The hypoallergen peanut genotypes with low levels of allergen proteins for Ara h 1 (4 µg/g), Ara h 2 (41 µg/g), Ara h 3 (22 µg/g), Ara h 6 (829 µg/g), and Ara h 8 (0.01 µg/g) have paved the way for their use in breeding and genomics studies. In addition, these hypoallergen peanut genotypes are available for use in cultivation and industry, thus opened up new vistas for fighting against peanut allergy problem across the world.Peanut allergy is one of the serious health concern and affects more than 1% of the world’s population mainly in Americas, Australia, and Europe. Peanut allergy is sometimes life-threatening and adversely affect the life quality of allergic individuals and their families. Consumption of hypoallergen peanuts is the best solution, however, not much effort has been made in this direction for identifying or developing hypoallergen peanut varieties. A highly diverse peanut germplasm panel was phenotyped using a recently developed monoclonal antibody-based ELISA protocol to quantify five major allergens. Results revealed a wide phenotypic variation for all the five allergens studied  Although there may be several cases of food allergy in India , largely due to the consumption of peanuts (50–62%) and tree nuts (15–30%) in USA  in USA . Protein) in USA . Peanut ) in USA . However) in USA . The thr) in USA . The dia) in USA .Of the 32 different types of proteins present in peanut seeds , 18 of tPossible solutions to peanut allergy include the development of vaccine or development of allergen-free peanut varieties. Much research has been focussed on diagnosis and cure to minimize the impact of allergens in the human population, however, reducing allergen proteins in peanut varieties and their products can be the best solution. Unfortunately, insufficient scientific information on a total number of allergen genes in the peanut genome and level of phenotypic variability in existing peanut germplasm hinders further research in this area. Therefore, the main objective of this research was to identify hypoallergen peanut lines by screening a large number of diverse germplasm in the peanut reference set . The hypArachis hypogaea) and 36 wild species and has fair representation for its two subspecies namely fastigiata (154) and hypogaea (95). The subspecies fastigiata was further classified into four botanical varieties namely Fastigiata (70 accessions), Peruviana (5 accessions), Vulgaris (78 accessions), and Aequitoriana (1 accession). Similarly, the other subspecies hypogaea was classified into two botanical varieties namely Hirsuta (2 accessions) and Hypogaea (93 accessions) (The peanut “reference set” consisting of 300 diverse accessions representing 51 countries  Supple that repessions) . These cSample preparation and protein isolation were performed following the protocol mentioned in . In brieSandwich format ELISA was used in the study. The peanut allergen proteins were first sandwiched between two antibodies, and then streptavidin-peroxidase was captured. Each peanut sample contains a different quantity of allergen proteins which makes very difficult to estimate the accurate amount of allergen proteins present in seed samples. Dilution is a vital step for ELISA experiment which in turn determines the values of detection range for antibody and target antigen concentrations.The estimation of peanut allergen through sandwich ELISA were performed according to the recently published protocol . Each alhttp://www.sigmaplot.co.uk/products/sigmaplot/sigmaplot-details.php). Hierarchical clustering was done using average allergens content of five major allergens  on the basis of similarity matrix generated using HCA . Dendextend statistical package  showed wide phenotypic variations for all the five allergens. Among cultivated accessions, the phenotypic variation was very high for all five major allergens namely Ara h 1 , Ara h 2 , Ara h 3 , Ara h 6 , and Ara h 8 (0.01–70 µg/g) as compared to the wild relatives i.e., Ara h 1 , Ara h 2 , Ara h 3 , Ara h 6 , and Ara h 8 (0.3–11 µg/g) Table 1.A. hypogaea ssp. hypogaea (96 accessions) and A. hypogaea ssp. fastigiata (148 accessions). In ssp. hypogaea, the allergen content ranged from 29 to 22,000 µg/g for Ara h 1, 560 to 62,350 µg/g for Ara h 2, 93 to 63,720 µg/g for Ara h 3, 3,264 to 57,504 µg/g for Ara h 6, and 0.5 to 20 µg/g for Ara h 8 whereas in ssp. fastigiata, the allergen content varied from Ara h 1 , Ara h 2 , Ara h 3 , Ara h 6 , and Ara h 8 (0.3–11 µg/g) , Ara h 2 , Ara h 3 , Ara h 6 , and Ara h 8 (5 µg/g)  for Ara h 1, ICG 13491 (41 µg/g) for Ara h 2, ICG 6375 (22 µg/g) for Ara h 3, and ICGV 1328 (829 µg/g) for Ara h 6 were identified from ssp. fastigiata  Table 1.Phenotyping of accessions from different agronomic types namely Spanish bunch (72 accessions), Valencia bunch (68 accessions), Virginia bunch (53 accessions), and Virginia runner (33 accessions) indicated significant variation Table 1.The reference set included 105 traditional cultivars/landraces, 52 breeding/research material, 67 advanced/improved cultivars, and 36 wild accessions. Except for Ara h 3 , the average amount of Ara h 1 (404 µg/g), Ara h 2 (606 µg/g), Ara h 6 , and Ara h 8 (2 µg/g) was lower in wild accessions as compared to all the biological status groups of cultivated gene pool Table 1.The phenotyped set of 300 accessions represent different geographical regions namely South Asia , Southeast Asia , West and Central Africa , East and Southern Africa , South America (72 accessions), North America (27 accessions), and 19 accessions from remaining South America. The average amount for Ara h 1 was lowest in accession representing South America  followed by North America , ESA , WCA , remaining South America , SEA , and SA . Likewise, the average amount of Ara h 2, Ara h 3, Ara h 6, and Ara h 8 were identified in SA , North America , WCA , and South America (4 µg/g), respectively  followed by Sudan , Senegal , USA , Brazil , Nigeria , Indonesia , China , Myanmar , and India , Ara h 2 , Ara h 3 , Ara h 6 , and Ara h 8 (0.01–70.12 µg/g). Some germplasm lines showed low allergen content for combinations of two to three allergens.A. hypogaea ssp. fastigiata except ICG 115 which belongs to Valencia bunch of the same subspecies  to 36,833 µg/g (ICGV 02038) among cultivated accessions while it ranged from 28 µg/g (ICG 8124) to 1,293 µg/g (ICG 11555) among wild accessions   to 77,042 µg/g (ICGV 7000) among cultivated accessions (264) while it ranged from 516 µg/g (ICG 8144) to 15,757 µg/g (ICG 13218) among wild germplasm accessions (.5 µg/g) Table 3.A. hypogaea ssp. fastigiata except ICG 6888 (68 µg/g) which belongs to Valencia bunch of the same subspecies (The phenotypic variation of Ara h 3 in cultivated genotypes ranged from 22 µg/g (ICG 6375) to 106,765 µg/g (ICG 12625) while in wild accessions ranged from 1,185 µg/g (ICG 8135) to 20,474 µg/g (ICG 1156) Table 1.A. hypogaea ssp. fastigiata.For Ara h 6, the phenotypic wide variation identified in cultivated genotypes, and it ranged from 829 µg/g (ICGV 01328) to 103,892 µg/g (ICGV 02194) compared to wild accessions ranged from 4,381 µg/g (ICG 8123) to 56,857 µg/g (ICG 8195) Table 1.A. hypogaea ssp. fastigiata.The phenotypic variation for Ara h 8 was very narrow in cultivated accessions and it ranged from 0.01 µg/g (ICG 334) to 70 µg/g (ICG 12921) compared to wild accessions ranging from 0.3 µg/g (ICG 13206) to 11.27 µg/g (ICG 8973) Table 1.Peanut allergy is now a global health problem and so far no permanent solution is available to deal with this menace. More importantly, the peanut is consumed in the form of several peanut-based products, therefore, making the life of an allergic person more complicated and difficult. Hypoallergen lines provide an alternative approach to avoid these adverse reaction caused by IgE . The skiNot much efforts have been done toward phenotyping a large peanut germplasm collections in the world. This is majorly due to lack of robust and high-throughput analytical assays to quantify major allergen proteins in peanut seeds. Recently our lab developed an ELISA based protocol to estimate major peanut allergens  using peanut seeds . By usinResults confirmed a wide variation of five major peanut allergens in the peanut reference set. This study used monoclonal antibodies for each allergen for phenotyping of ICRISAT reference set representing global diversity. These monoclonal antibodies also used to observe differences in specific peanut allergen profile in peanut flour and peanut-based products such as peanut butter, flour, and other confectionary preparations for clinical use . ScreeniPrevious limited efforts in phenotyping closely related germplasm lines have not yielded in the identification of hypo-allergen peanut lines. Keeping in mind this fact, we explored a large number of diverse germplasm lines for phenotyping using newly developed very precise protocol . As a refastigiata subspecies and Spanish bunch types and can be used for developing hypoallergen lines through marker-assisted selection (MAS) or clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas9 approach. One previous study reported that the landraces conserved genetic variation for edible oil properties and also suitable for biodiesel production in Algerian peanut landraces , Ara h 2 , Ara h 3 , Ara h 6 , and Ara h 8 (0.01–70.12 µg/g) as compared to other biological groups i.e., breeding/research material, advanced/improved cultivar, and wild accessions. The quantification of five major allergens through immunological assay showed that the landraces conserved hypoallergen feature. These accessions are ICG 442 (22.7 µg/g) for Ara h 1, ICG 13491 (41 µg/g) for Ara h 2, ICG 6375 (22 µg/g) for Ara h 3, ICG 15405 , and ICG 334 (0.01 µg/g). These accessions mostly belong to andraces . This fiandraces .The development and release of several improved cultivars with high yield potential, biotic and abiotic stresses resistance, and enhanced/improved nutritional quality features in peanut has successfully been developed by combining the plant breeding techniques and efficient phenotyping methods. One of the previous studies reported that there are no significant differences in the allergen content among different peanut agronomic types consumed in western countries . HoweverWide varieties of peanut are grown to meet need of oil, food, and industries. The identified hypoallergen peanut lines can directly be used for cultivation and use in industry. Further, the identification of functional variation through genomics will facilitate the development of diagnostic markers for different allergens. The diagnostic markers can be used for improving varieties through MAS while the genes can be now edited through CRISPR/Cas9. CRISPR/Cas9 system has proven to be successful in various crop species over past years including wheat, tobacco, rice, potato, tomato sorghum, orange, and maize . AlthougThe study identified several hypoallergen peanut lines for further study. These hypoallergen lines can be directly used for commercial cultivation in addition to further breeding research for developing improved peanut varieties by combining several other agronomic traits. The output of this study also encourages researchers to identify functional variation so that molecular breeding through MAS, MABC, and genome editing can be deployed for developing new hypoallergen lines in peanut. The results have shown great hope toward fighting peanut allergy and ensuring enhanced food safety and security for humans as well as promises good opportunity for economic gains by producers, processors, and