BACKGROUND

Fermentable, short chain carbohydrates (FODMAPs) have been identified as triggers for functional gastrointestinal symptoms. In addition, excess FODMAP consumption has been implicated in the onset of Crohn's disease, and animal studies suggest that a low glycemic index diet can impair absorption of fructose, a major dietary FODMAP. Such hypotheses cannot be tested without the ability to quantify FODMAP ingestion with a validated dietary assessment tool.

OBJECTIVE

To assess the validity and reproducibility of a 297-item comprehensive, semi-quantitative food frequency questionnaire (FFQ) in estimating intake of macro- and micronutrients, FODMAPs, and glycemic index/load.

METHODS

One hundred healthy participants were recruited to complete the FFQ on two occasions, plus four 1-week food diaries kept during a 12-month period. Participants exhibiting major dietary change during the study period or low energy reporting on the FFQ were excluded.

METHODS

Validation and reproducibility of the semi-quantitative FFQ by comparison with the mean of four 1-week food diaries.

METHODS

Validation was assessed using Wilcoxon signed rank test, Spearman's correlation, Bland-Altman, and weighted κ statistics. Reproducibility was examined using Shrout-Fleiss intraclass correlation coefficient.

RESULTS

Seventy-two participants fulfilled inclusion and exclusion criteria. Demographics of the participants were comparable with 2006 Australian Census data. Consistent with other reported FFQs, the FFQ overestimated nutrient intake by a mean 140% (range=95% to 249%). However, based on the other analyses performed, it demonstrated validity for intake of sugars, fiber, alcohol, glycemic index, glucose, FODMAPs, calcium, folate, phosphate, potassium, iron, and magnesium; moderate validation for energy, total fat, saturated fat, carbohydrates, sodium, thiamin, sucrose, and retinol; poor validation for protein, mono/polyunsaturated fat, starch, glycemic load, niacin, and zinc. Riboflavin intake was not validated. Intraclass correlation coefficients for reproducibility ranged from 0.352 to 0.928.

CONCLUSIONS

The FFQ was validated for assessment of a wide range of nutrients, including the new class of carbohydrates, FODMAPs, and glycemic index. This provides a useful tool for dietary research, particularly in the area of gastroenterological disorders.

The model nephrotoxin cyclosporin A was administered to male Wistar-derived rats daily for 9 days at a dose level of 45 mg/kg per day. Urine samples were collected daily and the excretion pattern of low molecular mass organic molecules in the urine was studied using 1H NMR spectroscopy and HPLC-TOF/MS. Distinct changes in the pattern of endogenous metabolites, as a result of the daily administration of cyclosporin A, were observed by 1H NMR from day 7 onwards. The NMR-detected markers included raised concentrations of glucose, acetate, trimethylamine and succinate and reduced amounts of trimethylamine-N-oxide. In parallel studies by HPLC-TOF/MS a reduction in the quantities of kynurenic acid, xanthurenic acid, citric acid and riboflavin present in the urines was noted, together with reductions in a number of as yet unidentified compounds. In addition, signals resulting from the polyethylene glycol, present in the dosing vehicle, and cyclosporin A metabolites were detected by MS. However, these were excluded from the subsequent multivariate data analysis in order to highlight only changes to the endogenous metabolites. Analysis of both the 1H NMR and HPLC-MS spectroscopic data using pattern recognition techniques clearly identified the onset of changes due to nephrotoxicity.

Riboflavin-sensitized photodynamic modification of collagen led to significant formation of cross-linked molecules. Sodium azide or 1,4-diazabicyclo(2,2,2)octane, which are known to be singlet oxygen quenchers, and catalase could not inhibit the modification. Surprisingly, the collagen modification was accelerated in the presence of superoxide dismutase. The aggregation was accompanied by the loss of tyrosine and histidine residues in the collagen. An inhibitory effect of dissolved oxygen on the modification of collagen was observed. Similarly, the loss of tyrosine residues in the irradiated collagen was inhibited in the presence of dissolved oxygen. Dityrosine formation was also observed with the loss of tyrosine. These results indicate that photodynamic modification of tyrosine probably contributes to the riboflavin-sensitized cross-linking of collagen through the formation of dityrosine.

