Leishmania vector sand flies in Mexico, and human cases were analyzed. Niche models were constructed using sand fly occurrence datapoints from Canada, USA, Mexico, Guatemala and Belize. Nine non-correlated bioclimatic and four topographic data layers were used as niche components using GARP in OpenModeller. Both B2 and A2 climate change scenarios were used with two general circulation models for each scenario (CSIRO and HadCM3), for 2020, 2050 and 2080. There was an increase in niche breadth to 2080 in both scenarios for all species with the exception of Lutzomyia vexator. The principal direction of niche centroid displacement was to the northwest (64%), while the elevation range decreased greatest for tropical, and least for broad-range species. Lutzomyia cruciata is the only epidemiologically important species with high niche identity with that of Leishmania spp. in Mexico. Continued landscape modification in future climate change will provide an increased opportunity for the geographic expansion of NCA sand flys' ENM and human exposure to vectors of Leishmaniases.Ecological niche models are useful tools to infer potential spatial and temporal distributions in vector species and to measure epidemiological risk for infectious diseases such as the Leishmaniases. The ecological niche of 28 North and Central American sand fly species, including those with epidemiological relevance, can be used to analyze the vector's ecology and its association with transmission risk, and plan integrated regional vector surveillance and control programs. In this study, we model the environmental requirements of the principal North and Central American phlebotomine species and analyze three niche characteristics over future climate change scenarios: i) potential change in niche breadth, ii) direction and magnitude of niche centroid shifts, iii) shifts in elevation range. Niche identity between confirmed or incriminated  Lutzomyia diabolica and Lutzomyia shannoni. Despite the fact that Lutzomyia olmeca olmeca is the only confirmed vector species in Mexico, the present study demonstrates a significant niche identity between Leishmania spp. and Lutzomyia cruciata.The present study models the niche of the most abundant sand fly species in North and Central America, including all proven and incriminated vectors of Leishmaniases, an important neglected tropical disease of the region. The expansion and elevation or centroid shifts of the species' niche are modeled for extreme (A2) and conservative (B2) climate change scenarios to 2020, 2050 and 2080. In climate change scenarios, models predict significant niche breadth changes in geographic space, principally in temperate sand fly species, while elevation shifts occur principally in tropical, and greatest, in vector species. Niche centroid shifts for individual species were predominately to the northwest, and secondarily to the northeast. The highest proportion of human population at-risk for contact with a vector species was with  Leishmania spp. patency Leishmaniases are an increasingly important disease group worldwide, based on case numbers, geographic expansion, socioeconomic implications, psychological impact, and immunosuppression due to HIV infection which re-activates Leishmania spp. are transmitted by female sand flies of the genus Lutzomyia in the New World LeishmaniaLutzomyia longipalpis and Lutzomyia evansi are confirmed primary VL vectors in several countries Lutzomyia olmeca olmeca is a confirmed vector of LCL in Mexico Lutzomyia anthophoraLutzomyia cruciataLutzomyia diabolicaLutzomyia ovallesiLutzomyia panamensisLutzomyia shannoniLutzomyia ylephiletorLeishmania spp. Leishmania have been isolated in NCA, and are responsible for all human and canine clinical manifestations: Leishmania mexicana, Leishmania braziliensis, Leishmania panamensis and Leishmania infantum chagasiDespite early studies on the taxonomy and geographic distribution of sand flies in NCA, knowledge regarding the biology, distribution, and ecology of new collections and species continues to be registered from only a few regions Generally, macroclimatic variables influence species distributions at coarse scales, topographic variables at regional scales, and land use and biotic interactions at finer scales Phlebotomus papatasiPhlebotomus orientalis, Phlebotomus martiniPhlebotomus alexandriLutzomyia whitmani, Lutzomyia intermedia and Lutzomyia migoneiEcological niche modeling (ENM) has already been used to project the geographic distribution potential of epidemiologically relevant Old World sand fly species: All tropical and temperate NCA species from Guatemala and Belize to Canada are modeled together in this study, and niche characteristics as well as epidemiological associations of relevant species are analyzed in two contrasting CC scenarios. We have focused on analyzing potential change in species' geographic ranges as predicted by macroclimatic changes at the coarse-grain level, since these provide greater model consistency and accuracy for climate circulation models and their bioclimatic variables The study area for model construction and projection includes Canada, USA, Mexico, Guatemala and Belize, limited by 14.07°N, 58.23°N and −136.15°W, −56.29°W. The region was divided into 7,536,074 pixels at a resolution of 30 arc-seconds (0.008333°≈1 km) for latitude and longitude. Ecological region categories were assigned using the World Wildlife Fund (WWF) shape files based en Terrestrial Eco-regions of the World A database was constructed from collections reported in published scientific literature, entomological collections housed in several academic institutions in Mexico (Universidad Autónoma de Yucatán (UADY), El Colegio de la Frontera Sur (ECOSUR), and Universidad Autónoma de Nuevo León (UANL)), the Instituto Nacional de Diagnóstico y Referencia Epidemiológica (InDRE), and author's unpublished collections . The datwww.worldclim.org; last accessed Nov, 2011) at a resolution of 30 arc-seconds http://eros.usgs.gov/products/elevation/gtopo30/gtopo30.html; last accessed Dec, 2011) were also used for ENM models.Thirteen environmental layers were used for the construction of ENM. Nine bioclimatic data layers  were obtained from the Worldclim- Global Climate Data  and Each ENM was evaluated using two tests: accuracy, a measure of performance, and the AUC (area under the receiver operating curve [ROC]), as a test of predictive ability. Both tests are based on two types of error: commission  and omission  Leishmania spp. (PEN) and each vector Lu. anthophora, Lu. cruciata, Lu. diabolica, Lu. longipalpis, Lu. olmeca olmeca, Lu. ovallesi, Lu. panamensis, Lu. shannoni and Lu. ylephiletor. Human cases of Leishmaniases from multiple Mexican states, Campeche (N = 8), Chiapas (N = 161), Guerrero (N = 10), Morelos (N = 2), Oaxaca (N = 3), Puebla (N = 4), Quintana Roo (N = 101), Tabasco (N = 15) and Veracruz (N = 37) were used as proxy to generate the PEN. A maximum-entropy-based algorithm, MaxEnt ver.13.2 . The centroid of each ENM (current+future) was connected between the previous and the azimuth of the trajectory measured for future scenarios. The elevation range change for each ENM was also measured with the spatial analyst tool  from ArcMap.Change in geographic niche breadth for each ENM was calculated using the proportion of occupied pixels/total number of pixels. The overlap for current, 2020, 2050, and the 2080 models was classified into three categories: 25–50%, 51–75% and 76–100%. A shift in the niche centroid was measured using the spatial analyst tool  of ESRI ArcMap 10.0 . After obtaining the ENM, population projections were calculated using Hawth's Analysis Tool version 3.27  .The total population growth rate for Mexico was generated using projections for fertility, mortality and international migration Hellinger's-based I” for all pairwise combinations of vector-Leishmania spp. The empirical measure of niche similarity between populations is compared to a null distribution to test whether they are significantly different from similarity generated from niche models constructed with data points extracted randomly from the distribution range of the compared species. The hypothesis of niche identity is rejected when the empirically observed value for “Hellinger's based I” is significantly lower than the values expected from the pseudoreplicate data sets We calculated the “Lu. shannoni , with the exception of the higher elevation areas of the Sierra Madre of Chiapas and the volcanic range in San Marcos, Guatemala . The foushannoni , Lu. crucruciata , Lu. olma olmeca , and Lut deleoni .Lutzomyia vexator (Lu. anthophora. The AUC ranged from 0.80 to 0.99 (42 with an AUC higher than 0.96) for both training and test data. The p-values for all species were highly significant in predicting the known distribution of species  and 12–21 species (160%), northward, particularly along the Pacific coast of NA  and external accuracy from 0.57 to 1.00 (20 with an accuracy = 1.00); the lowest accuracy values were calculated for  vexator  and Lu.  species . Tropicast of NA . Highestst of NA , Table 2Lu. vexator's ENM expanded over time as for all other species, while it contracted using the HadCM3 model. The geographic projection of this species' ENM diminished in the HadCM3 model, in northern regions, as well as fragments in the south. Average niche breadth increase was marginally greater in temperate and broad-range as compared with tropical species. Most tropical species had a greater increase in B2 than A2, while the opposite was observed for temperate species , niche breadth increased over time for all but one species . In the  species . Species species .Lu. cruciata had the lowest overlap with other species (11.4%), while Lutzomyia permira (80.1%) and Lutzomyia undulata (80.2%) had the highest overlap with other species.Changes in ENM overlap among sand fly species (11.41–80.20%) was variable according to distribution category and time period. Overlap between current and 2050 projections was lowest for temperate species (average 43.49%) and highest for tropical (67.44%) species . The ERSLu. longipalpis and Lu. panamensis, and to the northwest for all other ERS. The distance shift of ENM centroids was variable (47–940 km) according to species and time periods  had the greatest centroid shift, followed by Lutzomyia bispinosa  and Lu. anthophora (temperate). In contrast, Lu. shannoni  has the lowest centroid shift of all 28 species.There is a direction shift in ENM centroids for all species in all time periods and both CC scenarios; the majority of species shifted to the northwest (64.3%), followed by northeast (35.1%), and minimally to the southwest (0.6%). The direction shift was to the northeast for  periods . As expeLu. cruciata and Lu. longipalpis shift to lower elevations. The elevation range of all broad-range species, as well as Lu. anthophora (temperate) and Lutzomyia carpenteri  increased.Although the shift in elevation range is highly variable, the average range for all 28 species decreased in future CC scenarios. In general, the decrease in the A2 scenario was greater than in the B2 . The eleThe combined changes in niche breadth, elevation and centroid range and direction was analyzed focusing on 2050; the CSIRO model was run using the B2 scenario , and theLu. olmeca olmeca (average elevation range = 930 m), the second group includes Lu. ovallesi, Lu. panamensis, and Lu. ylephiletor , the third group contains only Lu. longipalpis , and the fourth group is composed of Lu. anthophora, Lu. cruciata, Lu. diabolica, and Lu. shannoni . In the A2 scenario, the average shift for the four groups was 1,058 m, 1,156 m, 1,319 m, and 1,590 