industry.All datasets generated for this study are included in the article/MP conceived the idea. MP, AP, HS, HU, and RV designed the experiments. AP performed the experiment. AP and HS analyzed the Data. AP, RV, HS, and MP wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacterial microcompartments are large, roughly icosahedral shells that assemble around enzymes and reactants involved in certain metabolic pathways in bacteria. Motivated by microcompartment assembly, we use coarse-grained computational and theoretical modeling to study the factors that control the size and morphology of a protein shell assembling around hundreds to thousands of molecules. We perform dynamical simulations of shell assembly in the presence and absence of cargo over a range of interaction strengths, subunit and cargo stoichiometries, and the shell spontaneous curvature. Depending on these parameters, we find that the presence of a cargo can either increase or decrease the size of a shell relative to its intrinsic spontaneous curvature, as seen in recent experiments. These features are controlled by a balance of kinetic and thermodynamic effects, and the shell size is assembly pathway dependent. We discuss implications of these results for synthetic biology efforts to target new enzymes to microcompartment interiors. Bacterial microcompartments are protein shells that encase enzymes and reactants to enable bacteria to perform vital reactions, such as breaking down chemicals for energy or converting the products of photosynthesis into sugars. Microcompartments are essential for many bacteria, including human pathogens. Thus, there is great interest in understanding how microcompartment shells assemble around their cargo (the interior enzymes and reactants), and what determines the structure and size of a microcompartment. These questions are difficult to answer with experiments alone, because most intermediates in the assembly process are too short-lived to characterize in experiments. Therefore, this article describes theoretical and computational models for microcompartments, which predict assembly pathways and how the sizes of assembled shells depend on factors such as protein interactions and concentrations. The simulations show that the properties of the cargo are an important factor for determining shell size, and suggest an explanation for recent experimental results showing that cargo can either increase or decrease shell size. In addition to helping to understand the natural behavior of microcompartments, the simulations provide guidance to researchers working to reengineer microcompartments to produce drugs or biofuels. Moet al. kBT . Despite the lack of symmetry, most shells are closed, meaning that every hexamer and pentamer subunit interacts with respectively six and five neighboring subunits. The yield and fraction of complete shells are shown in The shells assembled around cargo are larger and lack the perfect icosahedral symmetry of the intrinsic preferred shell geometry (T = 3 size formed by empty shells (32 subunits), demonstrating that the cargo can robustly increase shell size. As the shell-cargo interaction is increased within the two-step regime (εCC ≳ 1.55), there is a sequence of predominant assembly outcomes. Weak interactions lead to a disordered layer of shell subunits on the cargo globule, moderate interactions result in one complete shell, and overly strong interactions drive multiple nucleation events throughout the system. This over-nucleation decreases the mean shell size since the system becomes depleted of cargo and shell subunits. The one-step regime exhibits a similar sequence, except that instead of a disordered globule there is no nucleation for weak shell-cargo interactions.Pathway dependence. A striking feature of εCC (maximized over εSC). We see a dramatic increase in shell size as the cargo-cargo interactions increase beyond εCC = 1.5, when the system transitions to two-step assembly pathways. The maximum shell size eventually decreases for εCC ≳ 1.65 due to over-nucleation.Dependence on shell subunit stoichiometry. To determine the effects of shell subunit stoichiometry, we performed simulations with varying concentrations ρp of pentamers subunits at fixed hexamer concentration. As shown in εPH = 1.3εHH), we observe a modest decrease in shell size of about 50% with increasing pentamer concentration. In contrast, for strong pentamer-hexamer interactions (εPH = 2εHH), even small concentrations of pentamers lead to rapid pentamer insertion and shells that are close in size to the minimum T = 3 geometry. At low pentamer stoichiometries we observe very large shells containing approximately 140 subunits; the shell size saturates because it is limited by the droplet size and multi-nucleation events that occur for these relatively strong cargo-cargo and cargo-shell interactions (εCC = 1.65 and εSC = 8.5). In comparison, empty shells with ρp/ρh = 0.1 and εPH = 1.3 have a mean size of 39 subunits.Kinetics vs. thermodynamics. Our trajectories start from an out-of-equilibrium condition of unassembled subunits, and reorganization of complete shells is slow in comparison to assembly timescales. Therefore the ensemble of shells that we observe in finite-time simulations can depend on both kinetic and thermodynamic effects. We performed several analyses to assess the relative importance of kinetics and thermodynamics.εCC < 1.5 at the simulated cargo concentration) the two initial conditions produce identical results. Above phase coexistence the pre-equilibrated globule leads to larger globule sizes in comparison to the homogeneous initial condition, since shell assembly tends to arrest globule coalescence. Correspondingly, the pre-equilibrated globule initial condition produces larger shells than the homogeneous initial condition (εCC ≈ 1.5), since there is a large nucleation barrier to cargo coalescence.First, we investigated whether assembly morphologies depend on initial configurations. For notational clarity, we will refer to the initial condition for simulations described so far, in which shell subunits and cargo start from random positions, as the ‘homogeneous’ initial condition. We performed a second set of simulations started from a ‘pre-equilibrated globule’ initial condition, in which the cargo particles were allowed to completely phase separate before introduction of the shell subunits . Similarly, decreasing the pentamer concentration ρp reduces pentamer insertion and thus decreases shell size . Due to these two competing effects, the equilibrium theory predicts a nonmonotonic dependence of the equilibrium shell size on εSC. The equilibrium theory identifies other factors which affect the ratio of surface to bulk energy and thus shell size. For example, increasing the stoichiometric ratio of cargo to shell subunits decreases the cargo chemical potential thus favoring larger shells, consistent with a previous theoretical study on virus capsid assembly [By comparing results from the equilibrium model and simulation results from two sets of initial conditions, we determine that the effect of cargo on shell size arises from the competition of several effects. The first two are equilibrium effects. Firstly, because only hexamers interact with the cargo, increasing the shell-cargo interaction increases the chemical potential of pentamers in the shell relative to hexamers. As noted above, decreasing pentamer adsorption favors larger shells, since there are only 12 pentamers in a complete shell  shells assemble around these nascent droplets, subsequent coarsening of globule-shell complexes is arrested on relevant timescales, resulting in a broad, non-equilibrium distribution of shell sizes . Specifically, at each value of κs we performed a series of simulations in which the maximum size of the cargo globule was controlled by changing the system size with fixed total cargo concentration and hexamer chemical potential . We section . An examκs. We observe complete wrapping for all system sizes above this threshold. Below the threshold size, assembly stalls with one or more open seams remaining; examples of this configuration are shown for a low and high bending modulus in κs tend to exhibit adjacent vacancy pairs  simplifies toμh = ghh + ghc − μh, ΔGp as the free energy due to the 12 pentameric vacancies, ghh(εHH) as the hexamer-hexamer interaction free energy, ghc(εSC) as the hexamer-cargo free energy, and μh = kBT log(ρh) the chemical potential of unassembled hexamers at concentration ρh. The term 8πκs describes the bending energy of the complete shell. The minimum globule size n* corresponds to the locus of parameter values at which ΔΩwrap = 0, givingn* results in Δμh = −2.4 and ΔGp = 80.5kBT, or 6.7kBT per pentameric defect. Plugging in ρh = 10−3 subunits/ghc = −8.1kBT for εSC = 7.0  then rer et al. . Thus, tWe have used computational and theoretical modeling to investigate factors that control the assembly of a protein shell around a fluid cargo. We have focused on two limiting regimes of protein interaction geometries—high spontaneous curvature that drives the formation of small shells, and zero spontaneous curvature that favors assembly of flat sheets or polydisperse shells. In both regimes the presence of cargo can significantly alter the size distribution of assembled shells. For high spontaneous curvature, encapsulated cargo tends to increase shell size, whereas for shell proteins with low (or zero) spontaneous curvature cargo templating provides a mechanism to drive shell curvature and thus tends to reduce shell size. These results could provide a qualitative explanation for experimental observations on different systems in which full microcompartment shells were either larger or smaller than empty shells , 37, 38.β-carboxysomes  favors larger shells whereas a strong shell bending modulus favors shells closer to the preferred size. Although dynamical simulations exhibit similar qualitative trends to these equilibrium results, we observe significant kinetic effects as well. Fast cargo coalescence relative to rates of shell assembly favors larger shells, since closure of an assembling shell prevents further cargo aggregation. Thus, the shell size is strongly correlated to the assembly pathway, with two-step assembly leading to larger shells than single-step pathways. Although many factors likely control shell size in biological systems, this result is consistent with the observations of small empty shell assemblies , 31, 37 pathways , 40) tenpathways , 41).et al. [Our results for shell proteins without spontaneous curvature build upon Rotskoff and Geissler , which iet al.  suggestse.g., overexpressing pentamers would shift the size distribution toward smaller shells ; systems with moderate shell spontaneous curvature may exhibit less dramatic cargo effects. Also note that the effective shell spontaneous curvature depends on the stoichiometries of different shell protein species; r shells .These results have implications for targeting new core enzymes to BMC interiors. Recent experiments have shown that alternative cargoes can be targeted to BMC interiors by incorporating encapsulation peptides that mediate cargo-shell interactions, but that relatively small amounts of cargo were packaged –23, 96. S1 FigεSC at indicated values of εCC. Other parameters are εHH = 1.8, εPH/εHH = 1.3, ρp/ρh = 0.5, and κs = 10kBT.A complete shell is defined as a structure in which all pentamers and hexamers have respectively five and six interactions with neighbors. Results are shown as a function of (TIF)Click