Collagen has shown promise as a bioink for extrusion-based bioprinting, but further development of new collagen bioink formulations is necessary to improve their printability. Screening these formulations by measuring print accuracy is a costly and time consuming process. We hypothesized that rheological properties of the bioink before, during, and/or after gelation can be used to predict printability. In this study, we investigated the effects of riboflavin photocrosslinking and pH on type I collagen bioink rheology before, during, and after gelation and directly correlated these findings to the printability of each bioink formulation. From the riboflavin crosslinking study, results showed that riboflavin crosslinking increased the storage moduli of collagen bioinks, but the degree of improvement was less pronounced at higher collagen concentrations. Dots printed with collagen bioinks with riboflavin crosslinking exhibited smaller dot footprint areas than those printed with collagen bioinks without riboflavin crosslinking. From the pH study, results showed that gelation kinetics and final gel moduli were highly pH dependent and both exhibited maxima around pH 8. The shape fidelity of printed lines was highest at pH 8-9.5. The effect of riboflavin crosslinking and pH on cell viability was assessed using bovine chondrocytes. Cell viability in collagen gels was found to decrease after blue light activated riboflavin crosslinking but was not affected by pH. Correlations between rheological parameters and printability showed that the modulus associated with the bioink immediately after extrusion and before deposition was the best predictor of bioink printability. These findings will allow for the more rapid screening of collagen bioink formulations.

BACKGROUND

To be successful, dietary guidance needs to identify foods that are nutrient rich, affordable, and appealing. Analyses of dietary surveys on "what we eat in America" can now be supplemented by analyses of nutrient density as well as nutrient cost.

OBJECTIVE

To explore the relative contribution of 9 food groups to energy and nutrient intakes and to assess the relative cost of selected nutrients by major food group.

METHODS

Dietary intake data were provided by the 4 cycles of the continuous National Health and Nutrition Examination Survey (NHANES 2001-2002, 2003-2004, 2005-2006, and 2007-2008). Research on the nutritive value and cost of U.S. foods was made possible by the merging of the U.S. Department of Agriculture (USDA) Food and Nutrition Database for Dietary Studies (FNDDS 2.0) with the USDA food prices database. Nutrient densities were calculated per 100 kcal. Nutrient costs were calculated as the dollar cost of meeting 10% daily value for a given nutrient.

RESULTS

Despite their low energy contribution (10%-13% of energy), milk and milk products contributed 47% of calcium, 42% of retinol, and 65% of vitamin D to the diets of children and adults. Milk and milk products were among the top sources of riboflavin, phosphorous, and vitamin B(12). Cost analyses showed that milk and milk products were by far the lowest-cost source of dietary calcium and were among the lowest-cost sources of riboflavin and vitamin B(12). Vegetables and fruit were the lowest-cost sources of vitamin C, whereas dry beans and legumes were the lowest-cost sources of fiber.

CONCLUSIONS

The nutrients-per-calorie and nutrient cost metrics can help identify affordable nutrient-rich foods.

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.

OBJECTIVE

To investigate the dietary pattern and nutritional status of adolescent girls attending schools in Dhaka city and to examine the association with various social factors.

METHODS

Cross-sectional study.

METHODS

Girls high schools in Dhaka city.

METHODS

A total of 384 girls, aged from 10 to 16 years, who were students of classes VI to IX of 12 girls high schools in Dhaka city were selected by systematic random sampling. Nutrient intake was assessed using the 24-h recall method and the usual pattern of food intake was examined using a 7-day food frequency questionnaire.