m, respectively , followed by Lu. shannoni , Lu. cruciata , Lu. longipalpis , Lu. ylephiletor , Lu. ovallesi , Lu. anthophora , and Lu. olmeca olmeca . The rural population  exposed to sand flies will increase over time in both CC scenarios  of exposed population, while Lu. olmeca olmeca overlaps with the lowest (8.9%). The only sand fly with significant niche identity with Leishmania spp. was Lu. cruciata  diversity is highest Lu. shannoni is projected to have the greatest increase in range size over time, perhaps due to the heterogeneous landscape where this species occurs: aquatic mangrove habitats, arid vegetation of spiny forest, desert, grasslands and xerophilous brushland, temperate vegetation of conifer and cloud forest and perennial, deciduous, and sub-deciduous tropical forest Lu. shannoni's relative abundance and habitat adaptation (conserved and modified habitats) at least in Quintana Roo, Mexico. Interestingly, Lu. cruciata, which also inhabits a wide variety of landscapes and is also a potential vector of LeishmaniaThe geographic projection of niche breadth increase is uniform surrounding most NCA sand fly ENMs over time in both CC scenarios. This uniform increase depends on specific landscape components, biotic interactions, habitat modification, or other characteristics affecting population growth or the species' fitness, all of which affect the realized niche The predominant trend for ENM centroid shift to the northwest was consistent over time for most sand fly species. However, the centroid of temperate species, which have the greatest ENM breadth increase, shifted predominately to the south, a trend previously observed in European birds Lutzomyia intermedia has an increased body size and greater dispersal capacity at higher elevations Lu. anthophora, Lu. diabolica, Lu. olmeca olmeca and Lu. panamensis), while others shift to a lower range (Lu. cruciata). Interestingly, an increase in Leishmaniases incidence in higher elevations has been reported in various countries, although it is not clear whether this is due to a prevalence shift of vector species, an increase in parasite prevalence only in certain vector species, or increased human exposure and changes in biotic interactions at higher elevations In addition to potential increases in fundamental niche area and latitude/longitude centroid shifts, the elevation range for certain species have been reported, due to shifting or varying thermoclines and precipitation Lu. shannoni, which is the same species projected to have the greatest increase in total niche breadth. Lutzomyia cruciata, an ERS currently proposed to form a species complex Niche breadth increase and centroid shifts of potential vector species could contribute to an increase in parasite dispersal and hence an increase in human transmission hazard. Even though the fundamental niche is projected to expand, dispersal capacity of the species will depend upon genetic plasticity, the availability of dispersal routes, and host interactions www.dgepi.salud.gob.mx; last accessed Dec, 2012). However, there is no effective surveillance program and very poor knowledge by medical personnel of the diseases, and hence the total number of officially recognized cases may be much higher. Since rural populations are the principal group exposed to vector contact Lu. diabolica, Lu. shannoni, and Lu. cruciata.The public health Secretariat in Mexico recognizes only 17,000 Leishmaniases cases over the last twenty-two years  and Lu. olmeca olmeca (84.84%), based only on geographic overlap. Additional studies will be required to analyze landscape quality and its impact on niche overlap areas where vector, reservoir, and parasite species interact, in order to extend the use of niche identity analysis within fragmented landscapes.Ecological niche modeling of pathogens has been applied to a broad range of infectious and toxicity-related diseases in order to project potential shifts to the end of the present century: dengue fever and Lu. cruciata as an important vector of Leishmania spp. in México.The present analysis of the distribution of NCA sand fly species and their ENM shifts in climate change scenarios predicts range shifts which may modify vector-host interactions and relationships associated with habitat and future land use. Temperate sand fly species, and therefore those with least epidemiological importance, project the greatest ENM changes. Those changes projected for certain epidemiologically relevant tropical species support previous evidence for current, and also highlight future importance of Figure S1Ecological niche models for Brumptomyia hamata . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S2Ecological niche models for Brumptomyia mesai . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S3Ecological niche models for Lutzomyia beltrani . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S4Ecological niche models for Lutzomyia bispinosa . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S5Ecological niche models for Lutzomyia carpenteri . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S6Ecological niche models for Lutzomyia cayennensis . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S7Ecological niche models for Lutzomyia chiapanensis . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S8Ecological niche models for Lutzomyia cratifer . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S9Ecological niche models for Lutzomyia deleoni . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S10Ecological niche models for Lutzomyia dodgei . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S11Ecological niche models for Lutzomyia longipalpis . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S12Ecological niche models for Lutzomyia ovallesi . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S13Ecological niche models for Lutzomyia panamensis . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S14Ecological niche models for Lutzomyia permira . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S15Ecological niche models for Lutzomyia serrana . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S16Ecological niche models for Lutzomyia steatopyga . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S17Ecological niche models for Lutzomyia trinidadensis . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S18Ecological niche models for Lutzomyia undulata . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S19Ecological niche models for Lutzomyia ylephiletor . A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S20Ecological niche models for Lutzomyia anthophora (temperate). A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S21Ecological niche models for Lutzomyia californica (temperate). A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S22Ecological niche models for Lutzomyia diabolica (temperate). A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S23Ecological niche models for Lutzomyia stewarti (temperate). A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S24Ecological niche models for Lutzomyia texana (broad-range). A) A2 scenario, CSIRO model; B) A2 scenario, HadCM3 model; C) B2 scenario, CSIRO model and D) B2 scenario, HadCM3 model.(TIF)Click here for additional data file.Figure S25Proportion of pixels for sand fly ENM from current to 2080 in the A2 scenario. Predicted future scenarios using the CSIRO model (left) and HadCM3 model (right). Empty circles = tropical species; solid dot = temperate species; asterisk = broad-range species.(TIF)Click here for additional data file.Figure S26Changes in niche breadth, ENM centroid and elevational range in A2 scenario. The circle size represents the proportional distribution change, while the thickness is proportional to the overlap percentage between current and 2050 projections . The direction of the arrow represents the direction of change of the centroid position and its size represents its magnitude. The elevational range changes are represented by the bars . Epidemiologically important species are: 1) Lu. anthophora, 2) Lu. cruciata, 3) Lu. diabolica, 4) Lu. longipalpis, 5) Lu. olmeca olmeca, 6) Lu. ovallesi, 7) Lu. panamensis, 8) Lu. shannoni and 9) Lu. ylephiletor.(TIF)Click here for additional data file.Table S1Reference points for sand fly collection records used in ENM modeling of NCA species.(PDF)Click here for additional data file.Table S2Test accuracy, AUC (ROC curve) and statistical significance.(PDF)Click here for additional data file.Table S3Proportion of sand fly species' ENM overlap and their territorial projection of change to 2050.(PDF)Click here for additional data file.


URM11 and URM12 have overlapping expression patterns and are required for tRNA thiolation. The characterization of urm11 and urm12 mutants reveals that the lack of tRNA thiolation induces changes in general root architecture by influencing the rate of lateral root formation. In addition, they synergistically influence root hair cell growth. During the sulfur transfer reaction, URM proteins of different organisms interact with a thiouridylase, a protein-protein interaction that also takes place in Arabidopsis, since URM11 and URM12 interact with the Arabidopsis thiouridylase ROL5. Hence, the sulfur transfer reaction is conserved between distantly related species such as yeast, humans, and plants, and in Arabidopsis has an impact on root development.Ubiquitins are small peptides that allow for posttranslational modification of proteins. Ubiquitin-related modifier (URM) proteins belong to the class of ubiquitin-like proteins. A primary function of URM proteins has been shown to be the sulfur transfer reaction leading to thiolation of tRNAs, a process that is important for accurate and effective protein translation. Recent analyses revealed that the Arabidopsis genome codes for two URM proteins, URM11 and URM12, which both are active in the tRNA thiolation process. Here, we show that  Ubiquitins (Ub) are small peptides that allow posttranslational modification of proteins. Ubiquitylation is the reversible attachment of Ub to proteins involving activation, conjugation, and ligation of Ub via corresponding E1, E2, and E3 ligase activities, respectively In addition to Ub, ubiquitin-related modifiers (URMs) were identified that are not highly homologous to ubiquitin in respect to the amino acid sequence but share a β-grasp motif as typical structural feature of this type of protein. The primary identified function of URM and URM-related peptides in many different organisms such as archaea, yeast, and eukaryotes is as sulfur carriers in tRNA thiolation URM11 and URM12, were identified encoding proteins that are involved in tRNA thiolation cnx5/sir1 mutants show a severe growth defect, whereas the rol5 mutant is mainly affected in root growth and shows changes in cell wall architecture. The enhanced severity of the cnx5/sir1 mutant phenotype compared to rol5 is likely caused by a general defect in sulfur transfer reactions in this mutant that also affects molybdopterin biosynthesis Recently, two Arabidopsis genes, URM11 is expressed to a higher level than URM12, there is a significant synergistic interaction between the two proteins. Finally, the analysis of mutants including a urm11 urm12 double mutant shows that the lack of tRNA thiolation has an effect on the general root architecture but also on root cell development.This work presents a more detailed characterization of URM11 and URM12 of Arabidopsis. Our data support the finding that URM11 and URM12 are involved in tRNA thiolation. The protein interactions of the URM proteins are conserved in plants, as they interact with the thiouridylase ROL5. Even though URM genes,  