here for additional data file.S2 FigεSC at indicated values of εCC. Other parameters are as in Ratio of complete shells to the total number of shells with at least 32 subunits as a function of (TIF)Click here for additional data file.S3 FigQ6 of Ref. [r, and Qlm(r) is the (lmth) spherical harmonic of r. Results are normalized by the value for perfect icosahedral symmetry, Q6 = 0.663, and blue circles correspond to the complete shells from the simulations used for The bond order parameter  of Ref.  is shown(TIF)Click here for additional data file.S4 Fig(A) With εSC = 8.0, εHH = 2.0, ρp/ρh = 0.6, εPH/εHH = 1.5, and κs = 16kBT, small shells assemble and bud from the globule. At this moderate shell-cargo affinity, pentamers rapidly associate with adsorbed hexamers, driving high shell curvature. The final shells have 44-63 subunits, encapsulating 133-274 cargo particles. (B) With stronger shell-cargo interactions ), hexamers adsorb rapidly and exclude pentamers from the globule. Eventually there are 12 vacancies in the hexamer lattice that are filled by pentamers. The final shell has 104 subunits encapsulating 641 cargo particles. (C) Further increasing the shell cargo interaction ) leads to multiple nucleation events and polydisperse shell. The simulation results in four complete shells containing 37-92 subunits and 116-532 cargo particles.These snapshots are from Brownian dynamics simulations that used an alternative initial condition (described in the text), in which cargo particles were allowed to equilibrate before introduction of shell subunits. (TIF)Click here for additional data file.S5 FigεCC = 1.5, εHH = 2.0, εPH/εHH = 1.5, ρp/ρh = 0.5, and εangle = 1.0 (κs ≈ 16kBT).Other parameters are (TIF)Click here for additional data file.S6 Fig(A) Results are shown for parameters at which at least 1% of subunits are in shells, for εHH = 1.8, and shell bending modulus κs = 10kBT. Cargo and shell volume fractions are the same as in (B) Mean and standard deviation of the equilibrium shell size distribution as a function of cargo-cargo affinity, maximized over shell-cargo affinity. Other parameters are as in (A).(TIF)Click here for additional data file.S7 FigεHH = 1.8, κs = 10, εCC = 1.65, and εSC = 10.0.The theory parameters are calculated to approximately match the simulation parameters in (TIF)Click here for additional data file.S8 Figghc = −8.1  and (S2.12) with hexamer-cargo affinity see Ref. ), and κs(TIF)Click here for additional data file.S9 Figμc = 0.18, ΔGp = 80, and κs = 20kBT. Other parameters are from the calculations in Ref. [εSC = 7.0, εCC = 1.7, and εHH = 1.8.(A) Empty shells and (B) With cargo, under conditions of excess shell subunits (limiting cargo). Size distributions are obtained by solving Eq. (S2.12), with Δ in Ref.  for εSC (TIF)Click here for additional data file.S10 FigεHH = 1.8, εSC = 9.0, and εCC = 1.5.The shell has 98 hexamers and 12 pentamers, and other parameters are (TIF)Click here for additional data file.S1 VideoεHH = 2.6, ratio of pentamer/hexamer affinity εPH/εHH = 1.3, and pentamer/hexamer stoichiometric ratio ρp/ρh = 0.5.Parameters are hexamer-hexamer interaction strength (MP4)Click here for additional data file.S2 VideoεHH = 1.8, ratio of pentamer/hexamer affinity εPH/εHH = 1.3, ρp/ρh = 0.5, and shell-cargo affinity εSC = 8.75. Please be aware that this file is over 20MB.The final structure has 68 hexamers, 12 pentamers, and 408 encapsulated cargo particles. Other parameters are hexamer-hexamer interaction strength (MP4)Click here for additional data file.S3 VideoεHH = 1.8, εSC = 8.5, εPH/εHH = 1.3, and ρp/ρh = 0.5. Please be aware that this file is over 20MB.The final structure has 167 hexameters, 12 pentamers, and 1520 encapsulated cargo particles. Other parameters are (MP4)Click here for additional data file.S4 VideoThe final shell has 231 and 2261 hexamers and cargo particles respectively, as well as 12 pentameric vacancies. Please be aware that this file is over 20MB.(MP4)Click here for additional data file.S5 VideoThe final shell has 71 and 361 hexamers and cargo particles respectively, 8 pentameric vacancies, and 2 double vacancies. An example of a double vacancy is visible in the front of the final frame. Please be aware that this file is over 20MB.(MP4)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.


Limited data are available on the incidence of variations in nucleotide sequences of long terminal repeat (LTR) regions of Bovine Leukemia Virus (BLV). Consequently, the possible impact of SNPs on BLV LTR function are poorly elucidated. Thus, a detailed and representative study of full-length LTR sequences obtained from sixty-four BLV isolates from different geographical regions of Poland, Moldova, Croatia, Ukraine and Russia were analyzed for their genetic variability.Overlap extension PCR, sequencing and Bayesian phylogenetic reconstruction of LTR sequences were performed. These analyses were followed by detailed sequence comparison, estimation of genetic heterogeneity and identification of transcription factor binding site (TFBS) modifications.env gene classification of BLV into 10 genotypes, and further clustered analysed sequences into three genotypes - G4, G7 and G8. Additional molecular studies revealed the presence of 97 point mutations distributed at 89 positions throughout all 64 LTR sequences. The highest rate of variability was noted in U3 and U5 subregions. However, the variability in regulatory sequences (VR) was assessed as lower than the variability within non-regulatory sequences (VNR) for both, U3 and U5 subregions. In contrast, VR value for R subregion, as well as for the total LTR, was higher than the VNR suggesting the existence of positive selection. Twelve unique SNPs for these LTR sequences localized in regulatory and non-regulatory elements were identified. The presence of different types of substitutions lead to the abrogation of present or to the creation of additional TFBS.Phylogenetic analysis of curated LTR sequences and those available in the GenBank database reflected the acknowledged This study represents the largest study of LTR genetic variability of BLV field isolates from Eastern part of Europe. Phylogenetic analysis of LTRs supports the clustering BLV variants based on their geographic origin. The SNP screening showed variations modifying LTR regulatory sequences, as well as altering TFBS. These features warrant further exploration as they could be related to proviral load and distinctive regulation of BLV transcription and replication.The online version of this article (10.1186/s12985-018-1062-z) contains supplementary material, which is available to authorized users. Deltaretrovirus of the family Retroviridae. BLV virions contain a diploid RNA genome that is reverse transcribed into double-stranded DNA, which is ultimately integrated into the host genome in the form of a provirus. The BLV genome consists of gag, pol and env, structural genes and a region X which contains several open reading frames for Tax, Rex, R3 and G4 regulatory proteins [Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), a chronic, lymphoproliferative disease associated with persistent lymphocytosis and B-cell lymphomas. BLV, together with human T-cell leukemia viruses type 1 and 2 , belong to the genus Whole BLV genome is flanked at both 5′ and 3′ ends by long terminal repeat (LTR) sequences , 5. EachSeveral studies have demonstrated that sequence variations in retroviral LTRs may affect interactions with transcription factors, leading to altered expression of viral genes and ultimately virus replication. Indeed, it was shown that mutations in lentiviral LTRs caused distinct transcriptional activities of HIV-1 and small ruminant lentiviruses , 15. Genenv gene, with the identification of ten distinct genotypes thus far [Analysis of BLV LTR sequences is limited to few studies; however, the most extensive nucleotide analysis focused on the thus far –21. Signthus far . Recentlthus far . LTR varthus far . In addithus far .Sero-epidemiological surveys revealed that BLV infection is widely disseminated throughout the world, with a high prevalence in North and South America, as well as some Asiatic and Middle Eastern countries . Poland In this study, full-length LTR sequences obtained from 64 BLV isolates from different geographical regions of Poland, Moldova, Croatia, Ukraine and Russia were analyzed for their genetic variability. We have found new SNPs modifying LTR regulatory sequences and provided further evidence for positive selection pressure in particular regions of the LTR.Blood samples were collected from 64 cattle, serologically positive for BLV infection, as was diagnosed by different commercially available ELISA kits  or AGID (agar gel immunodiffusion) tests. The animals came from 47 herds, located in 19 geographically distinct regions in five countries: Poland, Moldova, Croatia, Ukraine and Russia. Available information about the isolates is summarized in Table 2O and 4.5% NaCl. Afterwards, the cells were washed twice with PBS, aliquoted (5 × 106 cells), and stored as pellet at − 80 °C until DNA extraction. Five archived samples collected in 2009 from BLV positive cows from Poland were stored as DNA samples at − 20 °C until further use. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s instructions. For each sample, genomic DNA concentration was measured using a nanophotometer (IMPLEN) and the samples were stored at − 20 °C until examination.PBLs were isolated from 10 mL of blood by centrifugation at 1500 g for 25 min. Erythrocytes were haemolysed by osmotic shock with Hs, 0.4 μM each primer, 2 mM MgCl2 and 0.5 U of KAPA Taq Polymerase. The amplification was run in Biometra T-Personal Thermocycler under following conditions: initial denaturation 95 °C 5 min, subsequent steps 95 °C 30 s, annealing 65 °C 45 s, extension 72 °C 1 min, 38 cycles total, final additional extension 72 °C 7 min, hold at 4 °C. The second fragment a 571 bp long was amplified using the following set of primers: P1: 5’-TGTATGAAAGATCATGCCGA-3′; P609: 5′- GACCCAAAATGCCGCCGAG-3′, at the same conditions except the annealing condition at 52 °C 45 s and a 40-cycle run for the amplified product.To amplify the BLV LTR sequences, an overlap extension PCR technique was developed. The general principle of the overlap extension PCR is shown in Fig. http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi. For all amplifications, the PCR products were visualized by 1.5% agarose gel electrophoresis with Simply Safe (EURx) staining (5 μl /100 ml) in 1X TAE buffer.Finally, both PCR fragments were used at equimolar proportions as template to generate final fragment 914 bp long using the following set of primers: P7843: 5’-ATTCTACCCCTAGGCGAGCC-3′ and P571: 5’-GTTAGGGTTCCGGGGTGATC-3′ and the same content of reaction mixture. Thermal conditions were as follows: initial denaturation 95 °C 5 min, subsequent steps 95 °C 35 s, annealing 64 °C 50 s, extension 72 °C 1 min 15 s, 38 cycles total, final additional extension 72 °C 9 min, hold at 4 °C. The sequences of all primers were designed using Primer 3 web-based software, found at The PCR products were purified using a NucleoSpin Extract II Kit (Marcherey Nagel GmbH & Co) and subsequently, three independent PCR amplification products of each BLV isolate were sent to Genomed SA Company  for DNA sequencing in both directions, using a 3730xl DNA Analyzer (Applied Biosystems) and a Big Dye Terminator v3.1 Cycle Sequencing Kit. Raw sequence data were viewed and proofread in Geneious Pro version 5.5.9 (Biomatters Ltd) , the reaThe sequences generated