The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA * chemie   metabolismus   MeSH
• flavinmononukleotid * chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce * MeSH
• simulace molekulární dynamiky MeSH
• světlo * MeSH
• Thermosynechococcus metabolismus   MeSH
• transkripční faktory metabolismus   chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA vazebné proteiny MeSH
• DNA * MeSH
• flavinmononukleotid * MeSH
• flaviny MeSH
• transkripční faktory MeSH

AIM: To evaluate the long-term impact of accelerated corneal cross-linking (A-CXL) on selected refractive and topographical parameters in eyes with progressive keratoconus. METHODS: 77 eyes with keratoconus in 54 patients treated with A-CXL (10 min "epi-off" protocol) were included in the analysis. Preoperative and postoperative (1, 3 and 5 years after A-CXL) values of the studied parameters were compared. RESULTS: In the cohort, there was an improvement in best corrected central visual acuity (BCCVA) 1 year (p = 0.004) and 3 years (p.

• Klíčová slova
• progressive keratoconus; accelerated corneal cross-linking; refractive and topographical parameters,
• MeSH
• dospělí MeSH
• fotochemoterapie MeSH
• fotosenzibilizující látky MeSH
• keratokonus * farmakoterapie   diagnóza   patofyziologie   metabolismus   MeSH
• kolagen metabolismus   MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• reagencia zkříženě vázaná * MeSH
• refrakce oka MeSH
• riboflavin terapeutické užití   MeSH
• rohovková topografie * MeSH
• zraková ostrost * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fotosenzibilizující látky MeSH
• kolagen MeSH
• reagencia zkříženě vázaná * MeSH
• riboflavin MeSH

The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100 mm, 1.7 μm) using a gradient elution with an analysis time of 10 min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000 nmol/L, folic acid 2.5-250 nmol/L, α-tocopherol 0.5-100 μmol/L and all-trans-retinol 25-2500 nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.

• MeSH
• alfa-tokoferol * krev   MeSH
• bariatrická chirurgie MeSH
• chromatografie kapalinová metody   MeSH
• kyselina listová * krev   MeSH
• lidé MeSH
• limita detekce MeSH
• lineární modely MeSH
• reprodukovatelnost výsledků MeSH
• riboflavin * krev   MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• vitamin A * krev   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-tokoferol * MeSH
• kyselina listová * MeSH
• riboflavin * MeSH
• vitamin A * MeSH

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

• Klíčová slova
• LOV2 domain, flavin cofactor, genetically encoded photosensitizers, miniSOG, singlet oxygen,
• MeSH
• aminokyseliny MeSH
• flavinmononukleotid chemie   MeSH
• flavoproteiny * chemie   metabolismus   MeSH
• fotosenzibilizující látky * MeSH
• proteinové domény MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• flavinmononukleotid MeSH
• flavoproteiny * MeSH
• fotosenzibilizující látky * MeSH