which were termed URM11 and URM12Proteins with significant homology to ubiquitin-related modifier (URM) proteins have been identified in a number of organisms. The genome of Arabidopsis harbors two urm1 mutant defective in tRNA thiolation URM11 and URM12urm1 mutant by URM and GFP-URM proteins was compared. The Δurm1 mutant transformed with cDNAs for URM11 or GFP-URM11 and GFP-URM12 constructs under the control of a constitutively active yeast promoter was analyzed for the presence of thiolated tRNAs. The binding of N-acryloylamino phenyl mercuric chloride (APM) to 2-thiouridine residues leads to the retardation of thiolated tRNAs in acrylamide gels, making them readily detectable. The thiolated tRNAs detectable in wild-type yeast but absent in the Δurm1 mutant are present in the Δurm1 mutant complemented with either GFP-URM11 or GFP-URM12 to an extent that is comparable to the complementation with URM11 (The involvement of URM11 and URM12 in the sulfur carrier process important for the thiolation of eukaryotic cytoplasmic transfer RNAs (tRNAs) was indicated by the successful complementation of the yeast Δth URM11 . This co35S:GFP-URM11 and 35S:GFP. Both the GFP-URM11 fusion protein as well as GFP alone localize to the cytoplasm and show a signal in the nucleus (urm11 urm12 double mutants (see below). Immunodetection by western blotting of proteins from a total plant extract or a purified nuclear preparation revealed the presence of HA-URM11 in both fractions that migrated at the expected mass of around 13 kDa. In contrast, the histone H3 protein was only detectable in the nuclear preparation and not in the total fraction, confirming a strong enrichment of nuclear proteins in the nuclear fraction  was used which contains a T-DNA insertion in the first intron. For URM12, two insertion lines were used; urm12-1 harbors the insertion 250 bp upstream of the start codon and urm12-2 in the first intron (urm12-1 line still produced URM12 mRNA (data not shown). By contrast, both urm11-1 and urm12-2 lack detectable levels of gene expression of URM11 and URM12, respectively . Finally, the lrx1 urm11-1 urm12-2 triple mutant was complemented with 35S:HA-URM11 and 35S:HA-URM12 constructs. Transgenic lines expressing either of the two URM genes developed an lrx1-like root hair phenotype  are found in phylogenetically distantly related species. They can have a low level of identity or similarity, as found for Arabidopsis URM11 and URM12 versus the yeast Urm1p or human Urm1 Δurm1 mutant urm11-1 urm12-2 double mutant are missing detectable levels of thiolated tRNAs but are viable. However, in both organisms, the growth process is affected. The Arabidopsis urm11-1 urm12-2 double mutant develops a modified root architecture with a lower lateral root density compared to the wild type. Lateral root formation is under the control of auxin and cytokinin, but is also strongly influenced by nutrient availability Δurm1 is impaired in pseudohyphal growth, a developmental response to nutrient limitation The thiolation of the uridine in the wobble position of tRNAs conferred by the protein network involving URM proteins is assumed to increase codon-anticodon accuracy, while blocking this tRNA modification is expected to have a negative impact on translation efficiency URM11 and URM12 also affect root hair development. Even though the urm11-1 urm12-2 double mutant does not show impaired root hair growth, it does suppress the root hair formation mutant lrx1. LRX1 is an extracellular protein that is involved in root hair cell wall formation rol5 mutant was initially isolated as a suppressor of lrx1lrx1 was tested in the lrx1 urm11-1 urm12-2 triple mutant. The comparable effect of rol5 and the urm11-1 urm12-2 double mutant suggests that interfering with tRNA modification is causing the suppression of lrx1. Since lrx1 is a cell wall formation mutant, suppression is likely induced by changes in cell wall structures. Indeed, the rol5 mutant was shown to induce modifications in cell wall structures In addition to lateral root formation, mutations in lrx1 root hair mutant phenotype rol5 and urm11-1 urm12-2 mutant lines, supporting the hypothesis that alterations in tRNA thiolation have an impact on TOR signaling.In Arabidopsis, inhibiting the TOR network by rapamycin, a macrocyclic lactone specifically inhibiting the TOR kinase urm1 mutant, it is quite probable that URM12 shows a localization pattern that is comparable to URM11. Since the HA-URM11 construct is under the control of the strong 35S promoter, a possible ectopic detection of HA-URM11 protein in the nucleus cannot be excluded. Yet, in a high-throughput analysis of yeast protein localization, Urm1p was also localized to the cytoplasm and the nucleus. Even though this localization was later changed to cytoplasmic , there seems no direct experimental evidence for this restriction (www.yeastgenome.org). The URM11 and URM12 interaction partner ROL5, however, appears to predominantly colocalize with mitochondria The data presented here support the view that the tRNA thiolation process is conserved across distantly related species. This assumption is corroborated by the analysis of URM protein function in Arabidopsis /URM11_rev(TTATCCACCATGCAAAGTGGAAAT) and URM12_for (GGATCCATGCAATTTACTCTTGAGTTCGGT)/URM12_rev (TCATCCACCGTGCAGAGTCGAAAT). The obtained fragments were cloned into pGEM-T easy (Promega) for sequencing. For obtaining the N-terminal GFP fusions, the correct cDNAs in pGEM-T easy constructs were digested with BamHI and a GFP-BamHI cassette NotI and cloned into the yeast overexpression vector pFL61 For complementation of the yeast ROL5, URM11 and URM12 were amplified with the primer pairs KpnI-At2g44270-1F(GGTACCATGGAGGCCAAGAACAAGAAAGCAG)/SmaI-At2g44270-1R(CCCGGGTTAGAAATCCAGAGATCCACATTG) for ROL5, XbaI-URM11-F (TCTAGAATGCAATTAACTCTTGAATTCG)/BamHI-URM11-R(GGATCCTTATCCACCATGCAAAGTGGAA) for URM11 and XbaI-URM12-F(TCTAGAATGCAATTTACTCTTGAGTTCGGTGGAG)/BamHI-URM2R (GGATCCTCATCCACCGTGCAGAGTCGAAATGAAA) for URM12. These fragments were then cloned into pGEM-T easy for sequencing. Subsequently, one of the ROL5 clones was digested with KpnI and SacI and cloned into pLEXA-N  cut with the same enzymes. The clones of URM11 and URM12 were digested with XbaI and BamHI and cloned into pGAD-HA  cut with the same enzymes.For the yeast two-hybrid experiment, cDNAs of URM11 and URM12, genomic clones were amplified using the primer pairs URM11_HA_gen_for (CTCGAGATGTACCCATACGATGTTCCAGATTACGCTATGCAATTAACTCTTGAATTCGGGTAC)/URM11_HA_gen_rev(TCTAGAGAAAGGACACTTAAAATTGATAAATACTCTAATATCA) and URM12_HA_gen_for (CTCGAGATGTACCCATACGATGTTCCAGATTACGCTATGCAATTTACTCTTGAGTTCGGGTACACTATTAC)/URM12_HA_gen_rev(TCTAGA TCATCCACCGTGCAGAGTCGAAATGAAAACTATA), respectively. For sequencing, the clones were ligated into pGEM-T easy. Correct clones were digested with XhoI and XbaI and cloned into the expression cassette of pART7 35S CaMV promoter and OCS terminator, cut with the same enzymes. For plant transformation, the 35S-HAURM-OCS cassettes were cut out with NotI and cloned into the plant transformation vector pBART For expressing HA-tagged versions of GFP fusion constructs for transient expression in plants were produced by XhoI digestion of pART7-HA-URM11 and insertion of an XhoI-GFP cassette. The GFP gene was amplified from the vector pMDC83GFP_XhoI_for(CTCGAGATGAGTAAAGGAGAAGAACTTTTC) and GFP_XhoI_rev_NOSTOP(CTCGAGGTGGTGGTGGTGGTGGTGTTT).The N-terminal genomic URM11:GUS construct was produced by PCR amplification of the promoter using the primers URM11Prom_F (TTGCGACTCGGATTGGTTAGAATC) and URM11Prom_R (CGTCGTTGATGTTTGCAGGAGG) and cloning into pENTR (Invitrogen). A correct clone was then used for cloning of the URM11 promoter into the gateway vector MDC164 containing the GUS gene The URM12 promoter:GUS construct, 1.8 kb of promoter sequence 5′ upstream of the ATG start codon was PCR-amplified using the primers URM12Prom_F (AAGCTTGGTAAATAATCTATAGTTTGTTTAC) and URM12Prom_R (TCTAGA CTTCAATGAAGTTTTGCGGTAAC) with an HindIII site and a XbaI at the 5′ and 3′ end, respectively. After digestion with HindIII and XbaI, the fragment was cloned into pGPTV-BarFor obtaining the Arabidopsis thaliana, accession Columbia. The lrx1 allele is described in urm11-1 and urm12-2 allele are the Salk lines 024513 and 070672.47.90, respectively. The urm12-1 allele not further used in this study is the line ET5108 and is of the accession Landsberg erecta.All plant lines used are in vitro and in soil as described Seedlings and plants were grown pBART and pGPTV-bar vectors) or 20 µg/mL hygromycin (pMDC164).Selection of transgenic plants produced by the standard floral dip method was done on 20 µg/mL basta  Arabidopsis 2-week old seedlings grown under sterile conditions following the protocol of For western blotting, proteins were prepared by mixing nuclear extracts or total seedling material with standard SDS-PAGE loading buffer prior to heat denaturation. SDS-PAGE, blotting onto nitrocellulose, and immunodetection by ECL technology was performed as described by Ringli GUS expression analysis were done with a Leica LZ M125 stereomicroscope. Data points of lateral root development represent ≥25 seedlings. The experiment was done several times. For the root hair phenotype, over 30 seedlings of each line were analyzed.Epidermal GFP fluorescence was analyzed using a Zeiss Imager Z1 microscope equipped with an Axiocam HRC. GFP fluorescence of yeast cells was analyzed with a Leica DM6000 equipped with a Leica BFC 350FX. Phenotypic observations and ACTIN2F (59-AATGAGCTTCGTATTGCTCC-39) and ACTIN2R (59GCACAGTGTGAGACACACC-39), URM11_rt_for (ATGCAATTAACTCTTGAATTCG)/URM11_rt_rev(TTATCCACCATGCAAAGTGGAA), and URM12_rt_for(ATGCAATTTACTCTTGAGTTCG)/URM12_rt_rev (TCATCCACCGTGCAGAGTCGAA). The ACTIN2 PCR was done with 25 cycles,.the URM PCRs with 30 cycles of amplification.Seedlings were grown vertically on half-strength MS Medium in a vertical orientation for 7 d as described above. The tissue of 120 entire seedlings was frozen in liquid nitrogen and grinded. The powder was then used for RNA extraction using the SV Total RNA isolation System kit (Promega). The reverse transcription was conducted with 500 ng of total RNA using the i_script kit (Biorad). One tenth of the obtained cDNA was then used for RT-PCR using the primer pairs Arabidopsis seedlings were grown vertically on plates for 14 d as described. Approximately 250 seedlings were used for extraction. The seedlings were grinded in liquid nitrogen and the material was extracted two times with 8 ml acidic phenol (Sigma), 0.8 ml chloroform and once with 4 ml acidic phenol, 0.4 ml chloroform.pFL61 constructs and His, Leu, Ade and Ura for growth of the wild type or Δurm1 mutant. The tRNA was extracted 2 times with 4 ml acidic phenol, 0.4 ml chloroform.Yeast strains were grown at 30°C in 50 ml liquid SD media supplemented with His, Leu, Ade for strains complemented with After extraction of the plant or yeast material, tRNA was purified with AX100 columns from MACHEREY NAGEL following manufacturer’s instructions. For analysis, the purified tRNA was separated on an acrylamide gel supplemented with N-acryloylamino phenyl mercuric chloride (APM) by the method adapted from URM11: At2g45695; URM12: At3g61113; LRX1: At1g12040; ROL5: At2g44270.The Arabidopsis genes discussed in this study have the following accession numbers: Figure S1Arabidopsis GFP-URM11 localizes to the cytoplasm and the nucleus. Transient transformation of 35S:GFP-URM11 and 35S:GFP into onion cells results in a comparable pattern of cytoplasmic and nuclear fluorescence. For each construct, at least 15 transformed cells were analyzed. Bar = 100 µm.(TIF)Click here for additional data file.