from 64 field isolates from Poland (52), Moldova (4), Russia (5), Croatia (2) and Ukraine 1) were aligned with 17 sequences of previously characterized BLV isolates from other geographical locations using ClustalW algorithm implemented in Geneious Pro 5.5.9  to non-regulatory sites (VNR) in LTR region, with the same interpretation of results. To compare variability (number of substitutions per nucleotide per strain) in VR versus VNR sequences of LTRs representing 64 BLV isolates, the Friedman test was applied. The following regulatory elements of the LTR were analyzed: TRE1 , TRE2 , TRE3 , PU.1/Spi-B, κB, GRE, TATA Box, PAS, CAP site, USF, DAS , Poly(A) and IRF. The remaining sequences within the LTR were considered as non-regulatory sequences. The level of variability between particular subregions of the LTR (U3-R-U5) was assessed using Wilcoxon matched pair test.To detect selection on noncoding DNA we used similar approach to Hahn et al. and Zhao et al. , 28. A shttp://multitf.dcode.org).In further study, the consensus sequence was calculated based on the multiple alignment and it was used to determined nucleotide variations within each LTR in respect to known BLV genotypes (G1-G10). To analyze the transcription factor binding site (TFBS) modifications related to specific mutations, the multiTF server was used  and U3/R .To identify the extent of genetic variations within the 64 LTR sequences, a consensus sequence was constructed A/C in the TRE2 of seven sequences, substitution T(− 65)C in the GRE seen in 28 sequences, and substitution of T(− 41)A, T(− 37)A and T(− 36)C in the TATA Box, noted in 4, 9 and 17 sequences, respectively.The remaining variations were noted in DAS regulatory element of R subregion, and were characterized by substitution of A(+ 150)G found in 8 sequences, T(+ 161)C found in 3 sequences, and substitution of TC(+ 188/9)CT and T(+ 190)C recognized in 11 sequences.Despite the high variability within the U5 subregion, no accumulation of specific mutations within regulatory elements was noted. Additionally, the LTR sequence analysis revealed the presence of insertions and deletions in LTR. Four deletions at position C(− 72)del of GRE, T(− 11)del of CAP site, TC(+ 188/9)del of BoxC and A(+ 320)del in non-regulatory site were observed. Two insertions at T (+ 191) ins and C (+ 192) ins were found in DAS and a non-regulatory site, respectively.R) versus non-regulatory (VNR) sequences was calculated and compared using Wilcoxon matched pair test. The respective results were shown in Table R) was lower than the variability within non-regulatory sequences (VNR) for both, U3 and U5 subregions. VR versus VNR was lower than 1 showing a high probability of negative selection (p< 0.05 for U3 and p< 0.0005 for U5). Conversely, VR value for R subregion, was higher than the VNR suggesting the existence of positive selection (p< 0.0001). For total LTR non-significant positive selection was predicted (p < 0.900).A substantial number of SNPs found in regulatory sequences of LTR suggested the possibility of positive selective pressure on BLV LTRs. To examine this hypothesis, the variability in regulatory .Based on the multiple sequence alignment of 64 sequences and 17 reference sequences, a phylogenetic tree was constructed using the Bayesian method. The topology, with high posterior probabilities, indicated that all sequences were clearly classified into ten distinct groups Fig. . This toAnalyzed sequences were segregated into three main groups, corresponding to the G4, G7 and G8 genotypes. Forty-five sequences representing Polish isolates and sequences from Moldova, Russia and Ukraine were clustered together within G4 genotype with a posterior probability of 0.62. Further, they were clearly separated into distinct subgroups, designated as G4-I, G4-II, G4-III and G4-IV. G4-III was cosmopolitan group including sequences from Poland, Ukraine, Russia and Moldova, while the remaining G4 subgroups represented only sequences from Poland.The genotype G8, with high posterior probability score 1.00, grouped nine sequences from Poland and two sequences from Croatia, creating two separated subgroups G8-I and G8-II, respectively. All eight remaining sequences were found to create a G7 genotype with high posterior value 1.00. Five out them exclusively represented sequences from Poland forming a G7-I subgroup, while three sequences from Moldova were assigned to the G7-II subgroup.R versus VNR sequences was further calculated to estimate any selection pressure on LTR regulatory elements in individual genotypes and subgroups detected in this study. The respective results were shown in Table p < 0.006039) and G8 (p < 0.000002). In particular, it was noted for the G4-III (p < 0.029411) cosmopolitan subgroup including Polish, Ukrainian, Moldavian and Russian sequences, as well as for G8-I (p < 0.011078) subgroup of Polish sequences. Positive selection was also detected in the U3 subregion (p < 0.000020) and total LTR (p < 0.000001) of G4-I subgroup which included Polish sequences.The variability in VBased on the analysis of the multiple sequence alignment of 64 and 102 BLV LTR sequences collected in GenBank database A and T(− 36)C in only Polish isolates; G(− 133)C, T(+ 161)C for only Moldovan isolates; C(+ 162)T for Russian isolates), while some were found in isolates from multiple areas (e.g G(− 170)A and T(− 65)C for Polish and Croatian isolates; A(− 203)G, G(− 202)A, A(+ 150)G for Polish and Moldavian isolates; T(+ 11)C for Polish, Ukrainian, Moldavian and Russian isolates.The detection of SNPs in regulatory elements suggested that there may be differential BLV expression in host infected with particular genotypes and/or subgroups. Five out of 9 SNPs identified in regulatory elements corresponded with G4, G7 and G8 genotypes. Another six out of 12 SNPs typical for these genotypes were identified in non-regulatory binding sites of the LTR.To analyze transcription factor binding site (TFBS) modifications related to genotype and subgroup-specific SNPs for G4, G7 and G8 genotypes we used multiTF software. The data were evaluated for potential cellular transcription factors binding sites using an in silico analysis, in conjunction with SNPs that possibly abrogated existing TFBS or created a new TFBS Table .Table 6PFive SNPs, identified in TREx2, GRE/TREx3, TATA Box, and DAS were contained XBP-3, GATA-1, CIIIB1, TMF, Pit-1a, TFIID, Sp1 and PR binding predicted sites as determined by in silico analysis. Variations disrupted putative sites for XBP-3, GATA-1, CIIIB1 and TMF, as well as possibly created new CREM-tau2, AP-1, GATA-1, NF-E2, RAR-gamma2 binding sites.Six other SNPs that were not identified in common regulatory binding motifs were present in TFIID, GATA-1, Pit-1a, Sp1, TBP, AFP1 and PR binding putative sites determined by in silico analysis. Variations disrupted the predicted sites for TFIID, GATA-1, TBP and AFP1, and may have created new AP-1, PR, ACF binding putative sites. Future studies are required to assess the functional capacity of BLV promoters from distinct genotypes with divergent TF binding sites.env gene was 94.5–97.7% amongst BLV isolates representative of the ten genotypes.In this study we determined the nucleotide sequences for LTRs of BLV proviruses isolated from naturally-infected cattle from Poland, Moldova, Ukraine, Russia and Croatia. The 64 sequences analyzed proved to be 96.3–98.2% identical to the other BLV sequences isolated from cattle living in Japan, Myanmar, North, Central and South America. This data revealed that the genetic distances between BLV LTR sequences were very low among infected cattle and were comparable to that observed for coding sequences within the BLV genome. It was further shown that the homology of the -binding protein (TBP). TBP binds other cellular factors, as well as RNA polymerase II, to initiate transcription from the DNA template. The site-directed mutants TA(− 41 to − 40)AT and TT(− 37 to − 38)TTT of the TATA Box were shown to have significantly stronger responsiveness to Tax protein in comparison to the wild type LTR [Comparison the 64 LTR sequences with the previously described sequences from American and Asian countries reveals numerous SNPs which conferred differences throughout most of the length of the LTR. We propose that some of these differences may account for noted variability in viral transcription and replication. Expression of BLV is dependent on three imperfect 21 bp repeats (TRE), which are critical elements for both basal expression and for transactivation by the virally-encoded Tax protein. These TRE motifs contain imperfectly conserved cyclic AMP-responsive elements (CRE), specifically AGACGTCA, TGACGGCA, and TGACCTCA sites, that repress transcriptional activation by cellular CREB/ATF factors. This strategy avoids TRE site recognition during a host immune response . We notetype LTR .The numerous DAS variations, located downstream to the transcription initiation site, C+ 134)T and A(+ 150)G in BoxA, C(+ 166)T in BoxB, TC(+ 188/9)CT and T(+ 190)C in BoxC and mutations outside of the boxes in DAS G(+ 156)A, T(+ 161)C; C(+ 162)T; A(+ 180)G, T(+ 182)C, C(+ 183)T and T(+ 184)C observed in this study might also introduce discrepancies in promoter activity. Specifically, these were included in three conserved boxes, which were found to be identical with HTLV-1 DRE 1, that is essential for up regulation of the HTLV-1 promoter , 33. Kas4T and A site. Constitutive expression of NF-κB in B cells can induce low level of transcription during BLV infection, which can be up-regulated following immunological activation and thus initiate a positive feedback loop involving the Tax protein . Two SNPcis-acting promoter control sequences contained within U3 region , R region can undergo continuous substitutions due to host selection pressure. This sequence variation of target transcription factor binding sites in R region may reduce the binding sites recognition and potentially be detrimental to the efficacy of transcription factors on the BLV LTR and/or hormone responsiveness. Since glucocorticoid (GR) and progesterone (PR) receptors are known to drive the activity of the HIV-1 and select endogenous retroviruses, we believe they may likely also modulate the activity of BLV 5′ LTR [In our work, we investigated possible natural selection over the entire BLV LTR sequence, as well as particular regions of the LTR , from 64 new BLV isolates and found evidence of negative selection for most of the regulatory elements. These results are consistent with the observations of Zhao et al., who showed significantly higher variability of non-regulatory than regulatory sequences in LTRs based on the analysis of 52 BLV isolates from USA, Japan and Costa Rica . NeverthV 5′ LTR , 34, 35.Another finding emerging from the in silico analysis is the possibility that many additional cellular transcription factors could contribute to the regulation of BLV transcription. We found that genotype and subgroup-specific SNPs can lead to creation or abrogation several transcription factors in the LTR. The putative binding sites for transcription factors, unique for particular genotypes/subgroups, were observed notably for AP-1, PR, CREM-tau2, NF-E2, RAR-gamma2, GATA-1 and ACF. We postulate that these observed variations can lead to genotype/subtype- specific differences in viral gene expression.In support of this concept, Jeeninga et al. revealed that the change from κB to GABP binding site in the LTR of HIV-1 subtype E did not lead to loss of promoter function; instead, a gain of GABP binding to this