Evidence is growing for the long-term effects of environmental factors during early-life on later disease susceptibility. It is believed that epigenetic mechanisms (changes in gene function not mediated by DNA sequence alteration), particularly DNA methylation, play a role in these processes. This paper reviews the current state of knowledge of the involvement of C1 metabolism and methyl donors and cofactors in maternal diet-induced DNA methylation changes in utero as an epigenetic mechanism. Methyl groups for DNA methylation are mostly derived from the diet and supplied through C1 metabolism by way of choline, betaine, methionine or folate, with involvement of riboflavin and vitamins B6 and B12 as cofactors. Mouse models have shown that epigenetic features, for example DNA methylation, can be altered by periconceptional nutritional interventions such as folate supplementation, thereby changing offspring phenotype. Evidence of early nutrient-induced epigenetic change in human subjects is scant, but it is known that during pregnancy C1 metabolism has to cope with high fetal demands for folate and choline needed for neural tube closure and normal development. Retrospective studies investigating the effect of famine or season during pregnancy indicate that variation in early environmental exposure in utero leads to differences in DNA methylation of offspring. This may affect gene expression in the offspring. Further research is needed to examine the real impact of maternal nutrient availability on DNA methylation in the developing fetus.

OBJECTIVE

The mechanical properties of corneal tissue are linked to prevalent ocular diseases and therapeutic procedures. Brillouin microscopy is a novel optical technology that enables three-dimensional mechanical imaging. In this study, the feasibility of this noncontact technique was tested for in situ quantitative assessment of the biomechanical properties of the cornea.

METHODS

Brillouin light-scattering involves a spectral shift proportional to the longitudinal modulus of elasticity of the tissue. A 532-nm single-frequency laser and a custom-developed ultrahigh-resolution spectrometer were used to measure the Brillouin frequency. Confocal scanning was used to perform Brillouin elasticity imaging of the corneas of whole bovine eyes. The longitudinal modulus of the bovine corneas was compared before and after riboflavin corneal collagen photo-cross-linking. The Brillouin measurements were then compared with conventional stress-strain mechanical test results.

RESULTS

High-resolution Brillouin images of the cornea were obtained, revealing a striking depth-dependent variation of the elastic modulus across the cornea. Along the central axis, the Brillouin frequency shift varied gradually from 8.2 GHz in the epithelium to 7.5 GHz near the endothelium. The coefficients of the down slope were measured to be approximately 1.09, 0.32, and 2.94 GHz/mm in the anterior, posterior, and innermost stroma, respectively. On riboflavin collagen cross-linking, marked changes in the axial Brillouin profiles (P < 0.001) were noted before and after cross-linking.

CONCLUSIONS

Brillouin imaging can assess the biomechanical properties of cornea in situ with high spatial resolution. This novel technique has the potential for use in clinical diagnostics and treatment monitoring.

The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.

The multidrug transporter ABCG2 (BCRP/MXR/ABCP) can actively extrude a broad range of endogenous and exogenous substrates across biological membranes. ABCG2 limits oral availability and mediates hepatobiliary and renal excretion of its substrates, and thus influences the pharmacokinetics of many drugs. Recent work, relying mainly on the use of Abcg2(-/-) mice, has revealed important contributions of ABCG2 to the blood-brain, blood-testis and blood-fetal barriers. Together, these functions indicate a primary biological role of ABCG2 in protecting the organism from a range of xenobiotics. In addition, several other physiological functions of ABCG2 have been observed, including extrusion of porphyrins and/or porphyrin conjugates from hematopoietic cells, liver and harderian gland, as well as secretion of vitamin B(2) (riboflavin) and possibly other vitamins (biotin, vitamin K) into breast milk. However, the physiological significance of these processes has been difficult to establish, indicating that there is still a lot to learn about this intriguing protein.