Effective antiviral agents and vaccines against EV71 are not available. However, Vero cell-based chemically inactivated EV71 vaccines could be developed soon based on the success of inactivated polio vaccine. Like poliovirus, EV71 has a positive single-stranded RNA genome of about 7400 nucleotides which contains a single open reading frame (ORF) flanked by conserved and untranslated regions at both the 5The universal amplification of the full length genome of EV71 regardless of its genetic diversity, and the subsequent construction of a human RNA polymerase I-driven reverse genetics (RG) system to produce pure virus stocks in Vero cell within 10 days were described. The rescued viruses were characterized by DNA sequencing, cytopathic effect (CPE) and indirect fluorescent assay (IFA) in comparison with the wild-type viruses. Moreover, the rescued viruses grew to high titers and retained the same immunogenicity as the wild-type viruses.We have established a simplified method to rescue RG EV71 virus from diverse clinical isolates with detailed genetic information and to prepare virus stocks in only 10 days. This method could accelerate EV71 vaccine development. There is also a short poly A tail following the 3′-UTR[Enterovirus 71 (EV71) belongs to the enterovirus genus of the picornavirus family, which also includes coxsackievirus A16 (CA16), poliovirus and echovirus[e 3′-UTR,3. The Ee 3′-UTR,5. Basede 3′-UTR-6. Predoe 3′-UTR.EV71 is a causative agent of hand, foot and mouth disease (HFMD), most frequently affecting infants and children below 6 years old. Infection with EV71 is usually mild but occasionally leads to neurological manifestations ranging from aseptic meningitis to acute flaccid paralysis and lethal brainstem encephalitis,10. Largin vitro from cDNA have been developed for many picornaviruses, including EV71[in vivo have been described for influenza virus[Reverse genetics (RG) permits the use of complementary DNA (cDNA) copies of viral RNA genome to rescue virus with detailed features of viral genetic diversity, antigenicity and virulence. RG systems using T7 or S6 RNA polymerase to synthesize infectious RNA ing EV71,15. In aza virus, HFMD[1za virus and EV71za virus.Here, we have developed a robust RT-PCR method for the universal amplification of whole EV71 genome and a novel human RNA polymerase I (hPolI)-driven RG system for the rapid production of pure and immunogenic stock in Vero cell. The implications of these findings are discussed.′ end for joining with hPolI promoter and murine terminator (mTer) during the In-Fusion reaction. The genomic RNAs of EV71 strains of different subgenogroups  isolated from 1970 to 2010 were efficiently amplified by RT-PCR using above primers. Moreover, to help combat and study the emerging EV71 strains from Malaysia, the unknown genomes of 5 strains #363 to #577  on day 21. In addition, the neutralization titers elicited by RG and wild-type of EV71-B5 were statistically identical. The results indicated that the RG viruses retained the immunogenicity of their original wild-type virus. Hence, the pure viruses rescued by this novel RG system have great potential advantages in vaccine production.To test whether the RG viruses retained the same immunogenicity as wild-type virus, two RG viruses B5-RG1 and B5-RG2 were randomly chosen for immunogenic study in mice. The formalin inactivated whole EV71-B5 RG and wild type viruses with AlPOIn conventional studies, the infectious clones of single-stranded positive RNA viruses with large genome, such as poliovirus and FMDV, were constructed by elaborate ligation of several subgenomic clones generated from multiple, segmented RT-PCR due to the difficulties in long template PCR amplification-21. To ain vivo transcription at the 5′ and 3′ ends of viral RNA transcripts , EV71-B2 , EV71-B4 (HFMD41 /SIN), EV71-B5 , EV71-C1 , EV71-C2 , EV71-C4 , EV71-C5 , and some unknown EV71 strains from Malaysia labeled #363, #366, #396, #482, and #557. All EV71 wild type strains were isolated from patients and propagated in RD cell. All reverse genetics viruses were rescued in Vero cells.Human rhabdomyosarcoma (RD) cell and Vero cell were maintained in Minimum Essential medium  containing 10% fetal bovine serum  at 37°C in the presence of 5% CO18 as primer. The full-length genomic cDNAs of EV71 were amplified using PfuUltra II Hotstart PCR Master Mix  and EV71 specific universal primers EV71-Uni-F (5′-GGCCGCCGGGTTATTTTAAAACAGCCTGTGGGTTG) and EV71-Uni-R (5′-CCGAAGTTGGGGGGGTTTTTTTTTTTTTTTTTTTTTTTTTTGCTATTCYGGTTATAAC). The reaction parameters were as follows: an initiation step at 94°C for 2 min, followed by 40 cycles  and a final extension step at 72°C for 5 min. The amplicons were analyzed by gel electrophoresis and purified using QIAquick gel Purification Kit (Qiagen).Viral RNA was extracted from culture supernatant using RNeasy mini kit (Qiagen). The reverse transcription reaction was performed for 1 hour at 42°C with RevertAid First Strand cDNA Synthesis Kit (Fermentas) using viral RNA as the template and oligo(dT)′-CCCCCCCAACTTCGGAGGTC) and pJET-hPol1mTer-r (5′- AATAACCCGGCGGCCCAAAATG). The EV71 genomic cDNA amplicons were joined with linearized pJET-hPolI/mTer using In-Fusion HD cloning Kit . 5 μl of the cloning reaction was transformed into 100 μl of competent XL1-Blue E. coli by heat shock. The recombinant plasmids were selected by PCR screening with primers pJET-f (5′-CGACTCACTATAGGGAGAGCGGC) and EV71-VP1-r (5′-GCYCCRTATTC AAGRTCTTTCTC), and their correctness and genotypes were confirmed by DNA sequencing and phylogenetic tree analysis, respectively.The human RNA polymerase I (hPolI) promoter and murine terminator cassette sequence were amplified from sap/pol vector and then inserted into pJET1.2 vector as described previously. The pla5 Vero cells in 2 ml medium in 6-well plates. The transfected cells were lysed after 3 days by 3 freeze-thaw cycles and the supernatant containing viruses was collected for further passage. The rescued viruses were harvested after 4 days and their titers were determined as 50% tissue culture infective dose (TCID50) according to cytopathic effect (CPE) in Vero cell using the Reed and Muench formula.A mixture of 1 μg of each recombinant plasmid and 2.5 μl Lipofectamine 2000 reagent (Invitrogen) was transfected into 5x1050 assay.The growth kinetics of rescued viruses was determined in subconfluent Vero cell culture in 1 ml medium in 24-well plates. The cells were incubated with rescued viruses at a multiplicity of infection (MOI) of 1 for 1 h at 37°C, and then washed with PBS 3 times and cultured in MEM with 2% FBS. The infected cells were frozen at day 2, 3, 4, 5, 6 and 7 post-infection and stored at -80°C. After 3 freeze-thaw cycles, the virus concentrations were titrated by TCIDVero cells transfected with the recombinant plasmids or infected with rescued RG virus were grown in 96-well plates. The cells were fixed with 4% PFA after 24 h and incubated with guinea pig anti EV71 serum (produced in house) diluted 1:500 for 30 min at 37°C. Then the cells were washed and incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-guinea pig IgG (Dako) diluted 1:200 for another 30 min. Finally, the cells were rinsed with PBS and visualized under a fluorescent microscope (Olympus).th day post-infection and purified by ultracentrifugation as described previously[in vitro microneutralization assay[50 of virus, and the neutralization antibody titer was determined as the highest dilution of serum that protected 50% of Vero cell cultures.The rescued and wild type viruses in Vero cell were harvested at 5eviously. The puron assay. BrieflyAll animal experiments were carried out in accordance with the Guides for Animal Experiments of the National Institute of Infectious Diseases (NIID), and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Temasek Life Sciences Laboratory, Singapore .50: 50% tissue culture infection dose; MEM: Minimum essential medium; Vero: African green monkey kidney cell.EV71: Enterovirus 71; HFMD: Hand foot and mouth disease; ORF: Open reading frame; UTR: Untranslated region; hPolI: Human RNA polymerase I; mTer: Murine terminator; RG: Reverse genetics; CPE: Cytopathic effect; IFA: Immunofluorescent assay; MOI: Multiplicity of infection; TCIDThe authors declare that they have no competing interests.TM performed the experiments. TM and TKK drafted the manuscript. TM and JK designed the study. JK supervised the work and edited the final version of this manuscript. All authors read and approved the final manuscript.(C) IFA identification of the rescued EV71-B5 RG viruses. The viral proteins were detected with mouse monoclonal antibodies 1D9 and 4B12 which were raised against VP1 and 3D of EV71, respectively. The pJET-hPolI/mTer-EV71-B5 plasmid transfected cells and RG EV71-B5 or wild-type B5 infected cells showed positive IFA signals at 24 h; while mock cells were negative.Click here for file