mutated κB binding site enhanced Tat-mediated gene expression and improved virus replication in select cell lines . We founThe SNP T(− 41)A in the TATA Box, typical for the G7-I subtype, harbors a shift from an GATA-1/TMF to NF-E2/RAR-gamma2 binding putative sites. Interestingly, transcription factor NF-E2 is known to bind with a specific DNA sequence in order to modulate transcription by RNA polymerase II. These newly determined, in silico predicted TFBS can be useful for further experiments directed at defining the physical interactions between viral and host regulatory proteins, and may help define the biochemical processes of basal transcription and Tax transactivation in B cells.Our study represents the largest study of the LTR genetic variability of BLV field isolates from Eastern part of Europe. The SNP screening showed variations modifying LTR regulatory sequences, as well as altering existing TFBS. Different sets of mutations for LTRs from different genotypes and subtypes suggests that multiple, geographically localized changes, may be involved in viral adaptation to recent selective pressures. These adaptations may underlie differential clinical outcomes of BLV infection, with potential implications for agricultural practice and economy –41.Additional file 1:Alignment of 60 representative full-length BLV LTR sequences. The labeled rectangles in the upper part of figure refer to the regulatory elements of LTR. Dots indicate identity with consensus sequence, generated based on the 81 sequences analyzed in this study. Abbreviations are included in the attached list of abbreviations. Classification of the sequences according to the genotypes/subtypes is shown in the left part of the figure. (ZIP 281 kb)Additional file 2:LTR sequences from a total of 102 BLV field isolates used in this study, with GenBank accession numbers and geographic origin. (XLSX 14 kb)


Figure 1. The calculation of C4CV using a typical 12‐s radial pulse wave, noninvasively recorded by a pressure sensor at a sampling rate of 400 Hz.N = 2324); p values were the result of the log‐rank test. The reference group for the log‐rank test is the smallest quartile of C4CV (<4.3%).2. Figure 2. Kaplan–Meier event rates of the composite endpoints of (a) macrovascular and (b) microvascular events. Macrovascular events combined the MACE, coronary artery disease, and severe peripheral artery disease. Microvascular events combined the major adverse kidney events, macroalbuminuria, retinopathy, and polyneuropathy (N = 2324); p values were the result of the log‐rank test. The reference group for log‐rank test is the smallest quartile of C4CV (<4.3%).3. Figure 3. Kaplan–Meier event rates of (a) MACE, (b) coronary artery disease, (c) severe peripheral artery disease, (d) major adverse kidney events, (e) Macroalbuminuria, (f) Retinopathy, and (g) Polyneuropathy  on MACE.N = 2324).5. Figure 5. Incidence of (a) macrovascular and (b) microvascular events according to the decile level of C4CV values. Macrovascular events combined the MACE, coronary artery disease, and severe peripheral artery disease. Microvascular events combined the major adverse kidney events, macroalbuminuria, retinopathy, and polyneuropathy (The figure captions were published in wrong order and should instead be the following:N = 2324); p values were the result of the log‐rank test. The reference group for the log‐rank test is the smallest quartile of C4CV (<4.3%).Figure 1. Kaplan–Meier event rates of the composite endpoints of (a) macrovascular and (b) microvascular events. Macrovascular events combined the MACE, coronary artery disease, and severe peripheral artery disease. Microvascular events combined the major adverse kidney events, macroalbuminuria, retinopathy, and polyneuropathy (N = 2324); p values were the result of the log‐rank test. The reference group for log‐rank test is the smallest quartile of C4CV (<4.3%).Figure 2. Kaplan–Meier event rates of (a) MACE, (b) coronary artery disease, (c) severe peripheral artery disease, (d) major adverse kidney events, (e) Macroalbuminuria, (f) Retinopathy, and (g) Polyneuropathy  on MACE.N = 2324).Figure 4. Incidence of (a) macrovascular and (b) microvascular events according to the decile level of C4CV values. Macrovascular events combined the MACE, coronary artery disease, and severe peripheral artery disease. Microvascular events combined the major adverse kidney events, macroalbuminuria, retinopathy, and polyneuropathy (Figure 5. The calculation of C4CV using a typical 12‐s radial pulse wave, noninvasively recorded by a pressure sensor at a sampling rate of 400 Hz.The publisher apologizes for the errors.


Allium genus plants, such as leek (Allium porrum), are rich sources of anti-inflammatory and anti-oxidant secondary metabolites; this is of interest because it demonstrates their suitability as pharmacological alternatives for inflammatory processes, including allergy treatment. The composition of methanolic leek extract (LE) was analyzed by GC–MS and LC–IT/MS, and the total phenolic content and antioxidant capacity were quantified by colorimetric methods. Its pharmacological potential was analyzed in human bronchial epithelial Calu-3 cells, human mast cells LAD2, and humanized rat basophiles RBL-2H3. LE exhibited a cytotoxic effect on Calu-3 cells and HumRBL-2H3 cells only at high concentrations and in a dose-dependent manner. Moreover, LE decreased the degranulation of LAD2 and HumRBL-2H3 cells. LE treatment also significantly prevented alterations in transepithelial electrical resistance values and mRNA levels of glutathione-S-transferase (GST), c-Jun, and NFκB after treatment with H2O2 in ALI-cultured Calu-3 cells. Finally, ALI-cultured Calu-3 cells treated with LE showed lower permeability to Ole e 1 compared to untreated cells. A reduction in IL-6 secretion in ALI-cultured Calu-3 cells treated with LE was also observed. In summary, the results obtained in this work suggest that A. porrum extract may have potential anti-allergic effects due to its antioxidant and anti-inflammatory properties. This study provides several important insights into how LE can protect against allergy. Plants have been used for medicinal purposes since prehistoric times. They synthetize a wide variety of active compounds, named phytochemicals, with a potential use in drug development. Over the last few years, there has been an increase in the demand and recognition of medicinal plants. In fact, since 1999, the World Health Organization (WHO) has published three monographs to provide scientific information on the safety, efficacy, and quality control of widely used medicinal plants [Ammi visnaga plant [To date, the World Allergy Organization (WAO) has estimated that between 10–40% of the global population is affected by some type of allergic diseases, and it is predicted that their prevalence will continue to increase worldwide, thus representing a major global public health issue . These iga plant .The relative efficacy of these drugs, together with their side effects and high cost, raise the urgent need to look for new bioactive compounds. Searching for new stabilizers of mast cells that prevent their activation and the subsequent releasing of pro-inflammatory mediators contained in their granules is one of the main goals of allergy treatment. Recent advances towards the discovery of the next generation of mast cell stabilizers include a phytochemical study of plant extracts such as phenolic compound , while pAllium genus plants—in particular A. cepa (onion) and A. sativum (garlic)—exhibit beneficial properties for human health; anti-microbial, antioxidant, anti-tumorigenic, and immunomodulatory properties have been found [Moreover, oxidative stress induced from environmental factors can contribute to allergic inflammatory response. In that case, antioxidant phytochemicals such as the flavonoids morin flavonoid—present in many herbs—or astragalin from green tea seeds and persimmon leaves may reduce airway inflammation by regulating reactive oxygen species (ROS) signaling . Previouen found ,16,17,18en found .A. porrum) is a hardy biennial crop that has been historically consumed since ancient Egypt, and today, it is widely used as a culinary ingredient in gastronomy throughout the world. A broad set of bioactive plant chemicals has been reported in leek [Leek  on both human bronchial epithelial and mast cells. Individual phytochemicals were identified in the methanolic LE and their antioxidant, anti-inflammatory, membrane permeability, and cytotoxic activities were assayed.A collection of a set of 48 phenolic compounds meeting commercially available standards was purchased from Sigma Aldrich Chemicals. All of them were of an analytical grade and were used without further purifications. Methanol (mass spectrometry grade) was purchased from Fluka and ultrapure water was obtained from Milli-Q apparatus. The standard solutions (10 µg/mL) were prepared in methanol.Allium ampeloprasum var. porrum) were collected from the fields of Jaca (Spain) in autumn 2016. Long white stems (approximately 200 g) were skinned, cut longitudinally into thin strips, freeze-dried, and then ground into powder. Samples were stored in sealed plastic bottles at −80 °C until used. The absence of LPS in the samples was confirmed by using the transfected cell line THP1-XBlue™ and the QUANTI-Blue™ assay (InvitroGen), following manufacturer instructions.Leeks (w/v) in 100% methanol using a Brason Ultrasonic Bath Model 1200  at room temperature for 30 min. After centrifugation at 5000× g for 15 min, samples were filtrated through a 0.4 µm filter, followed by a 0.2 µm filter. Three milliliters of liquid extract were used for yield determination. Solvent was removed in a Univapo 100H  vacuum centrifuge at room temperature. Dry extracts were stored at −80 °C to prevent oxidative damage until analysis.Leek extract (LE) was prepared by ultrasonication of dried samples . Total phenols were expressed as mg of gallic acid equivalents (GAE) per g dry weight and presented as the mean ± standard error of mean (SEM) of triplicate samples.Total phenols were determined using Folin–Ciocalteu’s method by Radovanović et al. with minor modifications, using gallic acid as the standard . Briefly2SO4 0.2 M, NaH2PO4 9.3 mM, (NH4)6MO7O24 1.3 mM) for 90 min at 95 °C. After cooling to room temperature, the increase of absorbance by the subsequent formation of a green phosphate/Mo (V) complex was measured at 695 nm in a UV-VIS 1800 spectrophotometer. Total antioxidant capacity was expressed as mg of ascorbic acid equivalents (AAE) per g of dry weight. Data were the mean values ± SEM of triplicate experiments.The total antioxidant activity of the LE was determined by the phosphomolybdenum method  based onControl and AbsSample are the absorbances of the DPPH radical in the absence and presence of LE, respectively. The effective concentration of the extract required to neutralize 50% (w/v) of DPPH free radicals (EC50) was expressed in μg/mL and interpolated from the percent inhibition plot against the extract concentration. Data were the mean values ± SEM of triplicate experiments.The radical scavenging activity (RSA) of the LE was determined by using the 2,2-Diphenyl-2-picrilhydrazil (DPPH) assay according to Brand-Williams et al., with some modifications . BrieflyThe analysis of the LE by GC–MS was carried out at the mass spectrometry research support center at the Complutense University of Madrid (Spain) following a standard method. Briefly, samples were dried under nitrogen and derivatized with 60 μL of