Protein R2, the small subunit of ribonucleotide reductase of Escherichia coli, contains an essential free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged by hydroxyurea, the enzyme is transformed into an inactive form, metR2. E. coli contains a NAD(P)H:flavin oxidoreductase, named Fre, absolutely required for the regeneration of the radical and the activation of metR2 into R2. Consequently, an E. coli mutant strain lacking an active fre gene is more sensitive to hydroxyurea during growth, demonstrating the physiological protective function of Fre from the loss of the radical. However, this gene is not essential, and we found that E. coli contains a second tyrosyl radical generating activity, also residing in a flavin reductase. The enzyme has been purified 200-fold to homogeneity and found to be identical to sulfite reductase. Pure sulfite reductase has the ability to catalyze the reduction of free riboflavin, FMN, or FAD by NADPH and thus, as Fre, to transfer electrons to the iron center of metR2, a key step during the activation reaction.

In a hospital-based case-control study of pancreatic cancer conducted in Athens (1991-92), 181 patients with histologically confirmed cancer of the exocrine pancreas were compared with hospital patient controls and hospital visitor controls, individually matched to the cases by hospital, age, gender, and interviewer in a 1:1:1 ratio. All interviews were conducted in person in the respective hospitals. Diet was ascertained through a semiquantitative food-frequency questionnaire. Nutrient intakes for individuals were estimated by multiplying the nutrient content of a selected typical portion-size for each specified food-item by the frequency that the food was used per month, and summing these estimates for all food items. Data were analyzed using conditional logistic regression, controlling for tobacco smoking and total energy intake as well as for mutual confounding influences among nutrients. Adjusted odds ratios (rate ratios) for pancreatic cancer, associated with particular nutritional variables, were expressed per increments approximately equal to the standard deviations of (the residual of) the respective nutrients, on a daily basis. The adjusted odds ratios (OR) and 95 percent confidence intervals (CI) compared with other patient and visitor controls respectively, were: for polyunsaturated fat, OR = 1.32 (CI = 1.07-1.63) and 1.21 (CI = 0.98-1.49); and for crude fibre, OR = 0.80 (CI = 0.64-1.00) and 0.65 (CI = 0.50-0.86). No substantial, statistically significant or consistent, independent associations were noted for total energy, total protein, total fat, saturated fat, monounsaturated fat, dietary cholesterol, total carbohydrates, sucrose, vitamin C, vitamin A, riboflavin, or calcium.

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photocarcinogenesis and photoaging, and an urgent need exists for improved strategies for skin photoprotection. The redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defense against environmental electrophilic insult, has recently emerged as an important determinant of cutaneous damage from solar UV, and the concept of pharmacological activation of Nrf2 has attracted considerable attention as a novel approach to skin photoprotection. In this study, we examined feasibility of using tanshinones, a novel class of phenanthrenequinone-based cytoprotective Nrf2 inducers derived from the medicinal plant Salvia miltiorrhiza, for protection of cultured human skin cells and reconstructed human skin against solar simulated UV. Using a dual luciferase reporter assay in human Hs27 dermal fibroblasts pronounced transcriptional activation of Nrf2 by four major tanshinones [tanshinone I (T-I), dihydrotanshinone (DHT), tanshinone IIA (T-II-A) and cryptotanshinone (CT)] was detected. In fibroblasts, the more potent tanshinones T-I and DHT caused a significant increase in Nrf2 protein half-life via blockage of ubiquitination, ultimately resulting in upregulated expression of cytoprotective Nrf2 target genes (GCLC, NQO1) with the elevation of cellular glutathione levels. Similar tanshinone-induced changes were also observed in HaCaT keratinocytes. T-I and DHT pretreatment caused significant suppression of skin cell death induced by solar simulated UV and riboflavin-sensitized UVA. Moreover, feasibility of tanshinone-based cutaneous photoprotection was tested employing a human skin reconstruct exposed to solar simulated UV (80 mJ/cm(2) UVB; 1.53 J/cm(2) UVA). The occurrence of markers of epidermal solar insult (cleaved procaspase 3, pycnotic nuclei, eosinophilic cytoplasm, acellular cavities) was significantly attenuated in DHT-treated reconstructs that displayed increased immunohistochemical staining for Nrf2 and γ-GCS together with the elevation of total glutathione levels. Taken together, our data suggest the feasibility of achieving tanshinone-based cutaneous Nrf2-activation and photoprotection.

Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.

This paper provides a general review on folate and vitamin B12 nutrition and metabolism and the metabolic interrelationship between these vitamins. The effects of some common polymorphisms in folate and vitamin B12 genes and the influence of vitamin B6 and riboflavin status on folate and vitamin B12 metabolism are also discussed.

BACKGROUND

Homocysteine is an independent risk factor for vascular disease and is associated with dementia in older people. Potential mechanisms include altered endothelial and hemostatic function.

OBJECTIVE

We aimed to determine the effects of folic acid plus vitamin B-12, riboflavin, and vitamin B-6 on homocysteine and cognitive function.

METHODS

This was a factorial 2 x 2 x 2, randomized, placebo-controlled, double-blind study with 3 active treatments: folic acid (2.5 mg) plus vitamin B-12 (500 microg), vitamin B-6 (25 mg), and riboflavin (25 mg). We studied 185 patients aged>>or=65 y with ischemic vascular disease. Outcome measures included plasma homocysteine, fibrinogen, and von Willebrand factor at 3 mo and cognitive change (determined with the use of the Letter Digit Coding Test and on the basis of the Telephone Interview of Cognitive Status) after 1 y.

RESULTS

The mean (+/-SD) baseline plasma homocysteine concentration was 16.5 +/- 6.4 micromol/L. This value was 5.0 (95% CI: 3.8, 6.2) micromol/L lower in patients given folic acid plus vitamin B-12 than in patients not given folic acid plus vitamin B-12 but did not change significantly with vitamin B-6 or riboflavin treatment. Homocysteine lowering with folic acid plus vitamin B-12 had no significant effect, relative to the 2 other treatments, on fibrinogen, von Willebrand factor, or cognitive performance as measured by the Letter Digit Coding Test (mean change: -1; 95% CI: -2.3, 1.4) and the Telephone Interview of Cognitive Status (-0.7; 95% CI: -1.7, 0.4).

CONCLUSIONS

Oral folic acid plus vitamin B-12 decreased homocysteine concentrations in elderly patients with vascular disease but was not associated with statistically significant beneficial effects on cognitive function over the short or medium term.

OBJECTIVE

To investigate the efficacy and safety of corneal collagen cross-linking (CXL) with photoactivated riboflavin (photoactivated chromophore for infectious keratitis [PACK]-CXL) in the management of infectious keratitis with corneal melting.

METHODS

Prospective clinical trial.

METHODS

Forty eyes from 40 patients with advanced infectious keratitis and coexisting corneal melting.

METHODS

Twenty-one patients (21 eyes) underwent PACK-CXL treatment in addition to antimicrobial therapy. The control group consisted of 19 patients (19 eyes) who received only antimicrobial therapy.

METHODS

The slit-lamp characteristics of the corneal ulceration, corrected distance visual acuity, duration until healing, and complications were documented in each group. The Mann-Whitney U test was used for statistical analysis. P values less than 0.05 were considered statistically significant.

RESULTS

The average time until healing was 39.76 ± 18.22 days in the PACK-CXL group and 46.05 ± 27.44 days in the control group (P = 0.68). After treatment and healing, corrected distance visual acuity was 1.64 ± 0.62 in the PACK-CXL group and 1.67 ± 0.48 in the control group (P = 0.68). The corneal ulceration's width and length was significantly bigger in the PACK-CXL group (P = 0.004 and P = 0.007). Three patients in the control group demonstrated corneal perforation; infection recurred in 1 of them. No serious complications occurred in the PACK-CXL group.

CONCLUSIONS

Corneal CXL with photoactivated riboflavin did not shorten the time to corneal healing; however, the complication rate was 21% in the control group, whereas there was no incidence of corneal perforation or recurrence of the infection in the PACK-CXL group. These results indicate that PACK-CXL may be an effective adjuvant therapy in the management of severe infectious keratitis associated with corneal melting.

