains of B. subtilis, overexpression was successful in C. glutamicum and S. davaonensis. Under the culture conditions tested, a maximum of 1.6 ± 0.2 µM (ca. 0.7 mg/l) and 34.9 ± 5.2 µM (ca. 14 mg/l) roseoflavin was produced with recombinant strains of C. glutamicum and S. davaonensis, respectively. In S. davaonensis the roseoflavin yield was increased by 78%.
CONCLUSIONS
The results of this study provide a sound basis for the development of an economical roseoflavin production process.
Topics: Anti-Bacterial Agents; Bacillus subtilis; Corynebacterium glutamicum; Metabolic Engineering; Riboflavin; Streptomyces
PubMed: 31451111
DOI: 10.1186/s12934-019-1181-2 -
Veterinary Medicine and Science Jul 2022This study was conducted to evaluate the effect of adding riboflavin to boar sperm freezing extender on the challenge of cryopreservation.
OBJECTIVES
This study was conducted to evaluate the effect of adding riboflavin to boar sperm freezing extender on the challenge of cryopreservation.
METHODS
Different concentrations (0, 5, 10, 15, 20 or 25 μM) of riboflavin were added to the freezing extender. Spermatozoa motility, membrane integrity, acrosomal integrity, mitochondrial membrane potential and enzyme activities were analysed once 10 min after thawing. Q-PCR was used to detect the mRNA expression of Caspase3, Bcl-2 and Bax.
RESULTS
The results showed that the addition of 10 μM riboflavin to boar sperm freezing extender significantly increased the frozen-thawed sperm progressive motility compared with the control group (p < 0.05). Activities of superoxide dismutase, glutathione peroxidase and catalase improved after adding riboflavin to the extender (p < 0.05). During freezing-thawing, the boar sperm mitochondrial membrane potential, acrosomal integrity, plasma membrane and DNA at 10 μM in the riboflavin group increased by 6.6%, 9.6%, 5.49% and 5.62% (p < 0.05), respectively, compared with the control group. The addition of 10 μM riboflavin to the extender significantly decreased the malondialdehyde (p < 0.05) content, whereas it increased the ATP content (p < 0.05) of boar sperm during freezing-thawing. Furthermore, the expression of Caspase-3 and Bax (p < 0.05) were significantly lower, whereas the expression of BCL-2 (p < 0.05) was greater than the control group when adding 10 μM riboflavin to the extender.
CONCLUSIONS
Riboflavin showed cryoprotective capacity to the freezing extender used for boar sperm during the process of freezing-thawing, and the optimal concentration of riboflavin for the frozen extender was 10 μM.
Topics: Animals; Antioxidants; Cryopreservation; Cryoprotective Agents; Male; Riboflavin; Semen Preservation; Sperm Motility; Spermatozoa; Swine; bcl-2-Associated X Protein
PubMed: 35561277
DOI: 10.1002/vms3.833 -
The Cochrane Database of Systematic... Mar 2015Keratoconus is a condition of the eye that affects approximately 1 in 2000 people. The disease leads to a gradual increase in corneal curvature and decrease in visual... (Review)
Review
BACKGROUND
Keratoconus is a condition of the eye that affects approximately 1 in 2000 people. The disease leads to a gradual increase in corneal curvature and decrease in visual acuity with consequent impact on quality of life. Collagen cross-linking (CXL) with ultraviolet A (UVA) light and riboflavin (vitamin B2) is a relatively new treatment that has been reported to slow or halt the progression of the disease in its early stages.
OBJECTIVES
The objective of this review was to assess whether there is evidence that CXL is an effective and safe treatment for halting the progression of keratoconus compared to no treatment.
SEARCH METHODS
We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2014, Issue 7), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to August 2014), EMBASE (January 1980 to August 2014), Latin American and Caribbean Health Sciences Literature Database (LILACS) (1982 to August 2014), Cumulative Index to Nursing and Allied Health Literature (CINAHL) (1982 to August 2014), OpenGrey (System for Information on Grey Literature in Europe) (www.opengrey.eu/), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organisation International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We used no date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 28 August 2014.
SELECTION CRITERIA
We included randomised controlled trials (RCTs) where CXL with UVA light and riboflavin was used to treat people with keratoconus and was compared to no treatment.
DATA COLLECTION AND ANALYSIS
Two review authors independently screened the search results, assessed trial quality, and extracted data using standard methodological procedures expected by Cochrane. Our primary outcomes were two indicators of progression at 12 months: increase in maximum keratometry of 1.5 dioptres (D) or more and deterioration in uncorrected visual acuity of more than 0.2 logMAR.