N,O-Bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane . Then, the derivatized samples were analyzed on a Varian CP3800 GC system  with a ZB-5MS plus column  coupled to a Saturn 2200 ion trap mass spectrometer , using helium as the starting mobile phase at 60 °C, gradually increasing to 220 °C at 16 min, and to 320 °C at 5 min, with 320 °C held for 6 min. The injection volume was 2 μL and the flow rate was set up to 1 mL/min. Compounds were identified by searching against the NIST library and considering an R-Match higher than 700.A. porrum  Mass Spectrometer  with an Electrospray Interfase (ESI).Phenolic acids and flavonoids were then analyzed by LC–IT/MS. The identification of compounds was performed using an Agilent 1100 HPLC system  connected to a Bruker Daltonics esquire 3000With regard to the LC analysis, separation was on a Zorbax Eclipse XDB-C18 column  running the following gradient of methanol (solvent B) versus 0.1% (v/v) aqueous formic acid (solvent A) as the starting mobile phase: 2% B (0–1 min), 2–50% B (1–13 min), 50–95% B (13–18 min), 95% B for 2 min (18–20 min), and returning to starting conditions 2% B in 1 min (20–21 min) to finally keep the re-equilibration with a total analysis time of 25 min [m/z 50–1300. With regard to the MS analysis, the ESI–IT/MS was conducted in a positive and negative ionization mode and operated according to defined conditions: Nitrogen gas temperature was 350 °C; drying gas flow rate was 11.5 mL/min; capillary voltage was ± 4000 V and nebulizing pressure was 25 psi. Mass spectra were recorded using the full scan mode in the range of For the study, the data acquisition software employed was Esquire Control 5.2 and the data analysis was performed using the DataAnalysis 3.1 software .5 cells/mL, ATCC No. HTB-55, Lot. 61449062) was grown in DMEM/F-12  supplemented with 10% (v/v) fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine, at 37 °C and 5% CO2 until confluence. Then, the cells (7.5 × 105 cells/mL) were grown at an air-liquid interface (ALI) on inserts  in DMEM F-12 supplemented with 5% (v/v) fetal bovine serum, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 2 mM of L-glutamine. Culturing cells at ALI led to the formation of polarized tight monolayers, which mimic a human respiratory epithelial barrier.A human bronchial epithelial Calu-3 cell line  as previously described [ALI-cultured Calu-3 cells were apically exposed to the LPS free-Ole e 1 (25 µg/mL) allergen or LE (5 μg/mL) for 16 h, or Hescribed . Epithel5 cells/mL) were grown in serum-free Stem Pro-34 media , containing nutrient supplements, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 100 ng/mL recombinant human stem cell factor  at 37 °C and 5% CO2. The culture medium was hemidepleted each week with a fresh medium.The human mast LAD2 cell line was kindly provided by Drs. Dean Metcalfe and Arnold Kirshenbaum  . Cells (4 cells/mL) were grown in 75 cm2 tissue culture flasks at 37 °C and 5% CO2 as previously described [Rat basophilic leukemia cells transfected with cDNA coding for the human high affinity IgE receptor chains (HumRBL-2H3) were kindly donated by Dr. Lothan Vogel . The cells were cultured in MEM supplemented with 5% fetal bovine serum, 100 UI/mL penicillin, and 100 µg/mL streptomycin. The cells  were added to each well. Samples were incubated for a further 4 h in the dark. Then, media were removed, and 100 μL of dimethyl sulfoxide:methanol (1:1) were added to dissolve the purple insoluble MTT formazan; the absorbance was measured at 570 nm in an iMark plate reader  using a 650 nm filter as a reference. Cell viability was expressed as the percentage (%) of dead cells relative to the untreated control cells. All determinations were performed in triplicate.Dry LE was dissolved at 10 mg/mL in a phosphate buffer with pH 7.4, and the possible cytotoxic effects on LAD2, HumRBL-2H3, and Calu-3 cell lines were investigated by conducting cell viability assays using two-fold serial dilutions. Cell viability was determined by the 3--2,5-diphenyltetrazolium bromide (MTT) assay . Cells wThe determination of human IL-6 levels in the supernatants of the ALI-cultured Calu-3 cells after exposure to Ole e 1 with or without LE was performed using the ELISA kit BD OptEIA , according to the manufacturer instructions.The permeability of ALI-cultured Calu-3 cells to the Ole e 1 allergen, in the presence of absence of LE, was analyzed by a Western blot using an anti-Ole e 1 rabbit polyclonal antiserum (1:5000) generated by Dr. F. Vivanco’s laboratory , as previously described . For thaSera were collected from Ole e 1-allergic patients in accordance with a protocol approved by the ethics committee of both Ciudad Real and Cordoba Hospital (Spain), and a written informed consent was obtained from all subjects. The inclusion criteria were a well-defined clinical history of allergy to olive pollen and specific IgE antibodies to the Ole e 1 allergen. Non-atopic individuals were included as negative controls.6 cells/mL) were incubated in 96 well plates for 24 h at 37 °C under 5% CO2. Passive sensitization was performed by overnight incubation with sera from Ole e 1-allergic patients and non-atopic individuals as control . After washing them by centrifugation at 200× g for 10 min, cells were treated with LE (5 µg/mL) or PBS for another 5 h. Degranulation of cells was induced by incubation with Ole e 1 (25 µg/mL) in Tyrode´s buffer with 50% of D2O for 30 min. The released β-hexosaminidase enzyme was measured in the supernatants using p-nitrophenyl-N-acetyl-β-D-glucosaminide  in 40 mM citric acid, pH 4.5. Optical density was measured in an ELISA reader at 405 nm. Data were expressed as the percentage (%) of total β-hexosaminidase content in the cells. The spontaneous release was subtracted from this total value. The assay was performed as previously described, with minor modifications . HumRBL-6) were seeded in 96 well plates and treated with LE (0.05–50 µg/mL) for 24 h. Then, cells were activated with compound 48/80 in Tyrode´s buffer at 5 μg/mL for 30 min and processed as described above.For non-IgE mediated LAD2 activation, cells , according to the manufacturer’s instructions. RNA concentration and integrity were assessed in a 2100B Bioanalyzer (Agilent Technology). First-stranded cDNA was synthetized using the Superscript III first-strand synthesis System . RT–PCR was performed on a 7900HT fast real-time PCR detection system (Applied Biosystems), with the Power SYBR Green PCR Master Mix (Applied Biosystems) and the specific primers described in t test, 1-way ANOVA, or 2-way ANOVA were used for determining statistical significance (p < 0.05). Data were expressed as the mean ± SEM of three independent experiments. Statistical analyses were performed using GraphPad Prism software, version 7.0e (GraphPad). A two-tailed Student’s Leek extract was obtained using methanol extraction with an average yield of 45.5% ± 1.5 from three independent extracts, and qualitative composition was studied by GC–MS. Twelve organic acids, several carbohydrates, and L-amino acids were identified .The total phenol content in LE was 2.3 ± 0.2 mg/g dry weight, expressed as GAE .m/z experimental values from the base peak chromatograms obtained in the positive ion mode as well as the fragments generated in the source.The determination of phenolic compounds of LE was carried out by LC–IT/MS . Performm/z 287) after the loss of glycosyl units, while the quercetin mono-, di-, and triglycosides were predicted to produce quercetin aglycone (m/z 303) after the loss of glycosyl units.Besides, the fragmentation patterns observed for some compounds were consistent with the loss of hexose units and the corresponding aglycons. Based on the MS fragmentation data in the ESI positive mode, kampherol di- and triglycosides were predicted to produce kaempherol aglycone -epicatechin, (+)-catechin, kaempferol-50 values) determined using DPPH. Using the phosphomolybdenum method, the total antioxidant activity of methanolic LE, expressed as mg of AAE per gram of dry extract, was 60.2 ± 3.1 . Another4. However, the cytotoxicity of the LE increased as the concentration did. At 22 dilution, LE showed the most cytotoxic activity on HumRBL-2H3 cells (cell viability was reduced to 25%), followed by Calu-3 (75% cell viability). No significant cytotoxic effect of LE was detected on the LAD2 cells at any of the tested concentrations.In order to evaluate the cytotoxic effect of the methanolic LE, a cell viability assay was performed on three cell lines using MTT: Humanized rat basophils (HumRBL-2H3), human mast LAD2 cells, and human bronchial epithelial cells . The results are shown in To assay the effect of LE on degranulation of effector cells, the human mast cell line LAD2 was treated with LE for 48 h before addition of the component 48/80, a polymer that promotes the activation and degranulation of mast cells through an IgE-independent pathway. As shown in Leek extract also decreased the IgE-mediated degranulation of HumRBL-2H3 cells promoted by the Ole e 1 allergen in eight out of eleven individual sera tested from olive pollen-allergic patients B. 2O2 oxidative stress/inflammatory stimulus on day 7. Changes in TEER values  were monitored at different time-points post-treatment , A. sativum (garlic), A. ascalonicum , and A. porrum (leek) are rich sources of secondary metabolites [Species from the abolites ,34. Amonabolites ,36. Seveabolites .Leek extract was obtained using methanol extraction since it has been reported as one of the best solvents to obtain plant materials in terms of yield, total phenol content, and antioxidant activity . The quaAllium genus—were 3.80 ± 0.42 mg EAG/g dry weight and 3.18 ± 0, 16 mg EAG/g dry weight, respectively [The total phenol content found in LE (2.3 ± 0.2 mg/g dry weight) was lower than previously reported by other authors. This difference can be explained in terms of genetic, environmental, and experimental factors such as plant variety, geographical region, climatic conditions or extraction procedure, among others. Bernaert et al. found that total phenol content varied from 5 to 15 mg GAE/g dry weight in the extract of the 30 leek varieties analyzed . Moreoveectively .Several flavonoids glycosides and steroidal saponins were found in LE. Beneficial effects on human health have been reported for phenolic components attributed to their antioxidant activity due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides ,48,49.Allium genus, ranging from 14.5 to 128 mg AAE/g [We found a high antioxidant activity of methanolic LE. Numerous studies have reported both the total antioxidant and radical scavenging activities of members of the mg AAE/g ,50 and 1mg AAE/g ,51.LE decreased the degranulation of mast cells through both an IgE-independent and -dependent pathway. The inhibitory effect of plant extracts on mast cell degranulation has been reported before. Yoo et al. demonstrated that aged black garlic extract inhibited the release of β-hexosaminidase on HumRBL-2H3 cells . Moreove2O2 on TEER values and promoted epithelial barrier maturation. The protective effect of plant extracts against oxidative stress/inflammatory stimuli has been reported for other species, including Capsicum annuum (pepper) [Alpinia katsumadai [Boswellia serrate [Since respiratory epithelial barrier disruption has been observed in allergic patients, and airborne pollutants are considered as important inductors of lung oxidative stress , resulti(pepper) , Alpiniatsumadai , and Bos serrate .Exposure to environmental pollutants induces oxidative stress via the activation of transcription factors such as NFĸB and c-Jun ,60. MoreAllium genus have also shown their ability to reduce the production of pro-inflammatory cytokines in vivo. According to Oliveira et al., extracts of onion were able to reduce IL-4 and IL-5 levels in bronchoalveolar lavage of a murine model of asthma [A. hookeri decreased levels of TNF-α in nasal mucosa of a murine model of allergic rhinitis [IL-6 is a pro-inflammatory cytokine found at high levels in asthmatic patients , and we f asthma  and Kim rhinitis .A. porrum extract may have potential anti-allergic effects due to its antioxidant and anti-inflammatory properties. More research is needed in order to elucidate the mechanisms and the bioactive compounds responsible for these actions.In summary, the results obtained in this work suggest that