The fruits (dates) of the date palm (Phoenix dactylifera L.) contain a high percentage of carbohydrate (total sugars, 44-88%), fat (0.2-0.5%), 15 salts and minerals, protein (2.3-5.6%), vitamins and a high percentage of dietary fibre (6.4-11.5%). The flesh of dates contains 0.2-0.5% oil, whereas the seed contains 7.7-9.7% oil. The weight of the seed is 5.6-14.2% of the date. The fatty acids occur in both flesh and seed as a range of saturated and unsaturated acids, the seeds containing 14 types of fatty acids, but only eight of these fatty acids occur in very low concentration in the flesh. Unsaturated fatty acids include palmitoleic, oleic, linoleic and linolenic acids. The oleic acid content of the seeds varies from 41.1 to 58.8%, which suggests that the seeds of date could be used as a source of oleic acid. There are at least 15 minerals in dates. The percentage of each mineral in dried dates varies from 0.1 to 916 mg/100 g date depending on the type of mineral. In many varieties, potassium can be found at a concentration as high as 0.9% in the flesh while it is as high as 0.5% in some seeds. Other minerals and salts that are found in various proportions include boron, calcium, cobalt, copper, fluorine, iron, magnesium, manganese, potassium, phosphorous, sodium and zinc. Additionally, the seeds contain aluminum, cadmium, chloride, lead and sulphur in various proportions. Dates contain elemental fluorine that is useful in protecting teeth against decay. Selenium, another element believed to help prevent cancer and important in immune function, is also found in dates. The protein in dates contains 23 types of amino acids, some of which are not present in the most popular fruits such as oranges, apples and bananas. Dates contain at least six vitamins including a small amount of vitamin C, and vitamins B(1) thiamine, B(2) riboflavin, nicotinic acid (niacin) and vitamin A. The dietary fibre of 14 varieties of dates has been shown to be as high as 6.4-11.5% depending on variety and degree of ripeness. Dates contain 0.5-3.9% pectin, which may have important health benefits. The world production of dates has increased 2.9 times over 40 years, whereas the world population has doubled. The total world export of dates increased by 1.71% over 40 years. In many ways, dates may be considered as an almost ideal food, providing a wide range of essential nutrients and potential health benefits.

BACKGROUND

Dietary intake during childhood and adolescence is of increasing interest due to its influence on adult health, particularly obesity, cardiovascular disease and diabetes. There is a need to develop and validate dietary assessment methods suitable for large epidemiologic studies of children and adolescents. Limited large scale dietary studies of youth have been undertaken in Australia, due partly to the lack of a suitable dietary intake tool. A self-administered, semi-quantitative food-frequency questionnaire (FFQ), the 'Australian Child and Adolescent Eating Survey' (ACAES), was developed for youth aged 9-16 years. This study evaluated reproducibility and comparative validity of the ACAES FFQ using assisted food records (FRs) as the reference method.

METHODS

The ACAES FFQ was completed twice (FFQ1 and FFQ2) at an interval of 5 months, along with four one-day assisted FRs. Validity was evaluated by comparing the average of the FRs with FFQ2 (n = 113) as well as with the average of FFQ1 and FFQ2 (n = 101). Reproducibility was evaluated by comparing FFQ1 and FFQ2 (n = 101). The two methods were compared using correlations, Kappa statistics and Bland-Altman plots.

RESULTS

Correlation coefficients for comparative validity ranged from 0.03 for retinol to 0.56 for magnesium for transformed, energy-adjusted, deattenuated nutrient data, with correlation coefficients greater than 0.40 for total fat, saturated fat, monounsaturated fat, carbohydrate, sugars, riboflavin, vitamin C, folate, beta-carotene, magnesium, calcium and iron. Correlation coefficients for reproducibility ranged from 0.18 for vitamin A to 0.50 for calcium for transformed, energy-adjusted, deattenuated nutrient data. The ACAES FFQ ranked individuals reasonably accurately, with the comparative validity analysis showing that over 50% of participants were classified within one quintile for all nutrients, with only a small percentage grossly misclassified (0-7%).

CONCLUSIONS

The ACAES FFQ is the first child and adolescent specific FFQ available for ranking the dietary intakes of Australian children and adolescents for a range of nutrients in epidemiologic research and public health interventions.

OBJECTIVE

To evaluate the safety and efficacy of higher fluence cornea collagen cross linking (CXL).

METHODS

Twenty-one patients with bilateral keratoconus had randomized CXL in one eye (group A) with 7 mw/cm(2) for 15 minutes; the other eye (group B) had the standard 3 mw/cm(2) for 30 minutes; 50 um PTK with the Eye-Q 400 Hz Excimer laser (Wavelight, Erlagen, Germany) was used for epithelial removal. The patients were evaluated postoperatively at the following intervals: day 1, day 4, month 1, month 3, and then every 6 months.