MAIN RESULTS
We included three RCTs conducted in Australia, the United Kingdom, and the United States that enrolled a total of 225 eyes and analysed 219 eyes. The total number of people enrolled was not clear in two of the studies. Only adults were enrolled into these studies. Out of the eyes analysed, 119 had CXL (all using the epithelium-off technique) and 100 served as controls. One of these studies only reported comparative data on review outcomes. All three studies were at high risk for performance bias (lack of masking), detection bias (only one trial attempted to mask outcome assessment), and attrition bias (incomplete follow-up). It was not possible to pool data due to differences in measuring and reporting outcomes. We identified a further three unpublished trials that potentially had enrolled a total of 195 participants.There was limited evidence on the risk of progression. Analysis of the first few participants followed up to one year in one study suggested that eyes given CXL were less likely to have an increase in maximum keratometry of 1.5 D or more at 12 months compared to eyes given no treatment, but the confidence intervals (CI) were wide and compatible with no effect or more progression in the CXL group (risk ratio (RR) 0.12, 95% CI 0.01 to 2.00, 19 eyes). The same study reported the number of eyes with an increase of 2 D or more at 36 months in the whole cohort with a RR of 0.03 favouring CXL (95% CI 0.00 to 0.43, 94 eyes). Another study reported "progression" at 18 months using a different definition; people receiving CXL were less likely to progress, but again the effect was uncertain (RR 0.14, 95% CI 0.01 to 2.61, 44 eyes). We judged this to be very low-quality evidence due to the risk of bias of included studies, imprecision, indirectness and publication bias but noted that the size of the potential effect was large.On average, treated eyes had a less steep cornea (approximately 2 D less steep) (mean difference (MD) -1.92, 95% CI -2.54 to -1.30, 94 eyes, 1 RCT, very low-quality evidence) and better uncorrected visual acuity (approximately 2 lines or 10 letters better) (MD -0.20, 95% CI -0.31 to -0.09, 94 eyes, 1 RCT, very low-quality evidence) at 12 months. None of the studies reported loss of 0.2 logMAR acuity. The data on corneal thickness were inconsistent. There were no data available on quality of life or costs. Adverse effects were not uncommon but mostly transient and of low clinical significance. In one trial, 3 out of 12 participants treated with CXL had an adverse effect including corneal oedema, anterior chamber inflammation, and recurrent corneal erosions. In one trial at 3 years 3 out of 50 participants experienced adverse events including mild diffuse corneal oedema and paracentral infiltrate, peripheral corneal vascularisation, and subepithelial infiltrates and anterior chamber inflammation. No adverse effects were reported in the control groups.
AUTHORS' CONCLUSIONS
The evidence for the use of CXL in the management of keratoconus is limited due the lack of properly conducted RCTs.
Topics: Adult; Collagen; Confidence Intervals; Cross-Linking Reagents; Disease Progression; Humans; Keratoconus; Photosensitizing Agents; Randomized Controlled Trials as Topic; Riboflavin; Ultraviolet Therapy
PubMed: 25803325
DOI: 10.1002/14651858.CD010621.pub2 -
International Journal of Molecular... Dec 2022is a dangerous hospital pathogen primarily due to its ability to form biofilms on different abiotic and biotic surfaces. The present study investigated the effect of...
is a dangerous hospital pathogen primarily due to its ability to form biofilms on different abiotic and biotic surfaces. The present study investigated the effect of riboflavin- and chlorophyllin-based antimicrobial photodynamic therapy, performed with near-ultraviolet or blue light on the viability of bacterial cells in biofilms and their structural stability, also determining the extent of photoinduced generation of intracellular reactive oxygen species as well as the ability of to form biofilms after the treatment. The efficacy of antimicrobial photodynamic therapy was compared with that of light alone and the role of the photosensitizer type on the photosensitization mechanism was demonstrated. We found that the antibacterial effect of riboflavin-based antimicrobial photodynamic therapy depends on the ability of photoactivated riboflavin to generate intracellular reactive oxygen species but does not depend on the concentration of riboflavin and pre-incubation time before irradiation. Moreover, our results suggest a clear interconnection between the inactivation efficiency of chlorophyllin-based antimicrobial photodynamic therapy and the sensitivity of biofilms to used light. In summary, all the analyzed results suggest that riboflavin-based antimicrobial photodynamic therapy and chlorophyllin-based antimicrobial photodynamic therapy have the potential to be applied as an antibacterial treatment against biofilms or as a preventive measure against biofilm formation.