Saccharomyces cerevisiae pyrophosphatase Fpy1p. Genetic and functional studies suggest that Fpy1p may play a key role in flavin metabolism and is the first-reported non-Nudix superfamily enzyme to display FAD pyrophosphatase activity. Characterization of mutant yeast strains found that deletion of fpy1 counteracts the adverse effects that are caused by deletion of flx1, a known mitochondrial FAD transporter. We show that Fpy1p is capable of hydrolyzing FAD, NAD(H), and ADP-ribose. The enzymatic activity of Fpy1p is dependent upon the presence of K+ and divalent metal cations, with similar kinetic parameters to those that have been reported for Nudix FAD pyrophosphatases. In addition, we report that the deletion of fpy1 intensifies the FMN-dependence of null mutants of the riboflavin kinase Fmn1p, demonstrate that fpy1 mutation abolishes the decreased fitness resulting from the deletion of the flx1 ORF, and offer a possible mechanism for the genetic interplay between fpy1, flx1 and fmn1.The flavin cofactors FMN and FAD are required for a wide variety of biological processes, however, little is known about their metabolism. Here, we report the cloning and biochemical characterization of the  The riboflavin derivatives flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are critical enzyme cofactors in all living organisms. FAD and FMN are required for a wide variety of metabolic processes, some of which include mitochondrial electron transport, antioxidant reduction, protein folding, chromatin remodeling, as well as the metabolism of nucleotides, amino acids, other cofactors, and numerous other biologically important compounds –4. PlantAnimalia, but has been characterized in yeast, bacteria and plants [Riboflavin is synthesized from GTP and ribulose-5-phosphate. This biosynthetic pathway is absent in humans and other d plants ,8–12. FMd plants ,10,13–16d plants ,15,17–22Enzymes that interconvert riboflavin, FMN, and FAD may critically affect the balance and availability of flavin cofactors throughout the cell. Despite this importance, little is known about these enzymes, their interplay with other metabolic processes, or their true cellular location. Previous studies found that eukaryotic mitochondria and cytosol each possess a distinct set of flavin-interconverting-enzymes, however, many of the catalysts whose activity is described in these studies have yet to be identified ,19,23–26Saccharomyces cerevisiae, FMN is synthesized by a single-known riboflavin kinase, Fmn1p [S. cerevisiae also contain only a single-known FAD synthetase, Fad1p [Yeast are an excellent model organism for the study of riboflavin metabolism. In e, Fmn1p . Resultse, Fmn1p . S. ceree, Fad1p . In conte, Fad1p . Fad1p he, Fad1p .While many organisms, including yeast, are capable of hydrolyzing FMN and FAD, few of the corresponding genes have been identified for the enzymes that underlie this activity ,26,30–36S. cerevisiae are able to utilize exogenous riboflavin in the synthesis of FMN and FAD. This activity suggests these organelles contain both a riboflavin uptake system as well as riboflavin kinase and FAD synthetase enzymes [In addition to interest in the identification/characterization of novel flavin-interconverting-enzymes, there is also a growing effort aimed at describing the mechanism by which the yeast mitochondria meet their metabolic requirement for flavin cofactors. Mitochondria that are isolated from  enzymes . Further enzymes .S. cerevisiae is believed to be facilitated by Flx1p [S. cerevisiae are grown on the fermentable sugar galactose, consistent with the characteristics of a FAD importer [flx1 mutants can be rescued by overexpressing FAD1, although the specific mechanism for this effect has not been put forward [S. cerevisiae may partially counteract a decrease in fitness that is observed by deletion of the flx1 ORF [flx1 mutant is similar to the effect of the FAD1 upregulation with respect to the FLX1 mutant, suggesting antagonistic functions of the two.Transport of FAD across the mitochondrial membrane in by Flx1p ,41, howeby Flx1p , howeverimporter ,41. Furtimporter ,40,41. T forward . Boone aflx1 ORF . The efffpy1 and the FMN biosynthetic gene fmn1, and present data suggesting a possible mechanism for this effect.In this work, we report the cloning and characterization of the protein encoded by the YMR178w gene. The protein, hereby designated Fpy1p, was expressed in a recombinant system, purified, and characterized as a potassium-dependent pyrophosphatase with the capability of hydrolyzing FAD, NAD(H), and ADP-ribose. Fpy1p is unrelated to those of the NUDIX superfamily and represents a fully novel type of pyrophosphatase, with sequence homologs fused to FAD synthetases in animals and plants. Using this system, we additionally confirm the finding of Boone  that mutfpy1) and the gene encoding mitochondrial FAD transporter, flx1 [fpy1 may play a role in the metabolism of flavin cofactors. Fpy1p has sequence homologs across multiple kingdoms of life. S. cerevisiae protein in representative mammal, fish, invertebrate, algal, and dicot/monocot plant species.Previous studies identified a positive interaction between the uncharacterized gene YMR178w  tag for two-step purification by IMAC chromatography. Separation by SDS-PAGE demonstrated high purification of a protein of ~35 kDa , which i+. The only substantial difference between the two molecules is the presence of a positive charge at the nicotinamide moiety, indicating electrostatic interactions may interfere with substrate binding. Additionally, when assays included free nicotinamide or lumiflavin as potential competitive inhibitors, the reaction was unaffected  hydroxide [2+ in solution. Therefore, the higher pH that was optimal with magnesium likely reflects the true pH optimum of the enzyme.Cobalt and magnesium ions were also found to differ in their ability to activate Fpy1p as a function of pH . Whereasydroxide , which wm was calculated as 16.1 ± 2.1 μM and 19.0 ± 2.7 μM in presence of Co2+ and Mg2+, respectively, while the Vmax values were 0.641 ± 0.027 μMol min-1 mg-1 and 0.34 ± 0.027 μMol min-1 mg-1, respectively. m values vary considerably from enzyme-to-enzyme, ATNUDX23 , the only pyrophosphatase verified to affect flavin homeostasis in vivo, has a Km of 9.1 ± 0.9 μM and kcat of 0.08 s-1 [Fpy1p was found to follow standard Michaelis-Menten kinetics when specific activity was plotted as a function of FAD concentration . The Km 0.08 s-1 , which afpy1 was acquired and the gene for fmn1, which encodes the dual-localized cytosolic/mitochondrial riboflavin kinase, was deleted in both Δfpy1 mutants and the wild-type parent yeast. In both backgrounds, the Δfmn1 mutation resulted in FMN auxotrophy, similar to what was previously described [fmn1 single mutant displays only a 6–7% reduction in growth relative to wild-type when grown on media supplemented with 4 mM FMN (p<0.05), and the growth of Δfpy1 is statistically indistinguishable from that of wild-type, however, the Δfmn1/Δfpy1 double mutant displays an 80% reduction in growth relative to wild-type under the same conditions , the abundance of these metabolites was determined in total cell extracts of stationary phase yeast grown on acetate  as an enzyme of unknown function that may play a role in flavin nucleotide metabolism based on the genetic interaction with the mitochondrial FAD transporter Flx1p . Analysithaliana .S. cerevisiae genome, possess a conserved 23-amino acid consensus sequence referred to as the Nudix box [While several enzymes have been cloned that are capable of hydrolyzing FAD, to our knowledge all are members of the Nudix superfamily of hydrolases –33,36,37udix box . Fpy1p, udix box . Unlike udix box .A previous study that used GFP fusion concluded that Fpy1p localizes to both the cytosol and nucleus . Since tfpy1 and the riboflavin kinase encoding gene fmn1. Whereas growth of the Δfmn1 deletion mutants can be restored to near wild type levels by supplementation with exogenous FMN [fpy1 in the Δfmn1 background reduces colony growth by 78% despite no discernible effect of the fpy1 deletion in the wild type background  reveal that both are increased in the double mutant as compared to the single mutant.Consistent with a role in FMN homeostasis, we found a strong negative genetic interaction between nous FMN  the deleckground . Such a flx1 mutants suffer from perturbation of the citric acid cycle and mitochondrial electron transport. The decrease in the mitochondrial FAD contents in the Δflx1 mutant perturbs the FAD-dependent mitochondrial metabolism, as shown by the reduced activities of the FAD-dependent enzymes lipoamide dehydrogenase and succinate dehydrogenase (citric acid cycle and the respiratory chain) [S. cerevisiae as a means of NADH mobilization [The available evidence, in particular the decreased activity of mitochondrial enzyme activities as observed here and elsewhere ,41, suggy chain) ,41, thatlization . In a prlization . As showlization .Similar to the NDI1-deficient mutant, the flx1 mutant has a more severe phenotype when grown on acetate then when grown on glucose, likely because of the defective the citric acid cycle in mitochondria, as described above for the NDI1 mutant. Our data suggest that the fpy mutation helps alleviate this phenotype because reduced hydrolysis of FAD and NAD(H) in the mutants lacking Fpy1p in the cytosol helps in two ways: (1) by preserving the FAD needed by the cytosolic NADH-producing dehydrogenases and the G3P shuttle, and (2) by preserving the NADH produced in the cytosol. Thus, our data suggest that the role of Fpy1, a novel and non-Nudix pyrophosphatase, may be coordinating the regulation of FAD and NAD(H) contents in response to metabolic needs of the cell during the stationary phase.Except as otherwise noted, all chemicals and custom PCR primers were obtained from Sigma Aldrich. YeastBuster reagent was obtained from Novagen . Yeast–Ura dropout supplement was obtained from Clontech . Synthetic Drop-out Mix, minus Lysine, Methionine was obtained from US Biological . FMN that was obtained from Sigma Aldrich was additionally purified as described previously .S. cerevisiae with putative proteins from other species was conducted as follows: using the BLASTp suite available from NCBI (http://www.ncbi.nlm.nih.gov/), with YMR178wp as the query sequence, sequence homologs were identified in multiple species; for further analysis, we selected the homologs from a representative mammal , fish , invertebrate animal , green alga (Chlamydomonas reinhardtii XP_001693086), dicot plant , and monocot plant . Alignments were performed using ClustalW2 software  in conjunction with shading using BoxShade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Domain structure