RESULTS

FOR GROUPS A AND B RESPECTIVELY, IN MEAN VALUES: uncorrected distance visual acuity (UDVA) improved from 20/60 to 20/38, and 20/62 to 20/40; best corrected visual acuity (BCVA) from 20/30 to 20/25 in both groups; mean sphere was reduced by 2.5 and 2.1 diopters; mean cylinder was reduced by 2.9 and 2.5 diopters on average; Steepest K was reduced from 49.5 to 46.1, and from 48.7 to 45.8 diopters. There was no ectasia progression in any of the cases during the follow-up time studied. There was no change in the endothelial cell count. All patients returned to full activities postoperatively within a month. Four cases from group A and five cases from group B had delayed epithelial healing (completed by postoperative day 9). No other adverse effects were noted in any of the cases studied. Mean follow-up was 46 months (18-56). Corneal optical coherence tomography (OCT) revealed diffused light scattered in anterior two-thirds of the cornea stroma, which was more intense and much broader in diameter in group A than in group B.

CONCLUSIONS

This novel technique offers similar clinical results in ectasia stabilization without any adverse effects noted.

OBJECTIVE

Collagen crosslinking using ultraviolet- A (UVA) -irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone.

METHODS

Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm2 and with UVA alone between 2 and 9 mW/cm2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypan-blue and Yopro-fluorescence staining.

RESULTS

An abrupt cytotoxic irradiance level was found at 0.5 mW/cm2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/cm2 after UVA-irradiation alone.

CONCLUSIONS

A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm2, which is reached in the clinical setting in human corneas down to a depth of 300 microm using the standard surface UVA-irradiance of 3 mW/cm2.

OBJECTIVE

To assess the effectiveness of ultraviolet A (UVA) irradiation-induced collagen cross-linking (CCL) on keratoconus (KC) progression.

METHODS

A patient with bilateral, progressive KC underwent UVA irradiation (3 mW/cm for 30 minutes) after topical 0.1% riboflavin drops over a deepithelialized cornea. Twelve months later, a topography-guided penetrating keratoplasty (PRK; wavelight 400 Hz Eye-Q excimer) was performed in 1 eye for a refractive error of -3.50 -4.00 x 155 by using an attempted treatment of -2.50 -3.00 x 155. At all postoperative follow-up visits to 18 months, uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), pachymetry, and topography were performed.

RESULTS

In the treated left eye, the UCVA after the UVA CCL improved from 20/100 to 20/80, and the BSCVA improved from 20/50 to 20/40. Eighteen months after the topography-guided PRK, the UCVA was 20/20, and the BSCVA was 20/15, with a refractive error of Plano -0.50 x 150. The cornea was clear, and the endothelial cell count remained unchanged. The untreated right mate eye continued to progress during the same period.

CONCLUSIONS

The significant clinical improvement and the apparent stability of more than a year after UVA CCL, and subsequent PRK compared with the untreated mate eye, seems to validate this treatment approach for KC. An adjusted nomogram may be considered in the ablation of cross-linked cornea tissue to avoid overcorrections.

Substantial evidence indicates that bioenergetic dysfunction plays either a primary or secondary role in the pathophysiology of cell death in neurodegenerative and neuromuscular disorders, and even in normal aging. Agents that ameliorate bioenergetic defects may therefore be useful in therapy. Creatine, which increases muscle and brain phosphocreatine concentrations, and may inhibit the activation of the mitochondrial permeability transition, protects against neuronal degeneration in transgenic murine models of amyotrophic lateral sclerosis and Huntington's disease and in chemically mediated neurotoxicity. Initial studies of creatine use in humans appear promising; however, further long-term, well-designed trials are needed. Coenzyme Q10, Gingko biloba, nicotinamide, riboflavin, carnitine, lipoic acid, and dichloroacetate are other agents which may have beneficial effects on energy metabolism, but the preclinical and clinical evidence for efficacy in neurological diseases remains limited. These compounds are widely used as dietary supplements; however, they must be subjected to rigorous evaluation through randomized, double-blinded trials to establish efficacy, cost-effectiveness and safety in neurological disorders.