RESULTS

The prevalence of undernutrition among the participants assessed as stunting was 10% overall with younger girls being less stunted (2%) than older girls (16%), whereas 16% were thin with relatively more of the younger girls (21%) being thin than of the older girls (12%). Based on the usual pattern of food intake, a substantial proportion of the girls did not consume eggs (26%), milk (35%) or dark green leafy vegetables (20%). By comparison, larger proportions consumed meat (50%) and fish (65%) at least four times a week. For the intake of energy and protein, only 9 and 17% of the girls, respectively, met the recommended daily allowance (RDA). For nearly 77% of the girls, the intake of fat was less than the recommendation. Intakes less than the RDA were found for iron (77% of the girls), calcium (79%), vitamin A (62%), vitamin C (67%), and riboflavin (96%). Based on the food consumption data, cereals were the major source of energy (57%), thiamin (67%), niacin (63%) and iron (37%). Animal sources supplied 50% of dietary protein. Cooking fats were the principal source of fat (67%) in the diet. Milk was the major contributor for riboflavin and preformed vitamin A (retinol). Leafy vegetables and fruits were the main sources of provitamin A (carotenes). The girls from families with less educated parents were more likely to be thin and short for their age. Those girls from families with lower incomes and less educated parents had a dietary pattern which tended to be poor with regard to egg, milk, meat and fruit, with lower intakes of protein, fat and riboflavin.

CONCLUSIONS

The findings indicate that the diets of these girls tended to be inadequate both for macronutrients and micronutrients, with significant health implications. There was also a relationship between the family income and the education of the parents with the nutritional status of the girls.

The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 degrees C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins.

OBJECTIVE

To evaluate the distribution of riboflavin in the corneal stroma, under varying concentrations and application time.

METHODS

In 54 porcine eyes, the central corneal epithelium was removed, and 0.035, 0.1, or 0.2% riboflavin-5-phosphate (in 20% Dextran T-500) was applied for 10, 20, or 30 min (3 x 6 corneas in each of the 3 groups). Trephined corneal buttons were examined using confocal fluorescence microscopy. Stromal riboflavin distribution and concentration was determined by measuring riboflavin fluorescence in optical sections at 10 microm intervals through the entire cornea. The procedure was repeated in 7 human corneal donor grafts using 0.1% riboflavin-5-phosphate for 20 or 30 min.

RESULTS

In porcine corneas, fluorescence intensity peaked within the first 50 microm followed by a steep decline to baseline. Increasing the riboflavin concentration from 0.1 to 0.2% did not increase stromal depth propagation, although a higher concentration in the anterior 200 microm was observed. Reducing the riboflavin application time from 30 to 20 min had no impact on corneal depth propagation or total riboflavin uptake. However, a 10-min further reduction of the application time caused a significantly reduced riboflavin uptake. In all human corneas, fluorescence peaked within the anterior 50 microm, followed by a steep decline to baseline over the next 200 microm; similar to the observations in porcine corneas. The human corneas imbibed more riboflavin compared to the porcine corneas.

CONCLUSIONS

In human and porcine corneas, riboflavin does not appear to fully load the corneal stroma using the current clinical procedure. Instead, the uptake appears to be limited to the anterior approximately 200 microm. Changes in application time and riboflavin concentration have only little influence on stromal depth diffusion.

Forty-three patients on chronic hemodialysis who before the present study had only received a low-dose supplement of folic and ascorbic acid were studied prospectively for one year. After baseline values were obtained in month one, increasing doses of postdialysis vitamin supplements were prescribed for the vitamins which were found to be insufficient in order to determine the minimal amount of oral postdialysis supplement necessary to normalize vitamin levels. According to our results no systematic supplement was indicated for biotin, riboflavin or vitamin B12. For folic acid and vitamin C, supplementation with lower doses than those prescribed in many dialysis units allowed optimal vitamin levels in the majority of patients; 2 to 3 mg/week (300 to 400 micrograms/day) of folic acid and of 1000 to 1500 mg/week (150 to 200 mg/day) of vitamin C was considered sufficient. A severe pyridoxine deficiency was present in most >> 80%) unsupplemented patients, either as judged by pyridoxal-5-phosphate determinations in plasma or determination of specific enzyme activation in erythrocytes (EGOTo and alpha-EGOT); a postdialysis supplement of at least 100 to 150 mg/week of pyridoxine hydrochloride >> 15 to 20 mg/day) corrects this deficiency. The activity of the thiamine-dependent enzyme transketolase in erythrocytes (ETKo) was insufficient in 35% and marginal in 21% of the patients, while whole blood thiamine determined simultaneously in 10 of the ETKo-deficient patients was within the normal range. These results suggest that in uremia insufficient transketolase activity may be related to inhibition of the enzymatic system rather than to true vitamin deficiency. On a long-term basis a supplement of 200 to 300 mg/week of thiamine hydrochloride (30 to 45 mg/day) restored ETKo to satisfactory levels in most patients; whether this supplement is to be recommended warrants further studies.