OBJECTIVE

Air puff systems have been presented recently to measure corneal biomechanical properties in vivo. In our study we tested the influence of several factors on corneal deformation to an air puff: IOP, corneal rigidity, dehydration, presence of sclera, and in vivo versus in vitro conditions.

METHODS

We used 14 freshly enucleated porcine eyes and five human donor eyes for in vitro experiments; nine human eyes were used for in vivo experiments. Corneal deformation was studied as a function of: IOP ranging from 15 to 45 mm Hg (in vitro); dehydration after riboflavin-dextran instillation (in vitro); corneal rigidity after standard ultraviolet (UV) corneal crosslinking (CXL, in vitro); boundary conditions, that is effect of the presence of the sclera (comparing corneal buttons and whole globes in vitro in pigs); and effect of ocular muscles (comparing human whole globes in vitro and in vivo). The temporal corneal deformation was characterized by the apex indentation across time, the maximal indentation depth, and the temporal symmetry (comparing inward versus outward deformation). The spatial corneal profile was characterized by the peak distance at maximal deformation.

RESULTS

Temporal and spatial deformation profiles were very sensitive to the IOP (P < 0.001). The sclera slightly affected the temporal symmetry, while the ocular muscles drastically changed the amount of corneal recovery. CXL produced a significant (P = 0.001) reduction of the cornea indentation (by a factor of 1.41), and a change in the temporal symmetry of the corneal deformation profile (by a factor of 1.65), indicating a change in the viscoelastic properties with treatment.

CONCLUSIONS

Corneal deformation following an air puff allows the measurement of dynamic properties, which are essential for the characterization of corneal biomechanics.

Folate plays a key role in DNA synthesis and methylation. Limited evidence suggests high intake may reduce risks of esophageal cancer overall; however, associations with esophageal cancer subtypes and Barrett's esophagus (BE), a precancerous lesion, remain unexplored. We evaluated the relation between intake of folate, B vitamins, and methyl-group donors (methionine, choline, betaine) from foods and supplements, polymorphisms in key folate-metabolizing genes, and risk of BE, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESCC) in 2 population-based case-control studies in Australia. BE patients without (n = 266) or with (n = 101) dysplasia were compared with population controls (n = 577); similarly, EAC (n = 636) or ESCC (n = 245) patients were compared with population controls (n = 1507) using multivariable adjusted logistic regression. Increasing intake of folate from foods was associated with reduced EAC risk (P-trend = 0.01) and mitigated the increased risks of ESCC associated with smoking and alcohol consumption. In contrast, high intake of folic acid from supplements was associated with a significantly elevated risk of BE with dysplasia. High intakes of riboflavin and methionine from food were associated with increased EAC risk, whereas increasing betaine intake was associated with reduced risks of BE without (P-trend = 0.004) or with dysplasia (P-trend = 0.02). Supplemental thiamin, riboflavin, niacin, and vitamin B-12 were associated with increased EAC risk. There were no consistent associations between genetic polymorphisms studied and BE or EAC risk. High intake of folate-containing foods may reduce risk of EAC, but our data raise the possibility that folic acid supplementation may increase risks of BE with dysplasia and EAC.

BACKGROUND

Riboflavin (vitamin B(2)) is the precursor for FAD, the cofactor for methylenetetrahydrofolate reductase (MTHFR). MTHFR catalyzes the formation of 5-methyltetrahydrofolate, which acts as a methyl donor for homocysteine remethylation. Individuals with the MTHFR 677C->>T mutation have increased plasma total homocysteine (tHcy) concentrations, particularly in association with low folate status. It has been proposed that riboflavin may act together with folate to lower plasma tHcy, particularly in individuals with the thermolabile MTHFR T variant.

METHODS

We measured B-vitamin status and plasma tHcy in 126 healthy individuals 20-63 years of age (42 CC, 42 CT, and 42 TT MTHFR genotypes) at baseline and after three interventions (4 months): placebo plus natural diet; daily 400 microg folic acid supplement plus natural diet; and increased dietary folate to 400 microg/day.