We evaluated the association between nutritional status and cognitive functioning in 260 noninstitutionalized men and women older than 60 years who had no known physical illnesses and were receiving no medications. Nutritional status was evaluated by three-day food records and also by biochemical determination of blood levels of specific nutrients. Cognitive status was evaluated by the Halstead-Reitan Categories Test (a nonverbal test of abstract thinking ability) and by the Wechsler Memory Test. Subjects with low blood levels of vitamins C or B12 scored worse on both tests. Subjects with low levels of riboflavin or folic acid scored worse on the categories test. These differences remained significant after controlling for age, gender, level of income, and amount of education. "Subclinical" malnutrition may play a small role in the depression of cognitive function detectable in some elderly individuals, or depressed cognitive function may result in reduced nutrient intake.

The Arabidopsis thaliana CORONATINE INSENSITIVE1 (COI1) gene encodes an F-box protein to assemble SCF(COI1) complexes essential for response to jasmonates (JAs), which are a family of plant signaling molecules required for many essential functions, including plant defense and reproduction. To better understand the molecular basis of JA action, we screened for suppressors of coi1 and isolated a coi1 suppressor1 (cos1) mutant. The cos1 mutation restores the coi1-related phenotypes, including defects in JA sensitivity, senescence, and plant defense responses. The COS1 gene was cloned through a map-based approach and found to encode lumazine synthase, a key component in the riboflavin pathway that is essential for diverse yet critical cellular processes. We demonstrated a novel function for the riboflavin pathway that acts downstream of COI1 in the JA signaling pathway and is required for suppression of the COI1-mediated root growth, senescence, and plant defense.

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

The mechanism of oxidative damage to the lens through intraocular photochemical generation of superoxide and its derivatization to other oxidants such as singlet oxygen, hydroxyl radical and hydrogen peroxide has been studied. Rat lenses when organ cultured aerobically in TC 199 containing additional amounts of riboflavin were damaged as demonstrated by an inhibition of the uptake of Rb 86 against a concentration gradient. The pump was not affected by light if the culture was conducted in the basal TC 199. However, light was observed to induce significant peroxidative degradation of the tissue lipids even in the basal medium, the degradation being indicated by the formation of malonaldehyde. Both the inhibition of the pump as well as the peroxidative degradation of the tissue lipids, were attenuated considerably by scavengers of superoxide and hydrogen peroxide. In addition, the lipid degradation was prevented by vitamins C and E. The results suggest that the photodynamic injury to the lens cation pump as well as to membrane lipids is incumbent upon an initial generation of superoxide and its derivatization to other oxidants. Thus, the ocular lens is susceptible to oxidative insult and physiological damage through photocatalytic generation of various oxygen radicals. Large concentrations of ascorbic acid in the aqueous humor seems to be able to provide significant protection against such an insult. Thus, this may be one of the functions of high concentration of ascorbic acid in the aqueous humor. The implication of oxidative stress has also been examined in the genesis of cataracts in vivo. Treatment with vitamin E of the Emory mouse led to a decrease in the rate of cataract progression suggesting that at least in some instances an oxidative stress could participate in the formation of cataracts. Oxygen radicals may inflict damage at multifarious biochemical sites. Human lens lipids were also shown to have an absorption maxima at 239 nm indicating their susceptibility to oxidative degradation. In addition the lipid extract has fluorescence similar to that of lipofuscins. The levels of MDA were higher in the brunescent cataracts as compared to that in the nonbrunescent cataracts. The implications of oxidative stress towards the genesis of cataracts in humans is being explored further.

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.

BACKGROUND

A medical device is being developed for the reduction of pathogens in PLT concentrates (PCs). The device uses broadband UV light and the compound riboflavin (vitamin B(2)).

METHODS

Pathogens were added to single-donor PLTs. After treatment, the infectivity of each pathogen was measured using established biologic assays. In vitro PLT performance was evaluated after treatment and after 5 days of storage using a panel of 10 in-vitro cell quality assays.

RESULTS

In studies with viral pathogens, the Pathogen Reduction Technology (PRT) system provided average log reduction factors of 4.46 +/- 0.39 for intracellular HIV, 5.93 +/- 0.20 for cells associated HIV, and 5.19 +/- 0.50 for West Nile virus. For the nonenveloped porcine parvovirus, a reduction factor greater than 5.0 log was observed. Staphylococcus epidermidis and Escherichia coli bacteria were also tested with observed reduction factors to the limits of detection of 4.0 log or greater. PLT cell quality was adequately maintained after treatment and during storage. Although P-selectin expression, glucose consumption, and lactate production increased relative to controls, this was not beyond accepted levels. The pH of treated PCs also decreased slightly relative to control PLTs on Days 1 and 5.