Nitric oxide (NO) and reactive oxygen species (ROS) play key roles in plant immunity. However, the regulatory mechanisms of the production of these radicals are not fully understood. Hypersensitive response (HR) cell death requires the simultaneous and balanced production of NO and ROS. In this study we indentified NbRibAencoding a bifunctional enzyme, guanosine triphosphate cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase, which participates in the biosynthesis of flavin, by screening genes related to mitogen-activated protein kinase-mediated cell death, using virus-induced gene silencing. Levels of endogenous riboflavin and its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important prosthetic groups for several enzymes participating in redox reactions, decreased in NbRibA-silenced Nicotiana benthamiana. Silencing NbRibA compromised not only HR cell death, but also the NO and ROS production induced by INF1 elicitin and a constitutively active form of NbMEK2 (NbMEK2DD), and also induced high susceptibility to oomycete Phytophthora infestans and ascomycete Colletotrichum orbiculare. Compromised radical production and HR cell death induced by INF1 in NbRibA-silenced leaves were rescued by adding riboflavin, FMN or FAD. These results indicate that flavin biosynthesis participates in regulating NO and ROS production, and HR cell death.

The effect of some B vitamins in chemical and thermal models of nociception in mice was investigated. The association thiamine/pyridoxine/cyanocobalamin (TPC, 20-200 mg/kg, i.p. or per os), thiamine, pyridoxine (50-200 mg/kg, i.p.) or riboflavin (3-100 mg/kg, i.p) induced an antinociceptive effect, not changed by naloxone (10 mg/kg, i.p.), in the acetic acid writhing model. Treatment for 7 days with thiamine/pyridoxine/cyanocobalamin (100 or 200 mg/kg, i.p.), thiamine (50 or 100 mg/kg) or pyridoxine (50 or 100 mg/kg) or acute treatment with riboflavin (6 or 12 mg/kg, i.p) inhibited the nociceptive response induced by formaldehyde. The B vitamins did not inhibit the nociceptive response in the hot-plate model. Both 7-day thiamine/pyridoxine/cyanocobalamin (100 mg/kg, i.p.) or acute riboflavin (25 or 50 mg/kg, i.p.) treatment partially reduced formaldehyde-induced hindpaw oedema. The B vitamins antinociceptive effect may involve inhibition of the synthesis and/or action of inflammatory mediators since it was not observed in the hot-plate model, was not reversed by naloxone, only the second phase of the formaldehyde-induced nociceptive response was inhibited, and formaldehyde-induced hindpaw oedema was reduced.

The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.

The wasp Campoletis sonorensis injects a polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. CsPDV protects the wasp egg and larvae by selectively disabling the host's cellular immune response, and by altering host physiology, growth, and development. Among the changes in host physiology brought about by CsPDV infection is a rapid, and specific decline in the translation of fat body mRNAs encoding selected major plasma proteins. Translational inhibition of the synthesis of all storage protein monomers, p82 (Riboflavin binding hexamer), and p74/p76 (arylphorin), occurs upon infection with CsPDV. Moreover, the prewandering peak of the plasma enzyme juvenile hormone esterase (JHE) was blocked by CsPDV injection. Northern blotting of fat body mRNA demonstrated that transcript levels of storage proteins were not affected by infection. Plasma titers of the iron binding proteins transferrin (p72) and ferritin (p24/26), and of the plasma juvenile hormone binding protein (p25) were not changed by CsPDV infection. That storage protein and JHE synthesis are translationally suppressed, while the synthesis of other plasma proteins continues apace, suggests that CsPDV infection may lead to translational discrimination among available mRNAs in CsPDV infected fat bodies. The effect of this translational discrimination is to shunt host resources away from larval growth and adult development, which presumably makes them available to the developing endoparasitoid.