The aim of this study is to apply accurate assays of vitamin D forms  were recorded. Hierarchical linear regression was used to predict neonatal 25(OH)D concentrations.2 and 25(OH)D3 forms compared to neonates  with a ratio of 1:3. The epimer concentrations, which contribute approximately 25% to the total vitamin D levels, were similar in mothers and neonates . No correlation was observed in mothers between the levels of the circulating form (25OHD3) and its active form. Neonatal 25(OH)D2 was best predicted by maternal characteristics, whereas 25(OH)D3 was strongly associated to maternal vitamin D forms . Maternal characteristics explained 12.2% of the neonatal 25(OH)D, maternal 25(OH)D concentrations explained 32.1%, while epimers contributed an additional 11.9%.Mothers had similar concentrations of 25(OH)DBy applying a novel highly specific vitamin D assay, the present study is the first to quantify 3-epi-25(OH)D concentrations in mother - newborn pairs. This accurate assay highlights a considerable proportion of vitamin D exists as epimers and a lack of correlation between the circulating and active forms. These results highlight the need for accurate measurements to appraise vitamin D status. Maternal characteristics and circulating forms of vitamin D, along with their epimers explain 56% of neonate vitamin D concentrations. The roles of active and epimer forms in the maternal - neonatal vitamin D relationship warrant further investigation. Vitamin D insufficiency and deficiency has been associated with a wide spectrum of diseases, ranging from neurological disorders to chronic inflammatory conditions . The res2D concentrations in a meta-analysis of mother-neonate studies suggest that measurement of vitamin D concentrations should go beyond the routinely measured 25(OH)D forms , double-hydroxylated , epimers ], in a Northern Greece cohort of pregnant women at term and their neonates, by applying a novel highly specific and accurate assay. A secondary aim was to predict neonatal vitamin D concentrations by means of maternal parameters.The primary aim of this study was to determine serum (mothers) and umbilical cord (neonates) concentrations of all vitamin D forms  compared to neonates  D F = 4.914, p =0.011] for neonates of mothers in the deficient, insufficient and sufficient group, respectively. This overall result was due to a difference between the deficient and sufficient groups (p =0.012), but not due to other comparisons .2D2 and 1α,25(OH)2D3 forms are concerned, only 1α,25(OH)2D3 was measured in mothers, at a very low concentration (0.06 ± 0.06 ng/mL). The 1α,25(OH)2D2 form was below the limit of quantitation. In line with previous reports [3] and the highly active 1α,25(OH)2D3 concentrations .The epimer concentrations D2 levels (75.6 ± 22.2% in mothers and 75.9 ± 23.9% in neonates); thus, the ratios of 25(OH)D3: 25(OH)D2 were 3:1, approximately, for both mother and neonates. A summary table of means, standard deviations and mean measurement errors for the primary forms of vitamins D2 and D3 along with the active forms and their epimers is provided in Additional file 2D3 and 3-epi-25(OH)D concentrations.Mean concentrations of vitamin D forms in mothers and neonates are illustrated in Figure  reports , no signThe hierarchical linear regression models for predicting neonatal 25(OH)D concentrations are detailed in Table 2 concentrations were best predicted from maternal characteristics (R2 = 0.253), whereas 25(OH)D3 was strongly linked to maternal vitamin D forms (R2 = 0.478). Maternal serum concentrations of PTH, Ca and P together only explained a small proportion of the neonatal 25(OH)D2 (R2 = 0.046) and an even smaller part of the 25(OH)D3 (R2 = 0.013). Neonatal vitamin D concentrations were calculated as the sum of 25(OH)D2 and 25(OH)D3. Circulating neonatal vitamin D concentrations in newborns followed the pattern of predicting 25(OH)D3, with maternal 25(OH)D2 and 25(OH)D3 explaining 32.1% of the neonatal vitamin D variance and epimer forms contributing an additional 11.9%. Therefore, all four maternal vitamin D forms combined  explained 44% of the neonatal vitamin D concentrations when controlled for other maternal characteristics such as age, UVB exposure, vitamin D and Ca intake and Ca, P and PTH concentrations. On the contrary, 1α,25(OH)2D3 did not make a contribution to the neonatal vitamin D concentrations. Predicting 25(OH)D in neonates, mother’s age showed statistical significance for the coefficients (β = -0.343). For 25(OH)D2, mother’s 25(OH)D2 concentrations showed statistical significance for the coefficients (β = 0.218) and 3-epi-25(OH)D3 (β = 0.596). These standardized β values can be used for weighting each individual’s measures on the IVs to obtain individual predicted score on the DV, respectively. Mother’s age was independent of vitamin D intake , negatively correlated with UVB exposure  and weakly negatively correlated with Ca intake . the neonatal 25(OH)DThe potential impact of vitamin D deficiency during pregnancy on maternal and neonatal health has attracted much interest in recent years. It has been suggested that maintaining adequate maternal stores of vitamin D during pregnancy is of vital importance for both mothers and neonates to ensure skeletal and extra-skeletal health.The results of this study come mainly from a population of pregnant women with vitamin D deficiency or insufficiency. Although the study was not designed for this3 being the most abundant circulating vitamin D form in both mothers and neonates. These results reflect previous reports of widespread vitamin D deficiency and insufficiency in Europe and the USA. However, the known cross-reactivity of many assays with the epimer forms suggests that levels reported in previous studies are overestimations. Furthermore, the conundrum of a mismatch between levels of the usually quantified circulating forms (25OHD) and the active form 2D) [3 and 1α,25(OH)2D3 once epimers have been differentiated. purpose, a high prevalence of maternal vitamin D insufficiency and deficiency was detected, in a sunny European area, such as Northern Greece. A similar pattern of distribution between maternal and neonatal 25(OH)D concentrations was observed, with 25(OH)D-(OH)2D)  have bee2 and 25(OH)D3, separately; serum total 25(OH)D has been designated as the functional indicator of vitamin D status [. However, the ability to accurately measure serum concentrations of 25(OH)D2 and 25(OH)D3 brings new potential to both observational and intervention studies. On a physiological basis, it could be hypothesised that maternal vitamin D active forms have an impact on the newborn, which, to a great extent, depends on the mother to form its dynamic vitamin D equilibrium. Therefore, these findings confirm current concerns regarding the maintenance of adequate maternal vitamin D status during pregnancy, since the reflection of maternal concentrations of these forms explains 32.1% in neonates. It should be noted that data on 25(OH)D2 and 25(OH)D3 concentrations exclude epimer forms; thus, caution should be paid when comparing them to other studies [At this time, there is no documented benefit in measuring 25(OH)DD status . However studies .2 is the only high-dose preparation available in many countries, potential differences in the ability of assays to accurately detect 25(OH)D2 and 25(OH)D3 are of clinical importance, in cases where supplementation is suggested. Moreover, when 25(OH)D results are reported as 25(OH)D2 and 25(OH)D3, vitamin D2 administration does reduce serum 25(OH)D3 concentrations [3 value in the setting of ergocalciferol supplementation does not constitute vitamin D deficiency. LC-MS/MS and the potential of accurate measurement of both bioactive forms of vitamin D could offer a valuable tool in daily practice, in order to avoid data misinterpretation, especially in conditions like pregnancy.Given that vitamin Dtrations . Until t2 and 3-epi-25(OH)D3 forms have been previously reported in infants [3 form, in a small cohort of healthy adults [2 in both mothers and neonates. Large inter-individual variances in the epimer content were noted in vitamin D concentrations, ranging between 0% and 100% with 63.8% of the mothers and 67.8% of the neonates showing epimer to total circulating concentration of 25% or less. Thus, the epimer-adjusted concentration is only applicable to conclusions at the aggregated level  and should not be used for making judgments at the individual level, unless 3-epi-25(OH)D2 and 3-epi-25(OH)D3 are clearly separated and excluded from the 25(OH)D measurements. On the other hand, based on present results, it becomes evident that 1α,25(OH)2D2 and 1α,25(OH)2D3 have minor contributions to the sum of vitamin D measurements in both mothers and infants.The findings of this study, using a novel assay with the ability not just to exclude but also to measure vitamin D epimers demonstrated that epimers comprise approximately 25% of the measured vitamin D concentrations in both mothers and neonates, following similar patterns of distribution. The presence of both 3-epi-25(OH)D infants . Our groy adults . These rBased on these findings, it could be hypothesized that assays that do not separate the 3-epi forms or have significant cross-reactivity with the epimer will, most likely, report erroneously high concentrations for both infants and adults, as 3-epi constitutes a substantial fraction of total 25(OH)D. This assay limitation should be considered by clinicians measuring vitamin D status in infants and mothers. LC-MS/MS, by measuring vitamin D isoforms separately, provides a ‘clear-cut’ view of vitamin D status. By excluding epimer concentrations, it appears that the mean vitamin D concentration in term pregnancies is considerably below the sufficiency threshold. Although the sample size of the present study was small for drawing conclusions, the accurate measurement of active vitamin D metabolites could offer a valuable tool in the establishment of a novel, realistic view of vitamin D status during pregnancy.3 concentrations, which constitutes the major neonatal vitamin D form. The addition of certain maternal parameters could offer additional prognostic value in this process, specifically in neonatal 25(OH)D2 concentrations. The additional analytical capacity enhances the predictive power with the epimers contributing 11.9% to an overall 44% explained variances in active vitamin D concentrations in neonates. This result significantly exceeded previous reports of 19% in a twin study, which investigated genetic versus maternal vitamin D concentrations in determining offspring vitamin D concentrations [Based on our primary results, regarding the accurate proportions of vitamin D metabolites in maternal circulation, we further investigated if there is an ability to predict neonatal 25(OH)D concentrations from maternal parameters. Our analysis showed that, apart from being a reliable marker of vitamin D maternal status, 25(OH)D comprises a significant parameter in predicting neonatal 25(OH)Dtrations . Overall2 and D3, hydroxylated derivatives and epimer forms. To the best of our knowledge, these data are unique to the literature on pregnancy. Second, as routine assays do not allow differentiation among the full range of different vitamin D forms, this novel assay allowed for a very detailed approach to the complex vitamin D metabolism in the mother - newborn bipole. Third, the study allowed the control for maternal characteristics such as age, UVB exposure, dietary intake and PTH, Ca and P concentrations, which afforded separating the explained variances for the active vitamin D