Agricultural production can be limited by low phosphorus (P) availability, with soil P being constrained by sorption and precipitation reactions making it less available for plant uptake. There are strong links between carbon (C) and nitrogen (N) availability and P cycling within soil P pools, with microorganisms being an integral component of soil P cycling mediating the availability of P to plants. Here we tested a conceptual model that proposes (i) the addition of readily-available organic substrates would increase the size of the microbial biomass thus exhausting the pool of easily-available P and (ii) this would cause the microbial biomass to access P from more recalcitrant pools. In this model it is hypothesised that the size of the microbial population is regulating access to less available P rather than the diversity of organisms contained within this biomass. To test this hypothesis we added mixtures of simple organic compounds that reflect typical root exudates at different C:N ratios to a soil microcosm experiment and assessed changes in soil P pools, microbial biomass and bacterial diversity measures. We report that low C:N ratio (C:N = 12.5:1) artificial root exudates increased the size of the microbial biomass while high C:N ratio (C:N = 50:1) artificial root exudates did not result in a similar increase in microbial biomass. Interestingly, addition of the root exudates did not alter bacterial diversity (measured via univariate diversity indices) but did alter bacterial community structure. Where C, N and P supply was sufficient to support plant growth the increase observed in microbial biomass occurred with a concurrent increase in plant yield. These soil P pools are broadly grouped as readily, moderately and sparingly  available pools [Phosphorus (P) is essential for plant growth and even though soils contain a large amount of total P, only a small fraction is available for plant uptake . P fertill roots . In mostle pools . P can ble pools , 7. ThesIt has been suggested that plants mediate the availability of P through release of organic anions  that assist in mobilising both organic and inorganic P , 9. A nui and Po) can be manipulated to increase plant available P. Microorganisms play a role in mediating the availability of nutrients to plants via two main avenues; (1) an increase in the size of the microbial biomass can result in the accumulation of a significant pool of P (2) the microbial biomass can drive the transformation of P from recalcitrant to more available P pools for plant uptake [–1 yr–1 for grassland soils and 7 kg P ha–1 yr–1 for arable soils [A second option to increase cycling of P from more recalcitrant pools into more available P pools is to consider how microbial cycling of recalcitrant P  of 5.0. Total soil C and N were 0.47 and 0.04% respectively resulting in a C:N ratio of 12.4. Total soil P was 42.0 mg kg-1 dry soil with a plant available P of 5.2 mg Colwell P kg-1 dry soil.Soil (0–10 cm) was collected from the uncultivated Ap horizon of a wheat paddock near the town of Beverley in the wheatbelt of Western Australia . The study was carried out on private land–the owner of this property gave permission to conduct the study on this site. The field study did not involve endangered or protected species. The soil is described as sand  with a pH (CaCl-1 soil (i.e. 13% w/w) prior to the experiment commencing resulting in a total of 30.75 g P applied as rock P per pot, and (3) Solution P treatment (SP) where 20.5 mg K2HPO4-P kg-1 soil was added every four weeks resulting in a total of 153.75 mg P being applied per pot. P application rates were different to reflect the difference in available P between easily available solution P and the less available P contained within rock P. Basal nutrients (with the exception of P) were added to all pots at the start of the experiment at concentrations (mg kg−1 soil) similar to that of Damon and Rengel [Lolium rigidum cv. Tetila) was germinated and transplanted after two weeks into each of the above sets of experimental pots at a rate of five seedlings per pot. A full set of pots was established for each of two specific root exudate treatments that were to be imposed over the duration of the experiment.Microcosms were prepared by placing 1.5 kg soil (sieved to <4 mm) into pots with a 12.5 cm diameter and 10cm height. Three sets of P conditions were imposed: (1) No P treatment (NP) where soil was not amended with P during the course of the experiment (i.e. control), (2) Rock P treatment (RP) where RP  was added at a rate 130 g kgd Rengel : N (33),–1 soil for each 4-week period and added as follows: 100, 200, 300 and 400 mg C kg–1 soil for weeks 1, 2, 3 and 4 respectively. This application regime was repeated every 4 weeks throughout the experiment and was chosen to mirror plant requirements as plant biomass was harvested at the end of each 4 week cycle. Thus