OBJECTIVE

Hyperhomocysteinemia is regarded as a public health problem of increasing importance likely to contribute to vascular disorders and premature mortality. Folate, cobalamin, pyridoxine, and riboflavin dietary deficiencies are currently regarded as causative factors. However, several investigations have indicated that the theory of vitamin B deprivation provides only a partial explanation for the observed abnormalities of sulfur-containing amino acids. We investigated the potential contributory role played by protein malnutrition.

METHODS

For that purpose, three cohorts of 20 adult patients presenting stage I, II, and III goiter underwent careful medical history, dietary inquiry, and clinical examination. Their overall health and nutrition states were assessed with classic anthropometry, measurement of vitamin B blood parameters, visceral protein markers, essential amino acids, total homocysteine, and cystathionine.

RESULTS

The concentrations of transthyretin, seven essential amino acids, and cystathionine progressively decreased as the thyroid gland increased. Methionine was the sole essential amino acid whose values did not change; total homocysteine was unique in that increased levels correlated negatively with transthyretin values. Taken together, the data point to a progressive deterioration of protein nutrition status impairing the transsulfuration pathway and is best explained by an acquired defect of cystathionine-beta-synthase activity.

CONCLUSIONS

Hyperhomocysteinemia may arise from the shrinking of endogenous nitrogen pools as a result of decreased protein intake or stress-induced increased losses. Raised total homocysteine may result from the attempt of the malnourished and/or stressed body to preserve methionine homeostasis.

BACKGROUND

Evidence from animal models suggests that redox homeostasis (the balance between oxidative stressors and antioxidants) and vascular health are important in the pathogenesis of sensorineural hearing loss (SNHL) and that dietary nutrients that have roles in these processes could influence the susceptibility to SNHL.

OBJECTIVE

To examine associations between total nutrient intakes and auditory function outcomes in an older human population.

METHODS

Descriptive characteristics and dietary data from food frequency questionnaires were collected in a cross-sectional study design and analyzed for associations with auditory function outcomes (i.e., otoacoustic emissions and pure tone audiometry measured in a sound-treated room by an audiologist).

METHODS

2111 adults, 49-99 yr of age

RESULTS

Higher carbohydrate, vitamin C, vitamin E, riboflavin, magnesium, and lycopene intakes were all significantly associated with larger TEOAE amplitude and better pure tone thresholds. Higher cholesterol, fat, and retinol intakes were significantly associated with lower TEOAE amplitude and worse pure tone thresholds.

CONCLUSIONS

These data suggest that nutrients with known roles in redox homeostasis and vascular health are associated with auditory function measures in a human population. Further investigation is warranted to determine direct and indirect influences of dietary intake on measures of auditory function and to explore which nutrients/nutrient combinations are predictive of SNHL.

In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given.

BACKGROUND

Identifying structurally similar proteins with different chain topologies can aid studies in homology modeling, protein folding, protein design, and protein evolution. These include circular permuted protein structures, and the more general cases of non-cyclic permutations between similar structures, which are related by non-topological rearrangement beyond circular permutation. We present a method based on an approximation algorithm that finds sequence-order independent structural alignments that are close to optimal. We formulate the structural alignment problem as a special case of the maximum-weight independent set problem, and solve this computationally intensive problem approximately by iteratively solving relaxations of a corresponding integer programming problem. The resulting structural alignment is sequence order independent. Our method is also insensitive to insertions, deletions, and gaps.

RESULTS

Using a novel similarity score and a statistical model for significance p-value, we are able to discover previously unknown circular permuted proteins between nucleoplasmin-core protein and auxin binding protein, between aspartate rasemase and 3-dehydrogenate dehydralase, as well as between migration inhibition factor and arginine repressor which involves an additional strand-swapping. We also report the finding of non-cyclic permuted protein structures existing in nature between AML1/core binding factor and ribofalvin synthase. Our method can be used for large scale alignment of protein structures regardless of the topology.