RESULTS

At baseline and after nutritional intervention, lower riboflavin status was associated with increased plasma tHcy concentrations. Plasma tHcy was 2.6 micromol/L higher in the lowest plasma riboflavin quartile compared with the highest (P <0.02) and was 4.2 micromol/L higher in the highest erythrocyte glutathione reductase activation coefficient (EGRAC) quartile compared with the lowest (P <0.001). This effect was not restricted to those with the T allele. Folic acid given as a 400 microg/day supplement appeared to exacerbate a tendency toward riboflavin deficiency, as suggested by an increase in the proportion of individuals with EGRAC>> or =1.4 from 52% to 65% after supplementation (P <0.05).

CONCLUSIONS

Folate and riboflavin interact to lower plasma tHcy, possibly by maximizing the catalytic activity of MTHFR. The effect may be unrelated to MTHFR genotype.

The role of riboflavin in cell maintenance and growth, and the mechanism by which it is absorbed into various human tissues and cell lines has been extensively studied over the past decade. Evidence suggests two absorption mechanisms, a saturable-active component that dominates at near physiological vitamin concentrations and a passive component that is revealed at oversupplemented riboflavin conditions. Various transport modulator studies consistently suggest a highly riboflavin specific, temperature-dependent active transport mechanism that is regulated by the Ca2+/calmodulin pathway. The PKA and PKG pathways have also been implicated in absorption regulation. The long-standing model that riboflavin absorption involves a carrier-mediated transporter has recently been challenged through studies suggesting a receptor-mediated endocytic component. The presence of a soluble, human riboflavin binding protein in the transport stratagem has been shown to play an important role in fetal development. The relationship of this binding protein with the riboflavin specific membrane bound protein, though currently not well defined, may involve a protein-protein interaction that plays a primary role in this proposed receptor-mediated component.

OBJECTIVE

To investigate biomechanical changes after corneal cross-linking (CXL) with riboflavin/ultraviolet-A (UVA) in keratoconus using the recently developed Ocular Response Analyzer (ORA, Reichert Technologies) software.

METHODS

Through use of the new ORA software (version 2.04), 37 new parameters derived from the best measurement signal with the highest wavescore of 4 measurements from 50 eyes of 46 patients with keratoconus were obtained before and 1 year after CXL. The parameters of 96 eyes from 96 age-matched, healthy individuals with a spherical equivalent refraction <3.00 diopters served as controls.

RESULTS

Corneal hysteresis (CH) and corneal resistance factor (CRF) before CXL were 7.38±1.42 mmHg and 6.16±1.42 mmHg, respectively, compared to 7.37±1.26 mmHg (P=.971) and 6.16±1.50 mmHg after CXL (P=.997), respectively. Based on these 37 new parameters, the area under peak 2 (p2area) showed a statistically significant increase from 1262.3±623.1 before CXL to 1704.3±732.3 1 year after CXL (35%; P=.001). The related value for the p2area of the healthy control group was 3374.9±1099.9. A significant negative correlation was observed between the p2area and the difference in CH-CRF values (r=-0.29, P=.001).

CONCLUSIONS

The area under peak 2 appears to be a more sensitive parameter to detect biomechanical changes after CXL than CH or CRF alone. After CXL, keratoconic corneas display altered biomechanical properties, which remain different to those observed in healthy corneas.

Folate and the metabolically related B-vitamins, vitamin B12 and riboflavin, have attracted much scientific and public health interest in recent years. Apart from a well established role in preventing neural tube defects (NTDs), evidence is emerging to support other potential roles for folate and/or related B-vitamins in protecting against cardiovascular disease (especially stroke), certain cancers, cognitive impairment and osteoporosis. However, typical folate intakes are sub-optimal, in that although adequate in preventing clinical folate deficiency (i.e. megaloblastic anaemia) in most people, they are generally insufficient to achieve a folate status associated with the lowest risk of NTDs. Natural food folates have a limited ability to enhance folate status as a result of their poor stability under typical cooking conditions and incomplete bioavailability when compared with the synthetic vitamin, folic acid (as found in supplements and fortified foods). Current folate recommendations to prevent NTDs (based primarily on folic acid supplementation) have been found to be ineffective in several European countries. In contrast, in North America and Chile, the policy of mandatory folic acid-fortification has proven itself in terms of lowering the prevalence of NTD, but remains controversial because of concerns regarding potential risks of chronic exposure to high-dose folic acid. In the case of vitamin B12, the achievement of an optimal status is particularly difficult for many older people because of the common problem of food-bound B12 malabsorption. Finally, there is evidence that riboflavin status is generally low in the UK population, and particularly so in younger women; this warrants further investigation.