concentrations over and above the shared variances by maternal characteristics. In combination, we were able to explain 56.1% in the variances in neonatal 25(OH)D concentrations. Limitations of the study were its rather small sample size and its cross-sectional design, which prevented prospective data to be collected throughout pregnancy. Moreover, measurement of vitamin D-binding protein (VDBP), a significant parameter of vitamin D dynamics in pregnancy, was not feasible.The present study has three major advantages. First, the data incorporate specific and accurate measurement of seven out of eight forms of vitamin D, including vitamin D3 and 3-epi-25(OH)D2 forms in both mothers and neonates, which contribute approximately 25% to the total vitamin D concentrations and iii) a relationship between maternal and neonatal concentrations leading to a prediction model. The accurate assay highlights a considerable proportion of vitamin D exists as epimers and there is a lack of correlation between the circulating and active forms. These results underscore the need for accurate measurements to appraise vitamin D status. The results, based on specific and accurate measurement, revealed that maternal characteristics and active forms of vitamin D, along with their epimers explain 56% of neonatal vitamin D concentrations. Further investigation, based on accurate measurements of vitamin D metabolites, is warranted to establish optimal concentrations during pregnancy, in an attempt to prevent maternal morbidity and developmental deficiencies.This study provided evidence for i) maternal and neonatal vitamin 25(OH)D concentrations in a sunny European area, which proved to be sub-optimal, ii) 3-epi-25(OH)D2; 1α,25(OH)2D3: 1-alpha-25-dihydroxyvitamin D3; 25(OH)D2: 25-hydroxyvitamin D2; 25(OH)D3: 25-hydroxyvitamin D3; 3-epi-25(OH)D2: 3-epimer-25-hydroxyvitamin D2; 3-epi-25(OH)D3: 3-epimer-25-hydroxyvitamin D3; BLQ: Below the limit of quantification; Ca: Calcium; DV: Dependent variables; BMI: Body mass index; IV: Independent variables; LC-MS/MS: Liquid chromatography tandem mass spectrometry; LLOQ: Lower limit of quantification; P: Phosphorous; PTH: Parathyroid hormone; SGA: Small-for-gestational age; UVB: Ultraviolet B; VDBP: Vitamin D-binding protein; VIF: Variance inflating factor1α,25(OH)2D2: 1-alpha-25-dihydroxyvitamin DThe authors declare that they have no competing interests.SNK, DGG, AP and DPN designed research; IS, HB, FP and VH conducted research; IS analysed the vitamin D forms; AP analyzed the data; SNK, DGG, AP and DPN wrote the paper; DPN and BCT had primary responsibility for final content. All authors read and approved the final manuscript.Descriptive statistics for the primary forms, active forms and epimers of maternal and neonatal 25(OH)D2 and 25(OH)D3.Click here for fileCorrelation matrix (Pearson’s r) for the model predicting neonatal 25(OH)D.Click here for file


Glycogen Storage Disease Type Ia (GSD Ia) is a group of autosomal recessive inborn errors of metabolism that is caused by deficiency in glucose-6-phosphatase,and it is the major subtype of Liver Glycogen Storage Disease(LGSD) cases. Patients afflicted with GSD Ia cannot maintain glucose homeostasis and manifest hypoglycemia, hepatomegaly, lactic academia. However, we cannot separate GSD Ia form LGSD through clinical manifestations and routine laboratory tests, except for analysis of G6PC gene mutation and assay of glucose-6-phosphatase enzyme activity, which is an invasive method. Therefore, the analysis of G6PC gene mutation is an important method for diagnosing GSD Ia.To investigate the G6PC gene mutations in patients with Glycogen Storage Disease type Ia (GSD Ia),and analyze the relationship of its genotype and phenotype.We diagnose 48 patients with LGSD by clinical manifestations, laboratory tests and glucagon test. The entire coding region of the G6PC gene from peripheral blood was screened by PCR combined with direct DNA sequencing. The G6PC genes of 50 unrelated healthy children were investigated as comparisons to rule out gene polymorphism. We used software named DNAMAN to analyze their conservatism through many species homology comparison.Among 48 patients, there were 41.67 %(20/48) of them were found to have G6PC gene mutations. Totally eight types of G6PC gene mutations were detected, which were 727G>T, R83H, H119L, L173P, I341N, V88Xfs254, C109T and W87Xfs270,and 727G>T and R83H were more frequent, with frequencies of 37.50%(15/40) and 22.50%(9/40), respectively. C109T and W87Xfs270 were never reported before. Our clinical and routine laboratory data showed that the patients manifested the same phenotype even with different genotype.Our study revealed 727G>T and R83H mutations were prevalence in Chinese patients with GSD Ia. C109T and W87Xfs270 might be novel pathogenic mutations. There was no clear relationship between genotype and phenotype.


Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting. During development, differentiation programs require global rearrangements in repression and activation of lineage-specific genes. Chromatin-based epigenetic mechanisms ensure correct integration of developmental signals at gene regulatory regions, allowing the action of transcription factors and maintaining novel expression states in derived cell populations. Polycomb group (PcG) proteins are transcriptional repressors that remodel chromatin through epigenetic modifications that prevent changes in cell identity by maintaining transcription patterns, throughout development and in adulthood ,2. They Vertebrate skeletal muscle formation constitutes an interesting model system to study the epigenetic signals and molecular mechanisms that govern cellular differentiation ,12. Prevmuscle creatine kinase (mCK) is a classic PRC2 target gene where its expression is associated with the displacement of PRC2-Ezh2 complex, myogenin (MyoG) shows a switch between PRC2-Ezh2/PRC2-Ezh1 complexes upon differentiation, suggesting a role of this dynamics in gene activation. In light of their different chromatin associations, we verified that a Msk1-dependent signalling that controls H3S28ph, is involved in the specific displacement of PRC2-Ezh2 from the MyoG and mCK regulatory regions, to result in muscle differentiation. This confirms the findings of previous reports that consider Ezh2 downregulation to be a necessary step in the myoblast-myotube transition [MyoG transcriptional activation via recruitment of MyoD transcriptional factor in post-mitotic myotubes.In the current work we report that two different PRC2 complexes contribute to skeletal muscle differentiation: PRC2-Ezh2, which is predominant in proliferating myoblasts, and PRC2-Ezh1, which contains Ezh1 but is devoid of Ezh2, and is specific for post-mitotic myotubes. Interestingly, these two PRC2 complexes are differentially associated with muscle regulatory regions. Indeed, while ansition ,14. SurpThus, our study reveals a novel important layer of PcG-mediated epigenetic regulation of skeletal muscle cell differentiation, in which the different dynamics and chromatin regulated switch between PRC2-Ezh2 and PRC2-Ezh1 complexes are coordinated to induce the transition from myoblast to myotube transcriptional programs.MyoG, mCK and myosin heavy chain IIB (MHCIIB), and subsequently to fuse into multinucleated terminally differentiated myotubes [It is known that decreasing levels of PcG Ezh2 protein activates the terminal myogenic program at the time of differentiation, thereby controlling skeletal muscle gene regulation ,14. Howemyotubes   upon differentiation (DM day 2)   Figure . These e) Figure . However) Figure  and 2IIIs Figure , whereass Figure .MyoG promoter in differentiating cells could play a role in the regulation of the proper transcriptional profile of this gene.Taken together, these results suggest that the binding of the PRC2-Ezh1 complex at the MyoG promoter, suggests evidence for an unexpected scenario in which signal-dependent changes in chromatin have to deal with two different PRC2 complexes. We decided to test the possibility that the previously reported H3K27/H3S28 methyl/phospho switch mechanism [MyoG and mCK regulatory regions. Concomitant with the activation of these two genes, levels of H3S28ph and another active mark, acetylated histone 3 (AcH3), peaked at the MyoG promoter   and the echanism ,21 couldr Figure  and mCK r Figure  in myotur Figure . InteresG Figure  and mCK s Figure . In contn Figure . Additior Figure , mCK enhr Figure  and mCK r Figure  as well r Figure . These eG Figure  and mCK s Figure . In contt Figure . The difG Figure  and mCK s Figure  but not r Figure . Thus, tMyoG and mCK regulatory regions during muscle differentiation, we ruled out the possibility that H3S10ph functions in muscle gene activation  tail peptides, unmodified or modified with K27me3 or modified with the double mark K27me3S28ph, and we incubated them with nuclear extracts prepared from C2C12 myoblasts and myotubes. In agreement with earlier findings , Ezh2, Sn Figure  occurs tn Figure , we nextn Figure . We concMyoG promoter upon gene activation  targeting Ezh1, and induced to differentiate for 48 h . The binding of RNA Pol II was reduced at the coding region A, but not at the promoter, in the Ezh1 but not the Ezh2 knockdown  cells, as it has been shown that these domains are not limited to these cells [Surprisingly, our data showed that a PcG protein, such as Ezh1, is recruited on muscle specific gene when it is activated. Indeed, previous reports provided evidences that other PcG proteins bind actively transcribed genes ,37. The se cells . Indeed,se cells ,39. The As Ezh1 methyltransferase activity on histones is found to be modest , it willFuture genome-wide analysis coupled to loss-of-function experiments will be required to address EZH1 function in myofibres.MyoG transcriptional activation, removal of PRC2-Ezh2 from this gene would be necessary to guarantee its activation. One way of doing this would be to reduce intracellular PcG levels. In regard to this, Juan et al. [in vitro experiments indicate that the Msk1-methyl/phospho switch pathway is specific to the PRC2-Ezh2 complex, while it appears that PRC2-Ezh1 is not regulated by this mechanism  were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS) . Differentiation