OBJECTIVES: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among older adults in developed countries. Although many risk factors are known, the pathogenesis of AMD is still unclear. However, oxidative stress probably plays a vital role in the process of AMD. The increasing prevalence of AMD, risk of vision loss, limited treatment of dry form, expensive treatment of wet form, and decreased quality of life are factors that lead to considering modifiable risk factors of AMD, such as nutrition. This is the first study describing the relationship between dietary habits, dietary nutrient intake and AMD in the Czech Republic. METHODS: In this research, a total of 93 cases with AMD and 58 controls without AMD and cataracts participated. All participants were ophthalmologically examined at the Clinic of Eye Treatments at the University Hospital Brno. Data were collected using a pre-tested self-report questionnaire in a face-to-face interview. Food consumption frequency was assessed by an 18-item semiquantitative food-frequency questionnaire (FFQ). Dietary nutrient intakes were calculated from a 24-hour recall. RESULTS: Patients with AMD compared with controls had significantly higher consumption of legumes and lower consumption of meat products, salt and salty products. In men, we found statistically significant differences in alcohol consumption. The case group consumed alcoholic beverages more frequently (median: 2 times a week) than the control group (median: 1-3 times a month). No differences in alcohol consumption were found in women. In comparison to the case group, the control group had a significantly higher dietary intake of energy (5,783.8 vs. 4,849.3 kJ/day; p = 0.002), proteins (65.3 vs. 52.3 g/day; p = 0.002), fats (57.6 vs. 49.4 g/day; p = 0.046), saturated fatty acids (21.7 vs. 18.9 g/day; p = 0.026), carbohydrates (150.4 vs. 127.1 g/day; p = 0.017), dietary fibre (13.2 vs. 11.3 g/day; p = 0.044), vitamin B2 (1.0 vs. 0.9 mg/day; p = 0.029), vitamin B3 (13.9 vs. 10.0 mg/day; p = 0.011), pantothenic acid (3.5 vs. 2.8 mg/day; p = 0.001), vitamin B6 (1.3 vs. 1.0 mg/day; p = 0.001), potassium (1,656.5 vs. 1,418.0 mg/day; p = 0.022), phosphorus (845.4 vs. 718.7 mg/day; p = 0.020), magnesium (176.5 vs. 143.0 mg/day; p = 0.012), copper (1.0 vs. 0.8 mg/day; p = 0.011), and zinc (7.1 vs. 6.1 mg/day; p = 0.012) counted from a 24-hour recall. CONCLUSIONS: According to FFQ, dietary habits in the patients with AMD and controls were similar. In men from the case group, we found statistically significant higher alcohol consumption. According to a 24-hour recall, the controls achieved recommended dietary intakes rather than cases. In comparison to the case group, the control group had a significantly higher dietary intake of energy, proteins, fats, saturated fatty acids, carbohydrates, dietary fibre, vitamin B2, vitamin B3, pantothenic acid, vitamin B6, potassium, phosphorus, magnesium, copper, and zinc.

• Klíčová slova
• age-related macular degeneration, antioxidants, dietary habits, food groups, oxidative stress,
• MeSH
• dieta MeSH
• dietní tuky MeSH
• draslík MeSH
• energetický příjem MeSH
• fosfor MeSH
• hořčík * MeSH
• kvalita života MeSH
• kyselina pantothenová MeSH
• lidé MeSH
• makulární degenerace * epidemiologie   chemicky indukované   MeSH
• mastné kyseliny MeSH
• měď MeSH
• niacinamid MeSH
• potravní vláknina MeSH
• přijímání potravy MeSH
• riboflavin MeSH
• senioři MeSH
• stravovací zvyklosti MeSH
• studie případů a kontrol MeSH
• vitamin B6 MeSH
• zinek MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dietní tuky MeSH
• draslík MeSH
• fosfor MeSH
• hořčík * MeSH
• kyselina pantothenová MeSH
• mastné kyseliny MeSH
• měď MeSH
• niacinamid MeSH
• potravní vláknina MeSH
• riboflavin MeSH
• vitamin B6 MeSH
• zinek MeSH

BACKGROUND: Pathogen reduction technology (PRT) may improve the safety of RBCs for transfusion. As the Czech Republic considers PRT, we asked what effects riboflavin and UV light PRT pre-freezing has on the post-thaw recovery and properties of cryopreserved RBCs (CRBCs) after deglycerolization and liquid storage. STUDY DESIGN AND METHODS: 24 Group O whole blood (WB) units were leukoreduced and then treated with riboflavin and UV light PRT (Mirasol, Terumo BCT, USA) before cryopreservation (T-CRBC); 20 similarly-collected units were untreated controls (C-CRBC). Units were processed to RBCs and then cryopreserved with 40% glycerol (wt/vol), frozen at -80°C, stored >118 days, reconstituted as deglycerolized RBC units in AS-3, and stored at 4 ± 2°C for 21 days. One treated unit sustained massive hemolysis during the post-thaw wash process and was removed from data analysis. The remaining units were assessed pre-PRT, post-PRT, and post-thaw-wash on days 0, 7, 14, and 21 for hematocrit, volume, hemoglobin per transfusion unit, pH, % hemolysis, hemoglobin in the supernatant, potassium, phosphorus, NH3 , osmolality, ATP, and 2,3-diphosphoglycerate. RESULTS: PRT with leukoreduction caused a 5% loss of RBC followed by a 24% freeze-thaw-wash related loss for a total 28% loss but treated units contained an average of 45 g of hemoglobin, meeting European Union guidelines for CRBC. T-CRBCs displayed higher post-wash hemolysis, potassium, and ammonia concentrations, and lower ATP at the end of storage. CONCLUSIONS: Cryopreserved RBCs from Riboflavin and UV light-treated WB meet the criteria for clinical use for 7 days after thawing and provide additional protection against infectious threats.