OBJECTIVE

We compared an iontophoresis riboflavin delivery technique for transepithelial corneal collagen crosslinking (I-CXL) with a conventional CXL (C-CXL).

METHODS

We designed three experimental sets using 152 New Zealand rabbits to study riboflavin application by iontophoresis using charged riboflavin solution (Ricrolin+) with a 1-mA current for 5 minutes. The first set was to compare riboflavin concentration measured by HPLC in corneas after iontophoresis or conventional riboflavin application. The second set was to analyze autofluorescence and stromal collagen modification immediately and 14 days after I-CXL or C-CXL, by using nonlinear two-photon microscopy (TP) and second harmonic generation (SHG). In the third set, physical modifications after I-CXL and C-CXL were evaluated by stress-strain measurements and by studying corneal resistance against collagenase digestion.

RESULTS

Based on HPLC analysis, we found that iontophoresis allowed riboflavin diffusion with 2-fold less riboflavin concentration than conventional application (936.2 ± 312.5 and 1708 ± 908.3 ng/mL, respectively, P < 0.05). Corneal TP and SHG imaging revealed that I-CXL and C-CXL resulted in a comparable increased anterior and median stromal autofluorescence and collagen packing. The stress at 10% strain showed a similar stiffness of corneas treated by I-CXL or C-CXL (631.9 ± 241.5 and 680.3 ± 216.4 kPa, respectively, P = 0.908). Moreover, we observed an increased resistance against corneal collagenase digestion after I-CXL and C-CXL (61.90% ± 5.28% and 72.21% ± 4.32% of remaining surface, respectively, P = 0.154).

CONCLUSIONS

This experimental study suggests that I-CXL is a promising alternative methodology for riboflavin delivery in crosslinking treatments, preserving the epithelium.

OBJECTIVE

To report four cases of severe keratitis after standard corneal collagen cross-linking (CXL) treatment for keratoconus.

METHODS

Four patients with progressive keratoconus from two different centers were treated by ultraviolet A (UVA) CXL, using riboflavin as a photosensitizer. The epithelium was removed over the central 8 to 9 mm of the cornea. Riboflavin 0.1% in dextran 20% was instilled every 2 minutes for 30 minutes before UVA exposure. The UV-X light source (IROC), calibrated at 3 mW/cm(2), was applied for 30 minutes while instillation was continued every 2 minutes. At the end of the treatment, a bandage contact lens was applied and topical treatment consisting of a combination of antibiotics and/or anti-inflammatory drops was initiated.

RESULTS

Patients experienced delayed (after more than 24 hours) symptoms and signs of inflammation. The eyes showed pronounced ciliary redness with cells in the anterior chamber and central keratic precipitates; multiple white infiltrates had developed at the edge and within the area of CXL. High-dose topical or subconjunctival corticosteroids led to rapid initial improvement of symptoms and signs. Herpes virus could not be detected on the ocular surface or on the anterior chamber tap of one patient.

CONCLUSIONS

We report four cases of keratitis and corneal scarring from a total of 117 eyes treated with CXL. The location of the scarring determined the amount of loss of visual acuity: in two eyes, there was a persistent decrease in best spectacle-corrected visual acuity.