was induced when cells reached approximately 80% confluency using DMEM containing ITS media supplement  or 2% horse serum (HS) (Euroclone). Human primary myoblasts from healthy donors were obtained from the Telethon BioBank . The cell lines were cultured in DMEM supplemented with 20% FBS , insulin 10 mg/ml, human fibroblast growth factor (hFGF) 25 ng/ml, human epidermal growth factor (hEGF) 10 ng/ml (proliferating medium), and then induced to differentiate by means of DMEM supplemented with 2% HS (differentiating medium). H89  was replaced every 24 h.Single muscle fibres were isolated by standard procedures. In brief, the hind limb muscles were digested with collagenase and single myofibres were cultured in GM1 , 0.5% chick embryo extract , and penicillin-streptomycin (GIBCO)) at 37°C in suspension for 72 h, and then plated on matrigel -coated dishes for satellite cell culture. Then, 3 days later, the fibres were removed and the medium replaced with proliferation medium . After 4-5 days, the medium was replaced with differentiation medium (DM: 2% HS and 0.5% chick embryo extract in DMEM).RNA was extracted from cells using TriReagent (Sigma) according to the manufacturer's instructions. cDNA synthesis was performed using the QuantiTect reverse transcription kit . Quantitative real-time PCR reactions were performed in triplicate using QuantiTect SYBR Green master mix (Qiagen) on a DNA Engine Opticon 2 machine (MJ Research) controlled by Opticon Monitor 2 software. C(T) values were calculated by Opticon Monitor 2 software. Gapdh, MHCIIB and mCK primers have been previously described . The remC2C12 cell line and satellite cells: siRNA EZH1 no. 1 (SI00997766), siRNA EZH1 no. 2 (SI00997773), siRNA SUZ12 no. 1 (SI01438416), siRNA SUZ12 no. 2 (SI01438402), as well as negative control siRNA  were purchased from Qiagen. The remaining siRNA sequences are as follows: siRNA Ezh2 no. 1: AAGGAAAGAACTGAAACTTA; siRNA Ezh2 no. 2: AAGCTGAAGCCTCCATGTTTA.Cells were transfected with HiPerfect (Qiagen) following the manufacturer's instructions. At 48 h after transfection the cells were induced to differentiate and collected at the indicated timepoints. All siRNAs were used at a final concentration of 20 nM.Human myoblasts: cells were transfected with DharmaFECT  following the manufacturer's instructions. At 48 h after transfection the cells were induced to differentiate and collected at the indicated timepoints. All siRNAs were used at a final concentration of 6 nM . The siRNA sequences are available upon request.Cells were harvested and washed twice with PBS. Cell lysis of total cell extracts was performed on ice in 50 mM Tris-HCl pH 8, 125 mM NaCl, 1% NP-40, 2 mM ethylenediaminetetra-acetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitory cocktail ) for 25 min. Insoluble material was pelleted by centrifugation at 16000 g for 3 min at 4°C. Protein concentration was determined using the Bradford assay . The proteins were denatured, reduced, separated by SDS-PAGE and transferred to nitrocellulose transfer membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween (Sigma) (TBST) and incubated with primary antibodies overnight at 4°C. Following three washes with TBST, membranes were incubated with the peroxidase-conjugated secondary antibody, in TBST with 2.5% non-fat dry milk, and immunoreactive proteins were detected using Supersignal West Dura HRP Detection Kit (Thermo-Scientific). For cytoplasmic and nuclear extracts preparation the cells were resuspended first in buffer A (10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.9, 10 mM KCl, 0.1 mM EDTA and 0.1 mM ethylene glycol tetra-acetic acid (EGTA)) supplemented with protease inhibitory cocktail (Roche), 1 mM dithiothreitol (DTT) and 1 mM PMSF. After incubation on ice for 10 min, NP-40 was added to a final concentration of 0.5% and the samples were vortexed for 5 s. Nuclei were pelleted at 13,200 rpm for 10 s and the cytoplasmic proteins were collected. The pellet was then washed five times with buffer A and resuspended in buffer C  and 1 mM PMSF). After 10 min on ice, the samples were sonicated and centrifuged at 13,200 rpm for 10 min and nuclear proteins were collected.ChIP was performed as previously described  using a crosslinking time of 10 min. Antibodies were coupled to Dynal magnetic beads (Invitrogen) by overnight incubation at 4°C. The following day, chromatin was added to antibody-bead complexes and incubated overnight at 4°C. The bound complexes were washed twice in Low Salt Solution, twice in High Salt Solution, once in LiCl and once in Tris/EDTA (TE) buffer. DNA was extracted from beads by standard phenol/chloroform extraction, precipitated and resuspended in 30 μl TE. To quantify the results, quantitative (q)PCR reactions were performed in triplicate  using QuantiTect SYBR Green master mix (Qiagen) on a DNA Engine Opticon 2 machine (MJ Research) controlled by Opticon Monitor 2 software. C(T) values were calculated by Opticon Monitor 2 software. To calculate relative enrichment the signal from the control immunoprecipitation experiment (Mock) was subtracted from that observed with the antibody of interest. Myoblasts values (GM) were set as 1 and values from differentiated cells in DM with or without inhibitor display relative enrichment or reduction to those observed in GM. ChIP primers are available upon request.For immunoblot: EZH2 3147) was from Cell Signaling . SUZ12 (46264), MyoG (12732), and MHCIIB (2064) were from Santa Cruz . β-Tubulin (T0198) was from Sigma. mCK antibody was kindly provided by Hidenori Ito . Ezh1 and EED antibodies were previously characterised ( was from). For ChSize exclusion chromatography was performed using C2C12 cell nuclear extracts on a Superose 6 PC 3.2/30 gel filtration column  using an AEKTA purifier system  in IP (300) buffer , Aprotinin, Leupeptin, 100 mM PMSF, 1 mM DTT). Immunodepletion was performed as described . BrieflyN-(9-fluorenyl)methoxycarbonyl)-based solid-phase synthesis. Peptides used for kinase assays corresponded to amino acids 21-33 of H3 containing an artificial Y at the C-terminus: H3 unmodified, ATKAARKSAPATGY; H3K27me3, ATKAARK(me3)SAPATGY; H3S28ph, ATKAARKS(ph)APATGY. Peptides used for precipitation experiments corresponded to amino acids 1-40 of H3 and contained a C-terminal non-native YCK sequence with the lysine biotinylated at the e-amino group: H3 unmodified, ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHR-YCK (biotin); H3K27me3, ARTKQTARKSTGGKAPRKQLATKAARK(me3)SAPATGGVKKPHR-YCK (biotin); H3K27me3S28ph, ARTKQTARKSTGGKAPRKQLATKAARK(me3)S(ph)APATGGVKKPHR-YCK (biotin).Histone H3 peptides were synthesised in unmodified and modified form using Fmoc (Recombinant MSK1 (Millipore) was used to phosphorylate H3 histone tail peptides (21-33). Kinase assays were performed according to the manufacturer's protocol by incubating 15 ng of MSK1 with 1 μg of peptide for 30 min at 30°C. The reaction was stopped by adding 0.5% phosphoric acid, spotted on P81 paper and washed three times with 0.5% phosphoric acid and once with acetone. Filter circles were air dried and counted in a scintillation counter.2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, protease inhibitor cocktail, PhosSTOP (Roche)) and sonicated on ice in a Branson Sonifier . Extract was left on ice for 30 min before centrifugation for 15 min at 16,000 g. The supernatant was supplemented with 0.1% Triton X-100 and used for precipitation experiments.For preparation of nuclear extracts, cells were lysed in buffer A , 0.075% NP-40). After incubation on ice for 15 min, nuclei were pelleted and washed once with buffer A without NP-40. The nuclear pellet was suspended in buffer B  were coupled to 50 μl streptavidin-coated paramagnetic beads in PBS/bovine serum albumin (BSA) (1 mg/ml) for 4 h at 4°C. Beads were washed three times with PD150  to remove unbound peptides. Peptide-bound beads were incubated with nuclear extract for 2 h and washed four times with PD300. Bound proteins were eluted with SDS sample buffer, separated by SDS-PAGE and analysed by immunoblotting.Cells were grown on coverslips, washed in PBS, fixed in 3.7% formaldehyde/PBS  and permeabilised in 0.2% Triton X-100/PBS . The coverslips were then washed in PBS, and blocked with 3% low-fat milk/PBS for 1 h at room temperature. Following overnight incubation with primary antibodies at 4°C, the coverslips were washed and incubated with secondary antibodies  for 60 min at 37°C, and then washed again and counterstained with 4',6-diamidino-2-phenylindole ). Pictures were captured using epifluorescence microscopy (Leica DM6000B) using Leica Application Suite software.6 cells per tube, washed with cold PBS 1 ×, fixed by 70% cold ethanol and incubated for 30 min on ice. After incubation, cells were washed with PBS 1 ×, resuspended in 0.5 ml of PBS 1 ×/RNase A (100 μg/ml) and incubated at 37°C for 30 min. Finally, propidium iodide (20 μg/ml) was added and the cells were incubated in the dark for 30 min at 4°C. The samples were then analyzed for the cell cycle profile and the cell death profile using a Becton Dickinson Instrument.C2C12 myoblasts were cultured in growing conditions and collected at 24 h, 48 h, 72 h and 96 h after plating. Cells were divided in aliquots of 1.2 × 10The authors declare that they have no competing interests.in vitro experiments. BB performed experiments and analyzed data. DP performed chromatography experiments. RK and DS purified and provided histone peptides. CM performed experiments on satellite muscle cells. RM purified and provided Ezh1 Ab. PLP and KH participated in critically reviewing the data. WF conceived the study and analyzed in vitro data and participated in manuscript preparation. VO conceived the study and wrote the manuscript. All authors read and approved the final manuscript.LS, ZJ and CP designed and performed experiments, analyzed data and wrote the manuscript. AS performed Dynamics of PRC2 components in human skeletal muscle and satellite cells. (A) Expression levels of hEzh2, hEzh1 and hSuz12 were measured by real-time PCR in human myoblasts grown in growth medium (GM) or differentiation medium (DM) . The transcription levels were normalised to hGapdh expression and represent the mean of three independent experiments ± SD. Fold enrichment