the root exudate amendment was increased week-by-week in each 4-week period to account for increasing plant requirements. Our design aimed to compare the effect of low C:N ratio (12.5:1) root exudates with high C:N ratio (50:1) root exudates on soil characteristics , plant characteristics  and microbial characteristics  as well as microbial diversity and community structure. The experiment was not designed to test whether amendment with artificial root exudates would have an effect as this has already been established  and thued , two C:N ratio treatments (C:N = 12.5:1 and C:N = 50:1) applied as described above with three replicates of each (n = 3). This experimental design resulted in 18 pots being destructively harvested at each of 6 time points resulting in a total of 108 experimental units. All pots were fully randomised in a glasshouse where the temperature was 22.6/17.1°C day/night and relative humidity was maintained at 64%. Pots were regularly checked and adjusted to 50% of soil water holding capacity with distilled water over the course of 20 weeks and had weekly amendments of C:N substrate mixes giving a C:N ratio of 12.5:1 or 50:1. Experimental pots were destructively harvested every 4 weeks over the course of 20 weeks. At each destructive sampling time point plant shoot material was first removed, root material was then removed from each pot and the remaining soil was sieved (4 mm) to homogenise it prior to sub-sampling for further soil analyses.After shoot and root material was removed the sieved soil was collected for analysis after each 4-week interval for soil Colwell P, mineral N, microbial biomass-C and -P (MB-C and MB-P); 10 g was preserved in liquid nitrogen and stored at –80°C for analysis of the abundance (quantitative polymerase chain reaction–qPCR) and diversity (ion torrent barcoded sequencing) of the bacterial community. Additional samples were collected after 20 weeks for measurement of soil P fractions.3 extractable P  extracted were recovered in the sequential order: resin-Pi, NaHCO3-Pi and Po, NaOH-Pi and–Po, NaOH, HCl-Pi and residual P. These P fractions were grouped into readily-available P , moderately-available P  and sparingly available P  as used by Guo and Yost [Soil samples were sequentially extracted for P using a modification of the Hedley procedure . In brieand Yost .Ryegrass was separated into root and shoot components which were oven-dried at 60°C for 3 d and weighed. Sub-samples of plant material (shoot and root separately) were then finely ground (<150 μm) prior to further analysis. A 100 mg sample from each replicate was digested in a nitric and perchloric acid mixture. The molybdo-vanado-phosphate method was used to determine the P concentration in the digest . PhosphoPartitioning of P into soil, accumulated plant biomass and microbial biomass pools using measured values was completed at the end of the 20-week incubation and expressed as percentages. Shifts in these partitions were statistically compared between exudate C:N ratios of 12.5:1 and 50:1 and run separately for each of the P treatments .2SO4 for 1 h. Non-fumigated soils were extracted by the same procedure. The oxidisable-C in the soil extracts was measured by an automated TOC Analyser . MB-C was calculated from the difference in total oxidisable-C between fumigated and non-fumigated soil with a correction factor of 0.45 [MB-C was determined by chloroform-fumigation extraction . Moist s of 0.45 .i) extracted by 0.5 M NaHCO3 (pH 8.5) from moist soil fumigated with CHCl3 and the amount extracted from unfumigated soil. Since some CHCI3-released P will be re-sorbed by soil during fumigation and extraction, an adjustment for this was made by incorporating a known quantity of Pi during extraction and correcting for recovery. MB-P was calculated from CHCl3-released Pi by dividing by 0.4, i.e. by assuming that 40% of the P in the biomass is rendered extractable as Pi by CHC13 [MB-P was determined from the difference between the amount of inorganic P  following the manufacturer’s instructions. Duplicate DNA extractions for each treatment were then pooled and quantified and the DNA extract was stored at– 40°C prior to further analysis. DNA was extracted in this manner from each of three experimental replicates prior to amplicon sequencing and quantitative PCR (qPCR).