CONCLUSIONS

The data indicate that the device successfully reduced the number of selected pathogens in PCs. Despite the fact that significant differences exist between treated and control in-vitro variables, it is speculated that the clinical effectiveness of both products will not be significantly different, based on comparison to historical data for products in routine clinical use today.

Animal source foods can provide a variety of micronutrients that are difficult to obtain in adequate quantities from plant source foods alone. In the 1980s, the Nutrition Collaborative Research Support Program identified six micronutrients that were particularly low in the primarily vegetarian diets of schoolchildren in rural Egypt, Kenya and Mexico: vitamin A, vitamin B-12, riboflavin, calcium, iron and zinc. Negative health outcomes associated with inadequate intake of these nutrients include anemia, poor growth, rickets, impaired cognitive performance, blindness, neuromuscular deficits and eventually, death. Animal source foods are particularly rich sources of all six of these nutrients, and relatively small amounts of these foods, added to a vegetarian diet, can substantially increase nutrient adequacy. Snacks designed for Kenyan schoolchildren provided more nutrients when animal and plant foods were combined. A snack that provided only 20% of a child's energy requirement could provide 38% of the calcium, 83% of the vitamin B-12 and 82% of the riboflavin requirements if milk was included. A similar snack that included ground beef rather than milk provided 86% of the zinc and 106% of the vitamin B-12 requirements, as well as 26% of the iron requirement. Food guides usually recommend several daily servings from animal source food groups (dairy products and meat or meat alternatives). An index that estimates nutrient adequacy based on adherence to such food guide recommendations may provide a useful method of quickly evaluating dietary quality in both developing and developed countries.

OBJECTIVE

To develop methods of collagen crosslinking the sclera to increase its biomechanical strength for the treatment of progressive myopia.

METHODS

Department of Ophthalmology, Technical University of Dresden, Dresden, Germany.

METHODS

Sagitally oriented scleral strips of 4.0 mm x 8.0 mm were prepared from 5 human postmortem eyes and 50 porcine cadaver eyes and treated with various crosslinking methods including physical crosslinking by combined riboflavin-ultraviolet A (UVA) or rose bengal/white-light irradiation and chemical crosslinking by incubation with glucose, ribose, glyceraldehyde, and glutaraldehyde solutions. Parallel scleral strips from the same eye were used as untreated controls. After crosslinking, stress-strain measurements of the treated and control scleras were performed using a microcomputer-controlled biomaterial tester.

RESULTS

A statistically significant increase in scleral rigidity was found after crosslinking with riboflavin-UVA, with a rise in stress in treated porcine (157%) and human (29%) sclera, and after treatment with glyceraldehyde, with a rise in stress in treated porcine (487%) and human (34%) sclera, and with glutaraldehyde, with a rise in stress in treated porcine (817%) and human sclera (122%) at 8% strain. The other crosslinking methods proved ineffective. The untreated human sclera had a 4-fold higher stiffness than porcine sclera.

CONCLUSIONS

Collagen crosslinking induced by riboflavin-UVA, glyceraldehyde, and glutaraldehyde led to a significant increase in biomechanical strength in human and porcine sclera. Using these methods, collagen crosslinking might become a treatment possibility for progressive myopia. Future animal and clinical studies must determine the best application methods and the long-term effects of increased crosslinking on scleral rigidity and prevent potential toxicity or serious side effects.

Mitochondrial fatty acid oxidation defects have been recognized since the early 1970s. The discovery rate has been rather constant, with 3-4 'new' disorders identified every decade and with the most recent example, ACAD9 deficiency, reported in 2007. In this presentation we will focus on three of the 'old' defects: medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, riboflavin responsive multiple acyl-CoA dehydrogenation (RR-MAD) deficiency, and short-chain acyl-CoA dehydrogenase (SCAD) deficiency. These disorders have been discussed in many publications and at countless conference presentations, and many questions relating to them have been answered. However, continuing clinical and pathophysiological research has raised many further questions, and new ideas and methodologies may be required to answer these. We will discuss these challenges. For MCAD deficiency the key question is why 80% of symptomatic patients are homozygous for the prevalent ACADM gene variation c.985A>> G whereas this is found in only approximately 50% of newborns with a positive screen. For RR-MAD deficiency, the challenge is to find the connection between variations in the ETFDH gene and the observed deficiency of a number of different mitochondrial dehydrogenases as well as deficiency of FAD and coenzyme Q(10). With SCAD deficiency, the challenge is to elucidate whether ACADS gene variations are disease-associated, especially when combined with other genetic/cellular/environmental factors, which may act synergistically.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.