illustrations were created using DOG 2.0 [Comparison of YMR178wp from  DOG 2.0 .PfuTurbo DNA polymerase  and the primer pair ATGGTGAAAGTAACTGCA-3’5’-AAAAAAGCAGGCTTC (forward), and GCTCTCCTGGTTCGAGAA-3’5’-AGAAAGCTGGGTC (reverse). Note that the primers comprise both the Ymr178w-specific region (plain text), and the sequence that is needed for further downstream amplification by attB primers (underlined). The resulting PCR fragment was re-amplified using attB primers (Invitrogen), then separated by agarose gel electrophoresis, and purified using Wizard PCR columns . The PCR product was sub-cloned by BP recombination into the pDONR221 vector (Invitrogen). The YMR178w open reading frame was transferred into the pYES-DEST52 destination vector from the pDONR221 construct via LR recombination. All cloning procedures were done in accordance with the manufacturer’s protocols. The constructs were verified by DNA sequencing.The YMR178w open reading frame was acquired in vector BG1805 from Open Biosystems . The open reading frame, excluding the stop codon, was amplified using S. cerevisiae strain Y258 (Open Biosystems). The expression vector was transformed into these yeast following the manufacturer’s protocol. Yeast carrying the expression vector were cultured at 30°C in synthetic–Ura dropout medium . Once OD600 reached 1.0–1.2, 3x expression medium  was added at 50% of the culture volume, and incubation was continued for six hours before harvesting cells at 5000 × g and 4°C for 10 minutes.The S.c. EasyComp Transformation Kit (Invitrogen) was used to generate chemically competent cells of the S. cerevisiae cells expressing the recombinant protein was prepared by resuspending the cell pellet in 1x Yeastbuster with THP solution at a ratio of 5 ml of Yeastbuster per 1 g wet weight of cell pellet. After incubating at room temperature for 20 minutes with gentle shaking, the extracts were cleared by centrifugation at 16,000 × g, 4°C for 20 minutes. The recombinant proteins were purified from the cell lysate using an Äkta FPLC system equipped with 1-ml IMAC columns  charged according to the manufacturer’s protocol. All chromatography steps were performed at 4°C. A column charged with Cu2+ was equilibrated with Binding Buffer  before loading the clarified cell lysates. Unbound proteins were removed by washing with 15 column volumes of Binding Buffer, followed by elution of bound proteins by a linear gradient of Binding Buffer to Elution Buffer  over 15 column volumes. Fpy1p eluted at ~125 mM imidazole. Fractions containing the desired protein were pooled and loaded directly onto a Ni2+-charged IMAC column equilibrated with Binding Buffer, which was then washed with 10 column volumes of Binding Buffer before elution with a linear gradient of Binding Buffer to Elution Buffer over 20 column volumes. The desired protein eluted at ~225 mM imidazole. Fractions containing Fpy1p were pooled and immediately desalted into Protein Storage Buffer  using Zeba™ Desalt Spin Columns . Desalted samples were aliquoted and stored at -80°C until use.Extract of the Molecular weight of the native protein was estimated by gel filtration chromatography on a Superdex 200 10/300 column  equilibrated with gel filtration buffer . A calibration curve was generated using the following gel filtration standards from Sigma Aldrich: Cytochrome c (12.4 kDa), Carbonic Anhydrase (29 kDa), Bovine Serum Albumin (66 kDa), Alcohol Dehydrogenase (150 kDa), and β-Amylase (200 kDa). Standards were detected by measuring absorbance at 280 nm in 100-μl fractions collected after the chromatographic separation, while the Fpy1p activity was detected by enzymatic assay.Unless otherwise indicated, the procedures described below were used. Initial reaction rates at steady state were measured. Product formation was proportional to enzyme concentration and time. Less than 10% of the substrates were consumed in reactions. Assay buffer was 100 mM Hepes-KOH, pH7.0, 200 mM KCl, and 1 mM THP. Metal ions were added as described under the individual results. Assay volume was 25 μl.18 column  linked to a 2475 fluorescence detector, and were measured by fluorescence detection using an excitation wavelength of 470 nm and an emission wavelength of 530 nm. The mobile phase contained 100 mM ammonium formate, 100 mM formic acid, and 20% methanol. Product formation was determined from fluorescence by comparison to standards. FMN and FAD concentrations in standard solutions were determined spectrophotometrically [18 column . Separation was performed using the gradient in Reactions were incubated at 30°C for 20 minutes, then quenched by addition of saturated formic acid to 5% of the assay volume. Precipitated protein was removed by centrifugation at 1500 x g for 15 minutes at 4°C. Reaction products and substrates were separated by reverse-phase chromatography using a Waters Alliance 2695 HPLC system with a Waters SunFire Ctrically . Alternatrically . Derivatm and kcat values were determined by varying the substrate concentration. The results were fit to the Michaelis-Menten equation using non-linear regression analysis software of the Enzyme Kinetics Module 1.2 in SigmaPlot 9.0.The Kfpy1, Δflx1, and Δflx1/Δfpy1 were isolated. To generate Δfmn1 deletions, one-step gene replacement was employed in the wild-type and Δfpy1 MATα backgrounds. The nourseothricin-resistance gene (NourS) was amplified from plasmid pAG25 [CGTGAAGCGGTCACAGACACGTGTTGTTGAAGTGTTGATG CAGCTGAAGCTTCGTACGC-3’5’- and TAAGAAAAACTACTAGCCTCATCACTCCCGCAGATCTCTA GCATAGGCCACTAGTGGATCTG-3’5’-, which include extensions (underlined) corresponding to the flanking sequences of fmn1. One step integration was performed to incorporate PCR products into the yeast genome to replace the fmn1 gene with the nourseothricin-resistance gene following the previously published protocol [5’-CAGGGGCATGATGTGACTGT-3’ and 5’- GTACTGATTAGGGGCAGGGC -3’) and absence of an amplicon with fmn1-specific primers (5’- AATACCTGCGCAACCAGGTC and 5’-CTTGACCCTGGCCCCATAAA-3’).Haploid deletion mutant strains for fpy1 in a BY4742 background (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) and flx1 in a BY4741 background (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) were acquired from Open Biosystems. Single colonies of each strain were picked from fresh YPD streak plates, patched together onto YPD agar, and incubated for 30° C to permit mating. After four hours, mated yeast were streaked onto selective SD/His/Ura/Leu plates , and the resultant heterozygous double mutant were grown at 30° C. Sporulation and random spore analysis were performed as described previously , with thid pAG25  using thprotocol . The tra2PO4, 1 g/l L-glutamic acid, 20 μg/ml histidine, 20 μg/ml uracil, 30 μg/ml leucine, 15 g/l bacto agar) or YPD plates (with FMN supplementation where applicable). After 48 and 72 hours for the YPD and SD-MSG plates, respectively, digital photographs were taken, including grid paper for size reference. Using ImageJ software [600 of 0.05, 1 cm path length. Cultures were grown at 30° C with shaking, and OD600 measurements were taken at regular intervals. For growth under hypoxic conditions, YPD medium was saturated with N2 gas and flasks were sealed with robber stopper. Yeast cells were incubated at 30°C without agitation, and OD600 measured as above.To determine the relative fitness of the mutants, single colonies of each strain were grown overnight in liquid YPD (with FMN supplementation where applicable) at 30° C with shaking. Overnight culture was diluted and spread on SD-MSG/His/Ura/Leu plates  was added to 200 μl mitochondrial suspension and vortexed to mix. Mitochondia were disrupted by five cycles of freezing and thawing, then centrifuged at 10,000 × g for 1 min at 4°C. The supernatant was saved, and the pellet was extracted in 600 μl of a N2-saturated acetonitrile and 50 mM ammonium acetate (3:1 v/v) before centrifugation at 10,000 × g for 5 min at 4°C. The resulting supernatant was pooled with the prior supernatant. Lipids were extracted with chloroform before the resulting aqueous phase from three preparations were pooled, snap frozen, and lyophilized for 15 h. Lyophilized samples were resuspended in 100 μl ice-cold 50 mM ammonium acetate, passed through a 0.45 μm filter, and analyzed by HPLC.NAD and NADH contents were determined using the method adapted from a previously published protocol . Briefly2 gas) was added to 200 μl cytosolic supernatant, vortexed to mix, and centrifuged at 10,000 × g for 1 min at 4°C. The remaining procedures were kept the same as above.NAD and NADH were isolated from cytosolic supernatant as above with the following changes: 1.5 ml ammonium acetate . Individual interactions were tested using a method adapted from Tarassov et al . Single FAD1, FMN1, and FLX1. Reference genes were TFC1, ALG9, and UBC1, based on previous recommendations [2, 0.25x SYBR Green, 50 nM ROX, 0.5 units Platinum Taq DNA polymerase , and 0.25 μM custom primers from Sigma Aldrich. Real-time PCR was performed using an Applied Biosystems 7500 Real Time PCR System. Amplification was performed using a 7-min initial denaturation step at 95° C, followed by 40 cycles of a 15-s denaturation step at 95° C and a 1-min annealing/extension step at 60° C. All reactions were carried out in triplicate, and amplicon quality was analyzed using the melting curve function carried out by the Applied Biosystems 7500 PCR System. Relative expression was determined from the CT values using the 2-ΔΔCT method [Wild-type and Δfpy1 yeast were grownhto log phase in YPD. Total RNA was extracted using the PureLink RNA Mini Kit , following the manufacturer’s instructions for extraction by enzymatic disruption, and including the optional on-column Purelink DNase treatment. Reverse transcription was performed on 2 μg of RNA using Omniscript Reverse Transcription Kit . The resulting cDNAs were diluted 1:10 in water and used in quantitative RT-PCR for determination of relative expression of ndations . PrimersT method .S1 FigS. cerevisiae with select proteins from other organisms. The species’ abbreviations are for Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; Dr, Danio rerio; Ce, Caenorhabditis elegans; Cr, Chlamydomonas reinhardtii; At, Arabidopsis thaliana; and Os, Oryza sativa. (B) Multiple sequence alignment of Fad1p from S. cerevisiae with same proteins as in (A). (C) Illustrated domain structures of the proteins in A and B. Blue denotes domains with sequence homology to Fad1p, while Red denotes domains with sequence homology to Fpy1p.(A) Multiple sequence alignment of Fpy1p from (TIF)Click here for additional data file.S2 Fig2 or (B) 10 mM MgCl2. Data is the average ± S.E. of three triplicate determinations. Curve is nonlinear fit to the Michaelis-Menten model using SigmaPlot 9.0.Measurements were made in the presence of either (A) 4 mM CoCl(TIF)Click here for additional data file.S3 Fig2. Data is the average ± S.E. of three triplicate determinations.For both panels, solid black bar is wild-type control, solid grey bar is Δfpy1 deletion mutant. (A) Real-time PCR analysis of genes involved in flavin metabolism in yeast grown in YPD. Shown is the fold change relative to the housekeeping gene TFC1, calculated as described under “Experimental Procedures.” Data is the average ± S.E. of three technical replicates. (B) Mitochondrial FAD pyrophosphatase activity of yeast grown in YP supplemented with 1% sodium acetate or 3% glycerol. Assays were carried out as described under “Experimental Procedures” with 50 mM FAD and 10 mM MgCl(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.