CONCLUSIONS

The approximation algorithm introduced in this work can find good solutions for the problem of protein structure alignment. Furthermore, this algorithm can detect topological differences between two spatially similar protein structures. The alignment between MIF and the arginine repressor demonstrates our algorithm's ability to detect structural similarities even when spatial rearrangement of structural units has occurred. The effectiveness of our method is also demonstrated by the discovery of previously unknown circular permutations. In addition, we report in this study the finding of a naturally occurring non-cyclic permuted protein between AML1/Core Binding Factor chain F and riboflavin synthase chain A.

Human plasma for therapeutic use, besides having optimal viral safety, must contain optimal levels of all coagulation factors and protease inhibitors to be clinically effective. Several new technologies for pathogen reduction of plasma (PRT) exist and are entering the stage of clinical testing. The main objective of this overview is to provide an update on the current states of three promising photoactive technologies that target pathogen nucleic acid for pathogen inactivation, applicable to single unit fresh-frozen plasma (FFP) and to highlight the experiences gained with classical pathogen reduction of pooled plasma using solvent-detergent (SD) treatment. It should be emphasized that none of the currently applied methods inactivate all types of pathogens and all have some effect on plasma quality when compared to fresh-frozen plasma. Pooled SD-plasma is the best documented clinical product, followed by methylene blue light treated (MBLT)-plasma. Recently, Psoralen light treated (PLT)-plasma has been introduced (CE-marked product in Europe) while Riboflavin light treated (RLT)-plasma is still under development. In principal, PRT for plasma not only differs in terms of the spectrum and log of pathogen reduction potential, but also in respect to the physicochemical/biological characteristics, and profiles of the adverse reactions, particularly in vulnerable patient groups. Therefore, an additional practical step such as oil extraction followed by chromatography to remove the solvent/detergent, and filtration or the use of some special absorbing matrix is required to reduce the residual photosensitive chemicals, their metabolites and photo adducts. This is required to improve the safety margin of the final product. Moreover, while it may be convenient to think that a combined pathogen reduction technology could improve the spectrum of known pathogens to be inactivated, one needs, in practice, to balance between the degree of pathogen reduction and the loss of some plasma protein activity. From the quality point of view, SD-plasma is a pooled standardized pharmaceutical product with extensive in-process control. However, both differences in production processes and the plasma source can influence final product quality. On the other hand, single unit plasma derived from nucleic acid PRT cannot be monitored by pharmaceutical process control and demonstrates the wide range of concentrations normally observed for plasma proteins. Pooling has the disadvantage that one single plasma unit can contaminate a whole pool, but this can be offset by several advantages that pooling and the SD process offer. Among these are reduction of a possible pathogen load by dilution and by neutralizing antibodies in the plasma pool, dilution and possible neutralization of antibodies and allergens which essentially eliminates transfusion-related acute lung injury (TRALI) and reduces allergic reactions significantly, removal of residual blood cells, cell fragments and bacteria, and removal of the largest von Willebrand-factor (vWF) molecules. On the other hand, some streamlining is required for technologies using single units of plasma, such as the use of plasma from male non-transfused donors to reduce TRALI and to avoid the O blood group in order to meet current specifications for FFP [Seghatchian J. What is happening? Are the current acceptance criteria for therapeutic plasma adequate? Transfus Apheresis Sci 2004; 31:67-79], and to exploit the potential benefit to inactivate residual lymphocytes and prevent transfusion-associated graft versus host disease. The cost effectiveness of pathogen inactivation is very low >> 2 million US dollar/life year saved), if however, non-infectious complications such as TRALI are taken into account; the cost for SDP is reduced to < 50,000 British pound/life year saved for those 48 years. Finally, from the therapeutic standpoint, two important questions still remain to be answered. First, whether the various pathogen reduced plasma products are clinically interchangeable and second, whether the conventional quality requirements of FFP are still adequate for the newer plasma products. These questions can only be answered by a head to head comparison, followed by large-scale clinical trials.