Methionine, folate, vitamin B(6), vitamin B(12), niacin, and riboflavin intakes may be related to breast carcinogenesis. These associations may vary by breast cancer type. Using the prospective cohort Shanghai Women's Health Study (1997-2008) including 718 Chinese breast cancer cases, the authors evaluated baseline dietary intake of these factors and breast cancer risk and whether the associations varied by menopausal status and estrogen receptor (ER) and progesterone receptor (PR) status. They estimated associations using hazard ratios and 95% confidence intervals from Cox proportional hazards regression models and stratified analyses by menopausal status and ER/PR status. Lowest quantile of intake was used as the comparison group. For postmenopausal women, dietary intakes of methionine and B vitamins were not associated with breast cancer risk. For premenopausal women, higher intake of folate was associated with decreased breast cancer risk (hazard ratio = 0.58, 95% confidence interval: 0.34, 0.99 for the highest vs. lowest quintile of intake). Only niacin intake was associated with ER+/PR+ breast cancer risk (hazard ratio = 1.62, 95% confidence interval: 1.07, 2.46; P for trend = 0.04 for the highest vs. lowest quartile of intake). Findings support the hypothesis that high folate intake may reduce breast cancer risk and that the association may vary by menopausal and ER/PR status.

The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5'-nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.

In situ gelling constructs, which form a hydrogel at the site of injection, offer the advantage of delivering cells and growth factors to the complex structure of the defect area for tissue engineering. In the present study, visible light crosslinkable hydrogel systems were presented using methacrylated glycol chitosan (MeGC) and three blue light initiators: camphorquinone (CQ), fluorescein (FR) and riboflavin (RF). A minimal irradiation time of 120 s was required to produce MeGC gels able to encapsulate cells with CQ or FR. Although prolonged irradiation up to 600 s improved the mechanical strength of CQ- or FR-initiated gels (compressive modulus 2.8 or 4.4 kPa, respectively), these conditions drastically reduced encapsulated chondrocyte viability to 5% and 25% for CQ and FR, respectively. Stable gels with 80-90% cell viability could be constructed using radiofrequency (RF) with only 40s irradiation time. Increasing irradiation time up to 300s significantly improved the compressive modulus of the RF-initiated MeGC gels up to 8.5 kPa without reducing cell viability. The swelling ratio and degradation rate were smaller at higher irradiation time. RF-photoinitiated hydrogels supported proliferation of encapsulated chondrocytes and extracellular matrix deposition. The feasibility of this photoinitiating system as in situ gelling hydrogels was further demonstrated in osteochondral and chondral defect models for potential cartilage tissue engineering. The MeGC hydrogels using RF offer a promising photoinitiating system in tissue engineering applications.

Although easily examined, abnormalities of the tongue can present a diagnostic and therapeutic dilemma for physicians. Recognition and diagnosis require a thorough history, including onset and duration, antecedent symptoms, and tobacco and alcohol use. Examination of tongue morphology and a careful assessment for lymphadenopathy are also important. Geographic tongue, fissured tongue, and hairy tongue are the most common tongue problems and do not require treatment. Median rhomboid glossitis is usually associated with a candidal infection and responds to topical antifungals. Atrophic glossitis is often linked to an underlying nutritional deficiency of iron, folic acid, vitamin B12, riboflavin, or niacin and resolves with correction of the underlying condition. Oral hairy leukoplakia, which can be a marker for underlying immunodeficiency, is caused by the Epstein-Barr virus and is treated with oral antivirals. Tongue growths usually require biopsy to differentiate benign lesions (e.g., granular cell tumors, fibromas, lymphoepithelial cysts) from premalignant leukoplakia or squamous cell carcinoma. Burning mouth syndrome often involves the tongue and has responded to treatment with alpha-lipoic acid, clonazepam, and cognitive behavior therapy in controlled trials. Several trials have also confirmed the effectiveness of surgical division of tongue-tie (ankyloglossia), in the context of optimizing the success of breastfeeding compared with education alone. Tongue lesions of unclear etiology may require biopsy or referral to an oral and maxillofacial surgeon, head and neck surgeon, or a dentist experienced in oral pathology.

5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420 in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidation, C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.