Topics: Acinetobacter baumannii; Reactive Oxygen Species; Anti-Infective Agents; Anti-Bacterial Agents; Photochemotherapy; Biofilms; Riboflavin
PubMed: 36614160
DOI: 10.3390/ijms24010722 -
PloS One 2015Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions...
Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria.
Topics: Bacteria; Bacterial Proteins; Biosynthetic Pathways; Evolution, Molecular; Flavin Mononucleotide; Genome, Bacterial; Membrane Transport Proteins; Phylogeny; Riboflavin; Riboswitch
PubMed: 25938806
DOI: 10.1371/journal.pone.0126124 -
Microbial Cell Factories Aug 2022Riboflavin is a precursor of FMN and FAD which act as coenzymes of numerous enzymes. Riboflavin is an important biotechnological commodity with annual market sales...
BACKGROUND
Riboflavin is a precursor of FMN and FAD which act as coenzymes of numerous enzymes. Riboflavin is an important biotechnological commodity with annual market sales exceeding nine billion US dollars. It is used primarily as a component of feed premixes, a food colorant, a component of multivitamin mixtures and medicines. Currently, industrial riboflavin production uses the bacterium, Bacillus subtilis, and the filamentous fungus, Ashbya gossypii, and utilizes glucose and/or oils as carbon substrates.
RESULTS
We studied riboflavin biosynthesis in the flavinogenic yeast Candida famata that is a genetically stable riboflavin overproducer. Here it was found that the wild type C. famata is characterized by robust growth on lactose and cheese whey and the engineered strains also overproduce riboflavin on whey. The riboflavin synthesis on whey was close to that obtained on glucose. To further enhance riboflavin production on whey, the gene of the transcription activator SEF1 was expressed under control of the lactose-induced promoter of the native β-galactosidase gene LAC4. These transformants produced elevated amounts of riboflavin on lactose and especially on whey. The strain with additional overexpression of gene RIB6 involved in conversion of ribulose-5-phosphate to riboflavin precursor had the highest titer of accumulated riboflavin in flasks during cultivation on whey. Activation of riboflavin synthesis was also obtained after overexpression of the GND1 gene that is involved in the synthesis of the riboflavin precursor ribulose-5-phosphate. The best engineered strains accumulated 2.5 g of riboflavin/L on whey supplemented only with (NH)SO during batch cultivation in bioreactor with high yield (more than 300 mg/g dry cell weight). The use of concentrated whey inhibited growth of wild-type and engineered strains of C. famata, so the mutants tolerant to concentrated whey were isolated.
CONCLUSIONS
Our data show that the waste of dairy industry is a promising substrate for riboflavin production by C. famata. Possibilities for using the engineered strains of C. famata to produce high-value commodity (riboflavin) from whey are discussed.
Topics: Candida; Cheese; Flavin Mononucleotide; Glucose; Lactose; Phosphates; Riboflavin; Whey
PubMed: 35964025
DOI: 10.1186/s12934-022-01888-0 -
Graefe's Archive For Clinical and... May 2021To analyze the effect of riboflavin UV-A illumination on mRNA and protein expression of healthy (HCFs) and keratoconus human corneal fibroblasts (KC-HCFs), concerning...
PURPOSE
To analyze the effect of riboflavin UV-A illumination on mRNA and protein expression of healthy (HCFs) and keratoconus human corneal fibroblasts (KC-HCFs), concerning the inflammatory markers NF-κB, iNOS, IL-6, and collagen 1 and 5 (Col 1/Col 5).
METHODS
Keratocytes were isolated from healthy (n = 3) and keratoconus (KC) corneas (n = 3) and were cultivated in basal medium with 5% fetal calf serum, which resulted in their transformation into human corneal fibroblasts (HCFs/KC-HCFs). Cells underwent 0.1% riboflavin UV-A illumination for 250 s (CXL). NF-κB, iNOS, IL-6, Col 1, and Col 5 expression was investigated by qPCR and Western blot analysis. IL-6 concentration of the cell culture supernatant and cell lysate was determined by ELISA.
RESULTS
In untreated KC-HCFs, NF-κB (p = 0.0002), iNOS (p = 0.0019), Col 1 (p = 0.0286), and Col 5 (p = 0.0054) mRNA expression was higher and IL-6 expression was lower (p = 0.0057), than in healthy controls. In HCFs, CXL led to an increased NF-κB (p = 0.0286) and IL-6 (p = 0.0057) mRNA expression. The IL-6 concentration in the cell culture supernatant was increased in HCFs (p = 0.0485) and KC-HCFs (p = 0.0485) after CXL. CXL increased intracellular IL-6 concentration only in KC-HCFs (p = 0.0357). In the HCF group (p = 0.0286), an increased Col 1 mRNA expression after CXL could be observed.