• Klíčová slova
• blood logistics, blood safety, blood storage, red cell additive solution-3,
• MeSH
• adenosintrifosfát MeSH
• draslík analýza   MeSH
• erytrocyty MeSH
• hemoglobiny analýza   MeSH
• hemolýza * MeSH
• konzervace krve MeSH
• kryoprezervace MeSH
• lidé MeSH
• riboflavin farmakologie   MeSH
• ultrafialové záření * MeSH
• zmrazování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• adenosintrifosfát MeSH
• draslík MeSH
• hemoglobiny MeSH
• riboflavin MeSH

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

• Klíčová slova
• FTIR spectroscopy, UV/vis spectroscopy, flavoproteins, genetic code expansion, kinetics, photosensory receptors, protein structural dynamics, signal transduction, site-specific vibrational probes, time-resolved methods,
• MeSH
• aminokyseliny * metabolismus   MeSH
• flavinmononukleotid * chemie   MeSH
• regulace genové exprese MeSH
• transkripční faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny * MeSH
• flavinmononukleotid * MeSH
• transkripční faktory MeSH

BACKGROUND: Pathogen reduction technology (PRT) is increasingly used in the preparation of platelets for therapeutic transfusion. As the Czech Republic considers PRT, we asked what effects PRT may have on the recovery and function of platelets after cryopreservation (CP), which we use in both military and civilian blood settings. STUDY DESIGN AND METHODS: 16 Group O apheresis platelets units were treated with PRT (Mirasol, Terumo BCT, USA) before freezing; 15 similarly collected units were frozen without PRT as controls. All units were processed with 5-6% DMSO, frozen at - 80 °C, stored > 14 days, and reconstituted in thawed AB plasma. After reconstitution, all units were assessed for: platelet count, mean platelet volume (MPV), platelet recovery, thromboelastography, thrombin generation time, endogenous thrombin potential (ETP), glucose, lactate, pH, pO2, pCO2, HCO3, CD41, CD42b, CD62, Annexin V, CCL5, CD62P, and aggregates > 2 mm and selected units for Kunicki score. RESULTS: PRT treated platelet units had lower platelet number (247 vs 278 ×109/U), reduced thromboelastographic MA (38 vs 62 mm) and demonstrated aggregates compared to untreated platelets. Plasma coagulation functions were largely unchanged. CONCLUSIONS: Samples from PRT units showed reduced platelet number, reduced function greater than the reduced number would cause, and aggregates. While the platelet numbers are sufficient to meet the European standard, marked platelets activation with weak clot strength suggest reduced effectiveness.

• Klíčová slova
• Cryopreservation, PRT, Platelets,
• MeSH
• konzervace krve MeSH
• kryoprezervace MeSH
• kyselina mléčná MeSH
• lidé MeSH
• riboflavin farmakologie   MeSH
• separace krevních složek * MeSH
• trombin MeSH
• trombocyty fyziologie   MeSH
• ultrafialové záření * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyselina mléčná MeSH
• riboflavin MeSH
• trombin MeSH

The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pKa1 = 6.6 and pKa2 = 9.2 at 2 μM FMN while it contained only a descending limb pKa of 9.7 at 50 μM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS-hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin.

• Klíčová slova
• FMN, NADH, Paracoccus denitrificans, dioxygen reduction,
• MeSH
• flavinmononukleotid chemie   MeSH
• flaviny chemie   MeSH
• katalýza MeSH
• kinetika MeSH
• NAD metabolismus   MeSH
• oxidace-redukce MeSH
• Paracoccus denitrificans * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• flavinmononukleotid MeSH
• flaviny MeSH
• NAD MeSH
• NADH oxidase MeSH Prohlížeč

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

• MeSH
• bakteriální proteiny chemie   MeSH
• flavinadenindinukleotid chemie   MeSH
• fotoreceptory mikroorganismů * chemie   MeSH
• glutamin * chemie   MeSH
• organické látky MeSH
• protony MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• světlo MeSH
• tyrosin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• flavinadenindinukleotid MeSH
• fotoreceptory mikroorganismů * MeSH
• glutamin * MeSH
• organické látky MeSH
• protony MeSH
• tyrosin MeSH