Secretory proteins such as apolipoprotein B-100 (apoB) undergo oxidative folding (formation of disulfide bonds) in the endoplasmic reticulum (ER) before secretion. Oxidative folding depends on flavoproteins in eukaryotes. Here, human liver (HepG2) cells were used to model effects of riboflavin concentrations in culture media on folding and secretion of apoB. Cells were cultured in media containing 3.1, 12.6, and 300 nmol/L of riboflavin, representing moderately deficient, physiological, and pharmacological plasma concentrations in humans, respectively. When cells were cultured in riboflavin-deficient medium, secretion of apoB decreased by >80% compared with controls cultured in physiological medium. The nuclear translocation of the transcription factor ATF-6 increased by >180% in riboflavin-deficient cells compared with physiological controls; this is consistent with ER stress. Nuclear translocation of ATF-6 was associated with activation of the unfolded protein response. Expression of stress-response genes coding for ubiquitin-activating enzyme 1, growth arrest and DNA damage inducible gene, and glucose regulated protein of 78 kDa was greater in riboflavin-deficient cells compared with other treatment groups. Finally, phosphorylation of the eukaryotic initiation factor (eukaryotic initiation factor 2alpha) increased in riboflavin-deficient cells, consistent with decreased translational activity. We conclude 1) that riboflavin deficiency causes ER stress and activation of unfolded protein response in HepG2 cells, and 2) that riboflavin deficiency decreases protein secretion in HepG2 cells. Decreased secretion of apoB in riboflavin-deficient cells might interfere with lipid homeostasis in vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) has been identified as a potential threat to the safety of blood products. The Mirasol Pathogen Reduction Technology System uses riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against MERS-CoV when tested in human plasma.

MERS-CoV (EMC strain) was used to inoculate plasma units that then underwent treatment with riboflavin and UV light. The infectious titers of MERS-CoV in the samples before and after treatment were determined by plaque assay on Vero cells. The treatments were initially performed in triplicate using pooled plasma (n = 3) and then repeated using individual plasma units (n = 6).

In both studies, riboflavin and UV light reduced the infectious titer of MERS-CoV below the limit of detection. The mean log reductions in the viral titers were ≥4.07 and ≥4.42 for the pooled and individual donor plasma, respectively.

Riboflavin and UV light effectively reduced the titer of MERS-CoV in human plasma products to below the limit of detection, suggesting that the treatment process may reduce the risk of transfusion transmission of MERS-CoV.

OBJECTIVE

To report the 4-year outcomes of corneal cross-linking (CXL) for progressive keratoconus in a population of different age groups.

METHODS

Retrospective, single-center, nonrandomized clinical study.

METHODS

Four hundred consecutive eyes treated with corneal CXL for progressive keratoconus from April 2006 through April 2010.

METHODS

After removal of the epithelium, the cornea was irrigated for 30 minutes with a solution of 0.1% riboflavin and 20% dextran, followed by irradiation with an ultraviolet A light of 3 mW/cm(2) for 30 minutes.

METHODS

Best-corrected visual acuity (BCVA), sphere and cylinder refraction, corneal topography, Scheimpflug tomography, and aberrometry were assessed at baseline and at 1, 6, 12, 24, 36, and 48 months after corneal CXL treatment. The compiled data were stratified according to age (group A, younger than 18 years; group B, 18-29 years; group C, 30-39 years; and group D, older than 40 years).

RESULTS

Comparative analysis included 400 eyes of 301 patients. Functional results showed a significant increase in BCVA in group A by a mean reduction of -0.11 logarithm of the minimum angle of resolution (logMAR) after 12 months, in group B by a mean reduction of -0.31 logMAR after 36 months, in group C by a mean reduction of -0.33 logMAR after 36 months, and in group D by a mean reduction of -0.26 logMAR after 36 months. Morphologic results showed an analogous regularization of corneal shape with a significant reduction of opposite sector index by a mean value of -0.53 at 12 months in group A, -1.14 at 36 months in group B, -1.10 at 36 months in group C, and -0.55 at 12 months for group D. Optical quality improvement was demonstrated by a mean significant reduction of coma -1.52 μm after 12 months in group A, -1.58 μm after 24 months in group B, -2.57 μm after 36 months for group C, and -0.25 μm after 36 months in group D.

CONCLUSIONS

Outcomes stratified by age indicate the efficacy of corneal CXL in stabilizing the progression of ectatic disease in all age groups and improving the functional and morphologic parameters in select groups. Results indicated better functional and morphologic results in the population between 18 and 39 years of age.

BACKGROUND

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