was calculated in comparison to myoblasts in GM. (B) Expression levels of Ezh2, Ezh1 and Suz12 were measured by real-time PCR in myofibre-derived satellite cells grown in GM or DM (72 h after differentiation induction). Pax7 was used as a control for these cells and myogenin (MyoG) and myosin heavy chain IIB (MHCIIB) were used as muscle differentiation controls. The transcription levels were normalised to Gapdh expression and represent the mean of three independent experiments ± SD. Fold enrichment was calculated in comparison to myoblasts in GM.Click here for fileC2C12-H89 treatment impairs muscle gene activation. (A) Schematic representation of the design of Msk1 inhibitor H89 treatment used in this study. (B) The effect of H89 treatment (5 μM and 10 μM) on C2C12 muscle cell differentiation was analysed in differentiation medium (DM) (48 h after treatment) by phase-contrast microscopy. (C) Expression levels of myogenin (MyoG) and muscle creatine kinase (mCK) were measured by real-time PCR in C2C12 myoblasts cultured in growth medium (GM) or DM (48 h after differentiation induction) with or without Msk1 inhibitor H89 (5 μM). Transcription levels were normalised to Gapdh expression. The data are shown as the average of three independent experiments, with error bars representing standard deviation. Fold enrichment was calculated in comparison to myoblasts in GM. (D) Immunoblot of MyoG and mCK from whole cell extracts of C2C12 myoblasts cultured in GM or DM (48 h after differentiation induction) with or without H89 (5 μM). β-Tubulin was used as a loading control. (E) Chromatin immunoprecipitation (ChIP) analyses of MyoG promoter, mCK enhancer and mCK promoter were performed on chromatin prepared from C2C12 cells cultured in GM or DM for 48 h after induction of differentiation, using histone H3 phosphorylation at serine 10 (H3S10ph) antibody. Levels of H3S10ph were normalised to histone H3 density. The precipitated DNA fragments were subjected to real-time PCR analysis. ChIP values are presented as relative enrichments to myoblasts. The values represent the mean ± SD of three independent experiments.Click here for fileEfficiency of Ezh1 and Ezh2 small interfering RNA (siRNA) in C2C12 cells. (A) Myoblasts were transfected with non-targeting siRNA (Nc = negative control) or siRNA against Ezh1 (siEzh1 no. 1 and siEzh1 no. 2), and the efficiency of siRNA was tested by real-time PCR in growth medium (GM) and in differentiation medium (DM) (48 h after differentiation induction). The transcription levels were normalised to Gapdh expression and represented as the average of three independent experiments ± SD. Fold enrichment was calculated in comparison to the negative control siRNA in GM. (B) Immunofluorescence for Ezh1 performed after delivery of siRNA into cells. Note the weak labelling in a high number of cells treated with Ezh1 siRNA (no. 2). Scale bar = 50 μm. (C) Myoblasts were transfected with non-targeting siRNA (Nc = negative control) or siRNA against Ezh2 (siEzh2 no. 1 and siEzh2 no. 2) and the efficiency of siRNA was tested by real-time PCR in GM and in DM (48 h after differentiation induction). The transcription levels were normalised to Gapdh expression and represented as the average of three independent experiments ± SD. Fold enrichment was calculated in comparison to the negative control siRNA in GM. (D) Immunofluorescence of Ezh2 48 h post transfection with siRNA (oligo no. 2). Scale bar = 100 μm.Click here for filemyogenin (MyoG) transcriptional activationEzh1-depleted human myoblasts and satellite cells show a delay in . (A) Human myoblasts were transfected with either non-targeting small interfering RNA (siRNA) (Nc = negative control) or siRNA against Ezh1. The effect of siRNA on cell morphology was analysed in growth medium (GM) (48 h after transfection) and in differentiation medium (DM) (2 and 4 days after differentiation induction) by phase-contrast microscopy. (B) The efficiency of siRNA for Ezh1 and the expression levels of MyoG were tested by real-time PCR in GM and in DM (2 days and 4 days after differentiation induction), in human myoblasts depleted for Ezh1. The transcription levels were normalised to Gapdh expression and are represented as the average of three independent experiments ± SD. Fold enrichment was calculated in comparison to negative control siRNA in GM. (C) Myofibre-derived satellite cells were transfected with non-targeting siRNA (Nc = negative control) or siRNA against Ezh1 (no. 1). The effect of siRNA on cell morphology was analysed in GM (48 h after transfection) and in DM (2 days after differentiation induction) by phase-contrast microscopy. (D) The efficiency of siRNA for Ezh1 and the expression levels of MyoG were tested by real-time PCR in GM and in DM (48 h after differentiation induction) in myofibre-derived satellite cells depleted for Ezh1 (oligo no. 1). The transcription levels were normalised to Gapdh expression. Fold enrichment was calculated as a percentage (%) of the negative control siRNA in GM.Click here for filemyogenin (MyoG) transcriptional activation in human myoblasts and in satellite cellsSuz12 small interfering RNA (siRNA) affects . (A) Human myoblasts were transfected either with non-targeting siRNA (Nc = negative control) or siRNA against Suz12. The effect of siRNA on cell morphology was analysed in growth medium (GM) (48 h after transfection) and in differentiation medium (DM) (2 and 4 days after differentiation induction) by phase-contrast microscopy. (B) The efficiency of siRNA for Suz12 and the expression levels of MyoG were tested by real-time PCR in GM and in DM (2 days and 4 days after differentiation induction), in human myoblasts depleted for Suz12. The transcription levels were normalised to Gapdh expression and are represented as the average of three independent experiments ± SD. Fold enrichment was calculated in comparison to the negative control siRNA in GM. (C) Myofibre-derived satellite cells were transfected with either non-targeting siRNA (Nc = negative control) or siRNA against Suz12 (no. 1). The effect of siRNA on cell morphology was analysed in GM (48 h after transfection) and in DM (2 days after differentiation induction) by phase-contrast microscopy. (D) The efficiency of siRNA for Suz12 and the expression levels of MyoG were tested by real-time PCR in GM and in DM (2 days after differentiation induction) in myofibre-derived satellite cells depleted for Suz12. The transcription levels were normalised to Gapdh expression. Fold enrichment was calculated in comparison to the negative control siRNA in GM.Click here for file


Data about usefulness of Statistical Process Control in the study of contact precautions (CP) dynamics for control of resistant bacteria are scarce.Retrospective cohort study. All admitted patients colonized or infected by MRSA, MDR-PAE and Abaumannii from 2005 to 2010 were included. Period I (01/2005-04/2008) without active surveillance; Period II (05/2008-12/2010), active surveillance in all patients admitted to the Intensive Care Unit and in readmitted previously colonized patients. Clonality was studied by PFGE. Charts: I-graph  and U-graph (CP per 10.000 patients days clustered by quarters).The average days between two consecutive CP in period I and period II were: 20 vs 31 days for MRSA, 41 vs 46 days for MDR-PAE and 53 vs 59 days for Ab, respectively. The average rate of patients under CP per 10.000 patient-days were: 3.19 vs 2.51 for MRSA, 1.40 vs 1.49 for PAE MR and 1.35 vs 1.09 for Ab (period I and II respectively). All outbreaks were coincident for special negative causes in graph I while the U graph only detected 2 out of 5 (type II error). All special positive causes was detected by graph I just after outbreak intervention (more days between consecutive CP). Although graph I often showed positive special causes in 5 out of 12, a type I error could not be ruled out.In a non endemic setting when few events are present, the I graph is more sensitive and less specific, while the U graph is more specific but less sensitive.None declared.


Contrast enhanced cardiac MR with T1 mapping enables quantification of diffuse fibrosis. T1 mapping is technically demanding since technical, physiological, and biochemical factors can interfere with T1 measurements. Contrast material dose, relaxivity, biodistribution, clearance, interaction with plasma proteins, and interference with co-medication must be taken into consideration. Gadobenate dimeglumine (Gd-BOPTA) has some weak protein binding capacities and higher molar relaxivity in plasma/blood resulting in shorter T1 times as compared to other extracellular gadolinium based contrast agents. To date it has not been assessed whether co-medication with other protein binding drugs alter myocardial T1 time. Therefore the aim of this study was to evaluate the interference of a typical protein binding drug (Ibuprofen) with Gd-BOPTA with respect to T1 times 50 vials were prepared with different concentrations of gadobenate dimeglumine (0-16 mmol/l), Ibuprofen (0-4.85 mmol/l) and human serum albumin (0-4 g/l) in physiologic NaCl solution and imaged at 1.5T with a spin echo sequence at multiple TRs to measure T1 values and calculate relaxivities. 10 volunteers  were imaged at 1.5T. T1 values for myocardium and blood pool were determined for various time points after administration of 0.15 mmol/kg gadobenate dimeglumine using a modified look-locker inversion-recovery sequence before and after administration of Ibuprofen over 24 hours. The partition coefficient was calculated as ΔR1myocardium/ΔR1blood, where R1=1/T1.In vivo there was no significant difference in post contrast T1 times of myocardium and blood, respectively and also in the partition coefficient between exam 1 and 2 (p>0.05). There was an excellent correlation of T1 times of myocardium and blood, respectively between exam 1 and 2 .In vitro no significant correlation was found between relaxivity and Ibuprofen concentration, neither in absence  nor in presence of albumine . Contrast enhanced T1 mapping with Gd-BOPTA is unaffected by co-medication with protein binding substances. Due to its excellent reproducibility it might be useful for follow-up exams.This study was sponsored in part by Guerbet Pharmaceutical Company Switzerland.