–1), 2 μL of template DNA and 5.6 μL sterile water [4 to 107 gene copies) with R2 values greater than 0.99. Efficiencies for all quantification reactions were 90–100%.Copy numbers of 16S ribosomal genes were determined by qPCR using the Applied Biosystems 7500 platform. Each 20 μL qPCR reaction contained 10 μL of Power SYBR Green Master Mix , 0.2 μL of the forward Eub338 (5’-ACTCCTACGGGAGGCAGCAG-3’) and reverse Eub518 (5’-ATTACCGCGGCTGCTGG-3’) primers at a concentration of 10 μM, 2 μL bovine serum albumin  as described previously [www.metagenomics.anl.gov).For each sample, approximately 300 base pairs of the V4/5 region of the bacterial 16S rRNA gene were amplified by PCR primer set 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) . Amplicoeviously . Sequenceviously . Raw demth edition; Lawes Trust, Harpenden, UK). Analysis of variance (ANOVA) was performed to determine whether P treatment and exudate C:N ratio significantly affected a range of univariate measures including soil characteristics , plant characteristics  and microbial characteristics  as well as measures of univariate bacterial richness and diversity . Data was checked for normality and skewed data was log transformed prior to analysis. Differences between means were then assessed using Tukey's HSD test with a significance cut-off of P = 0.05. Bacterial 16S rRNA gene abundance were log-transformed prior to ANOVA as values spanned several orders of magnitude. To determine whether the treatments significantly impacted the bacterial community structure, multivariate statistical analyses were performed in Primer 6 . Analyses were performed on data that had been generated using the QIIME pipeline as described above, with no transformation, and applying the Bray-Curtis measure of similarity. To visualise differences between treatments, ordinations were performed by principal coordinate (PCO) analysis. Tests of the null hypothesis, that there are no differences among a priori-defined groups, were performed by permutational multivariate analysis of variance (PERMANOVA). Relationships between changes in the bacterial community structure and individual soil characteristics were analysed using distance-based multivariate multiple regression (DistLM) with environmental measures subjected to a forward-selection procedure to develop a model to explain the variance in community structure data [The experiment consisted of three factors: P treatment, C:N ratio of applied artificial root exudates and time, each with three replications. Univariate statistical analyses were performed using GenStat  affected plant growth and plant P concentration. After 20 weeks, the solution P treatment with an exudate C:N ratio of 12.5:1 produced significantly more (P < 0.05) plant biomass (3.11 g dry weight) than any other treatment. Within each P treatment , application of exudates with a C:N ratio of 12.5:1 produced significantly more (P < 0.05) plant biomass than that when an exudate C:N ratio of 50:1 was applied (P treatment and exudate C:N ratio significantly ( applied .P < 0.001) affected by both P treatment and exudate C:N ratio. After 20 weeks there was a significant effect (P < 0.01) of exudate C:N amendment on plant P content for both RP and SP treatments but not for the NP treatment. However, the SP treatment where an exudate C:N ratio of 12.5:1 was applied had significantly higher (P < 0.001) plant P content  than the average 5.20 mg P per total plant biomass produced across other treatments (P > 0.05) between C:N exudate treatments for both NP (0.035 mg P plant biomass-1 day-1 ± 0.001) and RP (0.048 mg P plant biomass-1 day-1 ± 0.005) treatments; for the SP treatment amending with a C:N ratio of 12.5:1 resulted in a significantly greater (P < 0.05) overall rate of P uptake at a rate of 0.178 mg P plant biomass-1 day-1) compared to 0.054 mg P plant biomass-1 day-1 where a C:N ratio of 50:1 was applied across the experimental time period.Plant P content was significantly (eatments . The ratP < 0.05) effect of P treatment and exudate C:N ratio on plant-available P as measured by Colwell P (P < 0.05) initially (within the first 6 weeks) with easily released P from the added RP being released into solution and P supply being greater than plant demand for P. In time there was less P released from the RP and thus the soil Colwell P decreased significantly (P < 0.05) from 6 weeks until a steady state was reached about 12 weeks. In the NP treatment soil Colwell P increased significantly (P < 0.05) as the soil was wetted up and microbial activity commenced. Thus soil Colwell P was significantly higher (P < 0.05) after 6 weeks–the increase in Colwell P in the NP treatment was much less than for the RP treatment. With less P being available for release from soil organic matter and with plant growth increasing soil Colwell P decreased significantly (P < 0.05) from 6 weeks onwards until a steady state low soil Colwell P was reached at approximately 12 weeks. In the SP treatment where soluble P was added continuously the generally high soil Colwell P measured reflects this.Overall there was a significant  less plant-available P in the SP treatment (32.2 mg Colwell P kg-1 dry soil). The difference in plant-available P within the RP treatments was less pronounced than the SP treatment, but still significant (P < 0.05) with application of an exudate C:N ratio of 50:1 resulting in 16.2 mg Colwell P kg-1 dry soil compared to 12.3 mg Colwell P kg-1 dry soil with