CONCLUSION
Our study confirmed altered gene expression in untreated KC-HCFs compared to untreated HCFs. Riboflavin UV-A illumination affected gene expression only in HCFs. Increased IL-6 concentration in the cell culture supernatant and cell lysate indicate a secondary inflammatory response of HCFs and KC-HCFs to riboflavin UV-A illumination.
Topics: Collagen; Cornea; Cross-Linking Reagents; Fibroblasts; Humans; Interleukin-6; Keratoconus; Lighting; Photochemotherapy; Photosensitizing Agents; Riboflavin; Ultraviolet Rays
PubMed: 33443628
DOI: 10.1007/s00417-020-05058-z -
ACS Applied Bio Materials May 2023Bioengineered corneal tissue is a promising therapeutic modality for the treatment of corneal blindness as a substitute for cadaveric graft tissue. In this study, we...
Bioengineered corneal tissue is a promising therapeutic modality for the treatment of corneal blindness as a substitute for cadaveric graft tissue. In this study, we fabricated a collagen gel using ultraviolet-A (UV-A) light and riboflavin as a photosensitizer (PhotoCol-RB) as an -forming matrix to fill corneal wounds and create a cohesive interface between the crosslinked gel and adjacent collagen. The PhotoCol-RB gels supported corneal epithelialization and exhibited higher transparency compared to physically crosslinked collagen. We showed that different riboflavin concentrations yielded gels with different mechanical and biological properties. experiments using human corneal epithelial cells (hCECs) showed that hCECs are able to proliferate on the gel and express corneal cell markers such as cytokeratin 12 (CK12) and tight junctions (ZO-1). Using an burst assay, we also showed that the PhotoCol-RB gels are able to seal corneal perforations. organ culture of the gels filling lamellar keratectomy wounds showed that the epithelium that regenerated over the PhotoCol-RB gels formed a multilayer compared to just a double layer for those that grew over physically cross-linked collagen. These gels can be formed either directly on the wound site to conform to the geometry of a defect, or can be preformed and then applied to the corneal wound. Our results indicate that PhotoCol-RB gels merit further investigation as a way to stabilize and repair deep and perforating corneal wounds.
Topics: Humans; Collagen; Cornea; Regeneration; Riboflavin; Gels
PubMed: 37126648
DOI: 10.1021/acsabm.3c00015 -
BMC Infectious Diseases Jun 2024Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from...
BACKGROUND
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes.
METHODS
Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm).
RESULTS
We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M.S. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and HO. Since schistosomes are damaged by HO, we determined if SmNPP5 could impede HO production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M.S.
CONCLUSIONS
The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.
Topics: Riboflavin; Flavin Mononucleotide; Animals; Flavin-Adenine Dinucleotide; Schistosoma mansoni; Mice; Humans; Schistosomiasis mansoni
PubMed: 38918706
DOI: 10.1186/s12879-024-09538-z -
Balkan Medical Journal Jul 2022To evaluate the clinical, pathological, and genetic features of patients with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD).
AIMS
To evaluate the clinical, pathological, and genetic features of patients with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD).
METHODS
Thirty-one patients with RR-MADD admitted to our hospital from January 2005 to November 2020 were enrolled, and their clinical data were collected. Pathological characteristics of the muscle tissue and possible pathogenic gene mutations were analyzed.
RESULTS
The most common clinical features in all patients were symmetrical proximal muscle weakness. Laboratory examination revealed elevated levels of creatine kinase, homocysteine, and uric acid, acylcarnitines, and organic acid. The muscle biopsy revealed typical pathological changes like lipid deposition. Genetic analysis identified ETFDH mutations in 29 patients, among which one had homozygotes, 19 had compound heterozygotes, 7 had heterozygous mutations, and 2 had heterozygous mutations of both ETFDH and ETFA. Two patients had no pathogenic gene mutations. All patients were treated with riboflavin, and their symptoms improved, which was consistent with the diagnosis of RR-MADD.
CONCLUSION
The clinical manifestations and genetic test results of patients with RR-MADD are heterogeneous. Therefore, a comprehensive analysis of clinical, pathological, and genetic testing is essential for the early diagnosis of RR-MADD.
Topics: Electron-Transferring Flavoproteins; Humans; Iron-Sulfur Proteins; Multiple Acyl Coenzyme A Dehydrogenase Deficiency; Oxidoreductases Acting on CH-NH Group Donors; Riboflavin
PubMed: 35734957
DOI: 10.4274/balkanmedj.galenos.2022.2022-1-127