BACKGROUND

Pathogen reduction technologies for platelet (PLT) components offer a means to address continued viral transmission risks and imperfect bacterial detection systems. The efficacy of apheresis PLTs treated with riboflavin (vitamin B2) plus ultraviolet (UV) light (Mirasol, Navigant Biotechnologies) was investigated in a single-blind, crossover study in comparison to untreated PLTs.

METHODS

Normal subjects (n = 24) donated PLTs by apheresis on two occasions at least 2 weeks apart. Units were randomized to control or test arms, the latter receiving the addition of 28 mL of 500 micromol per L B2 and exposure to 6.2 J per mL UV light. PLTs were stored for 5 days with biochemical and hematologic analyses performed before and after illumination on Day 0 and at the end of storage. An aliquot of each unit was radiolabeled and returned to determine recovery and survival.

RESULTS

The PLT content of treated units was maintained from Day 0 (4.1 x 10(11) +/- 0.4 x 10(11)) to Day 5 (4.0 x 10(11) +/- 0.4 x 10(11)). Treatment with B2 plus UV light was associated with an increase in lactate production with concomitant increases in glucose consumption. pH (control, 7.38 +/- 0.07; test, 7.02 +/- 0.10) was well maintained throughout storage. Recovery of treated PLTs (50.0 +/- 18.9%) was reduced from that of control PLTs (66.5 +/- 13.4%); survival was similarly shortened (104 +/- 26 hr vs. 142 +/- 26 h; p < 0.001).

CONCLUSIONS

PLTs treated with B2 plus UV light demonstrate some alterations in in vitro measures but retain in vitro and in vivo capabilities similar to pathogen-reduced and licensed PLT components that have been shown to have useful clinical applicability. The recovery, survival, and metabolic properties of Mirasol PLTs should provide sufficient hemostatic support in thrombocytopenia to justify patient clinical trials.

Of the 617 genes from Buchnera aphidicola, the obligate bacterial symbiont of the pea aphid, 23% were differentially expressed in embryos compared to adults. Genes involved in flagellar apparatus and riboflavin synthesis exhibited particularly robust upregulation in embryos, suggesting functional differences between the symbiosis in the adult and embryo insect.

BACKGROUND

Pathogen reduction of platelets (PRT-PLTs) using riboflavin and ultraviolet light treatment has undergone Phase 1 and 2 studies examining efficacy and safety. This randomized controlled clinical trial (RCT) assessed the efficacy and safety of PRT-PLTs using the 1-hour corrected count increment (CCI(1hour) ) as the primary outcome.

METHODS

A noninferiority RCT was performed where patients with chemotherapy-induced thrombocytopenia (six centers) were randomly allocated to receive PRT-PLTs (Mirasol PRT, CaridianBCT Biotechnologies) or reference platelet (PLT) products. The treatment period was 28 days followed by a 28-day follow-up (safety) period. The primary outcome was the CCI(1hour) determined using up to the first eight on-protocol PLT transfusions given during the treatment period.

RESULTS

A total of 118 patients were randomly assigned (60 to PRT-PLTs; 58 to reference). Four patients per group did not require PLT transfusions leaving 110 patients in the analysis (56 PRT-PLTs; 54 reference). A total of 541 on-protocol PLT transfusions were given (303 PRT-PLTs; 238 reference). The least square mean CCI was 11,725 (standard error [SE], 1.140) for PRT-PLTs and 16,939 (SE, 1.149) for the reference group (difference, -5214; 95% confidence interval, -7542 to -2887; p<0.0001 for a test of the null hypothesis of no difference between the two groups).

CONCLUSIONS

The study failed to show noninferiority of PRT-PLTs based on predefined CCI criteria. PLT and red blood cell utilization in the two groups was not significantly different suggesting that the slightly lower CCIs (PRT-PLTs) did not increase blood product utilization. Safety data showed similar findings in the two groups. Further studies are required to determine if the lower CCI observed with PRT-PLTs translates into an increased risk of bleeding.