Micronutrient deficiencies and infectious diseases often coexist and exhibit complex interactions leading to the vicious cycle of malnutrition and infections among underprivileged populations of the developing countries, particularly in preschool children. Several micronutrients such as vitamin A, beta-carotene, folic acid, vitamin B12 vitamin C, riboflavin, iron, zinc, and selenium, have immunomodulating functions and thus influence the susceptibility of a host to infectious diseases and the course and outcome of such diseases. Certain of these micronutrients also possess antioxidant functions that not only regulate immune homeostasis of the host, but also alter the genome of the microbes, particularly in viruses, resulting in grave consequences like resurgence of old infectious diseases or the emergence of new infections. These micronutrient infection and immune function interactions and their clinical and public health relevance in developing countries are briefly reviewed in this article.

Mucosal-associated invariant T (MAIT) cells are a unique T cell subset in mammals. They are present at high frequencies at mucosal tissue sites and have an intrinsic capacity to respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells detects the non-polymorphic antigen-presenting molecule major histocompatibility complex class I-related protein 1 (MR1), which can bind microorganism-derived riboflavin metabolites. The striking evolutionary conservation in both the MR1 molecule and the MAIT T cell receptor suggests that strong selective pressures maintain this T cell pattern recognition system which detects microbial infection.

OBJECTIVE

To determine whether corneal collagen cross-linking with riboflavin (C3-R) augments the effect of inferior-segment Intacs (Addition Technology) in the treatment of keratoconus.

METHODS

Private practice, Beverly Hills, California, USA.

METHODS

A retrospective nonrandomized comparative case series comprised 12 eyes of 9 patients who had inferior-segment Intacs placement without C3-R (Intacs-only group) and 13 eyes of 12 patients who had inferior-segment Intacs placement combined with C3-R (Intacs with C3-R group). The 2 groups were matched preoperatively. All patients had inferior-segment Intacs placed with the incision in the steep axis of manifest refraction. Corneal collagen cross-linking with riboflavin was performed after the Intacs segments were inserted. Outcome measures were topographic keratometry values and the lower-upper (L-U) ratio, which is a topographic measure of the degree of keratoconus. Preoperative data were compared to results 1 day postoperatively and measurements at the last postoperative visit.

RESULTS

The Intacs with C3-R group had a significantly greater reduction in cylinder than the Intacs-only group (P<.05). Steep and average keratometry were reduced significantly more in the Intacs with C3-R group (P<.05). There was a greater reduction in L-U ratio in the Intacs with C3-R group (P<.05).

CONCLUSIONS

The addition of C3-R to the Intacs procedure resulted in greater keratoconus improvements than Intacs insertion alone.

BACKGROUND

A comprehensive update of the classification of all available kinases was carried out. This survey presents a complete global picture of this large functional class of proteins and confirms the soundness of our initial kinase classification scheme.

RESULTS

The new survey found the total number of kinase sequences in the protein database has increased more than three-fold (from 17,310 to 59,402), and the number of determined kinase structures increased two-fold (from 359 to 702) in the past three years. However, the framework of the original two-tier classification scheme (in families and fold groups) remains sufficient to describe all available kinases. Overall, the kinase sequences were classified into 25 families of homologous proteins, wherein 22 families (approximately 98.8% of all sequences) for which three-dimensional structures are known fall into 10 fold groups. These fold groups not only include some of the most widely spread proteins folds, such as the Rossmann-like fold, ferredoxin-like fold, TIM-barrel fold, and antiparallel beta-barrel fold, but also all major classes (all alpha, all beta, alpha+beta, alpha/beta) of protein structures. Fold predictions are made for remaining kinase families without a close homolog with solved structure. We also highlight two novel kinase structural folds, riboflavin kinase and dihydroxyacetone kinase, which have recently been characterized. Two protein families previously annotated as kinases are removed from the classification based on new experimental data.

CONCLUSIONS

Structural annotations of all kinase families are now revealed, including fold descriptions for all globular kinases, making this the first large functional class of proteins with a comprehensive structural annotation. Potential uses for this classification include deduction of protein function, structural fold, or enzymatic mechanism of poorly studied or newly discovered kinases based on proteins in the same family.