application of an exudate C:N ratio of 12.5:1.Assessing differences after the 20 week incubation there was a significant effect of exudate C:N ratio on plant-available P in the RP and SP treatments but not the NP treatment. The NP treatment had the lowest plant-available P levels at 5.3 mg Colwell P kgP < 0.05) resin extractable P (readily-available P) in soil at the end of the experiment (for treatments receiving exudates with C:N ratios of 12.5:1 and 50:1) compared to the start of the experiment. Resin and NaHCO3 extractable P fractions (readily-available P) in the NP treatment did not significantly differ (P > 0.05) between exudate C:N ratios of 12.5:1 and 50:1 at the end of the experiment. For RP, the HCl-extractable fraction (moderately-available P fraction) was significantly higher (P < 0.001) than the NaOH extractable P (moderately-available P) both at the start of the incubation and after 20 weeks of incubation with the exudate C:N ratios of 12.5:1 and 50:1. There were no significant differences (P > 0.01) in the NaOH or HCl-extractable fractions at the start of the experiment and after incubation with exudate C:N ratios of 12.5: or 50:1 for 20 weeks. For SP, treatments receiving exudates with a C:N ratio of 50:1 had significantly higher (P < 0.01) soil resin extractable P and NaHCO3 extractable P (P < 0.05) after 20 weeks when compared to soil at the start of the incubation and soil incubated for 20 weeks with an exudate C:N ratio 12.5:1.Total P and soil P fractions were assessed at the start of the experiment and after 20 weeks of incubation with exudate C:N substrates . The NP P < 0.01) were observed for soils incubated with exudate C:N ratios 12.5:1 and 50:1 after 20 weeks compared to HCl-extractable P at the start of the incubation. The residual or sparingly available P fraction did not differ significantly (P > 0.05) between soil at the start of the incubation and soil incubated for 20 weeks with exudate C:N ratios of 12.5:1 and 50:1 for NP, RP and SP treatments (Significantly lower levels of HCl-extractable P (eatments .P < 0.05) soil mineral N; however the effect of P treatment was only significant where an exudate C:N ratio of 12.5:1 was applied. Within each P treatment , soil mineral N concentration was significantly (P < 0.05) greater where an exudate C:N ratio of 12.5:1 was applied compared to an exudate C:N ratio of 50:1, with the greatest effect within the RP treatment (After 20 weeks of incubation P treatment and exudate C:N ratio significantly affected (reatment . AmendmeP < 0.005) affected soil MB-C. The MB-C was significantly (p<0.005) higher after 20 weeks at 322 mg C kg-1 dry soil in the SP (with the application of a C:N ratio of 12.5:1) compared to the RP and NP treatments, with RP also having a significantly higher MB-C (274 mg C kg-1 dry soil weight and a C:N ratio of 12.5:1) than the NP treatment which had the lowest MB-C at 146 mg C kg-1 dry soil with the application of a C:N ratio of 50:1. Within each P treatment, application of an exudate C:N ratio of 12.5:1 produced significantly higher MB-C than application of a C:N ratio of 50:1 (P < 0.001) (After 20 weeks P treatment and exudate C:N ratio significantly (< 0.001) .P < 0.001) higher (11 mg P kg–1 dry soil) MB-P than the RP (8.5 mg P kg–1 dry soil) and NP (6.6 mg P kg–1 dry soil) treatments. Within each P treatment, application of an exudate C:N ratio of 12.5:1 had significantly (P < 0.001) higher MB-P than application of an exudate C:N ratio of 50:1 (data not shown). Both P treatment and exudate C:N ratio also significantly (P < 0.001) affected bacterial 16S rRNA abundance. After 20 weeks, the SP treatment had a significantly (P < 0.001) greater bacterial abundance at 1.45 x 1010 16S gene copies g–1 dry soil than the NP or RP treatments. Within each P treatment, application of an exudate C:N ratio of 12.5:1 had a significantly greater 16S rRNA abundance than application of an exudate C:N ratio of 50:1 (P < 0.001) (MB-P followed the same trend as MB-C and after 20 weeks of incubation the SP treatment (with application of an exudate C:N ratio of 12.5:1) had significantly (< 0.001) .P < 0.05) while a significantly lower level of microbial P but similar level of plant biomass P was observed with application of an exudate C:N ratio of 50:1. In both NP and SP treatments, significantly (P < 0.05) more P was taken up into the plant and microbial biomass pools with application of an exudate with a C:N ratio of 12.5:1 compared to an exudate with a C:N ratio of 50:1. Unlike the NP treatment, the SP treatment with application of an exudate C:N ratio of 12.5:1 had a significantly higher portion of P in the plant biomass P compared to microbial P pool (P < 0.05). RP treatments for both exudate C:N ratios did not significantly differ (P < 0.05) between microbial and plant biomass P pools. For all NP, SP and RP treatments, soil P had significantly higher (P < 0.01) levels of P compared to either microbial and plant P at both exudate C:N ratios.Based on the P budgets at the end of the 20-week incubation , the NP P > 0.1) across all measures (univariate and multivariate). As such, the results outlined in the following section detail differences observed at the end of the 20-week incubation only. The obtained sequence data represented a diverse soil community, however observed operational taxonomic unit (OTU) richness estimators did not differ significantly (P > 0.1) among treatments (1456 ± 281 OTUs identified) including the Chao1 richness index (3363 ± 351). Coverage estimators were generally high (Good’s coverage 0.911 ± 0.01) and not significantly different (P > 0.1) across treatments indicating similar deep recovery of the community constituents across the treatments. Diversity did not differ significantly (P > 0.1) across treatments as measured by the Shannon index (7.35 ± 0.37) and Faith’s Phylogenetic Diversity (107.7 ± 10.8).Diversity and community structure at the start of the incubation did not significantly differ (P < 0.01) effect on the bacterial community structure (P < 0.001) to that where an exudate C:N ratio of 50:1 was applied. For each exudate C:N ratio the community structure was significantly different (P < 0.01) between P treatments, however the effect was greatest for RP compared to either NP of SP where the community structure differences between exudate C:N ratios of 12.5:1 and 50:1 was not as pronounced  where an exudate C:N ratio of 12.5:1 was applied compared to an exudate C:N ratio of 50:1. In contrast Acidobacteria and Beta- and Gamma-proteobacteria were significantly more abundant (P < 0.05) where an exudate C:N ratio of 50:1 was applied compared to an exudate C:N ratio of 12.5:1. Where SP was applied again Bacteriodetes was significantly more abundant (P < 0.05) where an exudate C:N ratio of 12.5:1 was applied compared to an exudate C:N ratio of 50:1, however Actinobacteria, and Beta-proteobacteria were also significantly more abundant (P < 0.05). Only Gamma-proteobacteria were significantly more abundant (P < 0.05) where an exudate C:N ratio of 50:1 was applied compared to an exudate C:N ratio of 12.5:1. For both NP and SP there was no significant effect (P > 0.05) of exudate C:N ratio on Chloroflexi, Firmicutes and Verrucmicrobiota. Where RP was applied Bacteroidetes, Actinobacteria and Beta-proteobacteria were significantly more abundant (P < 0.05) where an exudate C:N ratio of 12.5:1 was applied and Acidobacteria Firmicutes, Delta- and Gamma-proteobacteria were significantly more abundant (P < 0.05) where an exudate C:N ratio of 50:1 was applied. There was no significant effect (P > 0.05) of exudate C:N ratio on the abundance of Alpha-proteobacteria, Chloroflexi or Verrucomicrobia  increased the size of the microbial biomass while also increasing plant yield, at least where P supply was sufficient to support plant growth. However, implications for P cycling were variable with no change in the size of more recalcitrant P pools under any of the experimental conditions applied over the course of our 20-week incubation time and thus our data do not support the hypothesis that measurable P was released from more recalcitrant P pools. We do report an increase in plant yield with application of a low C:N ratio artificial root exudate when combined with the supply of an easily-available P source (SP treatment) or a less readily available but significant source of P (RP treatment) which supplied the plant with its C, N and P requirements. Therefore, despite the fact that we did not observe a decrease in more recalcitrant pools Plant root exudates impact the abundance, structure and diversity of rhizosphere microbial communities , 29, 30 Although there was no impact of C:N ratio of the artificial root exudate substrates on univariate measures of diversity, we found that both the P treatment and C:N ratio of the artificial root exudate significantly impacted bacterial community structure. The impact of RP on bacterial community structure observed here agrees with previous findings which illustrated the impact of minerals , 52 and 3-extractable P pool significantly increased (P < 0.05) where SP was added compared to NP and RP treatments which is likely related to the addition of soluble P into the system as application of P fertiliser increases the labile and moderately-available P pools [In planted systems, both plant roots and microbial biomass will initially take up the most easily-available forms of P; therefore mechanisms to increase the size of this pool are likely to result in increased plant yield. Movement of P from recalcitrant to more available P pools has been shown following the addition of easily decomposed organic material for example manures , 54. Sim P pools .Across all P treatments  the relative abundances of Acidobacteria, Actinobacteria, Bacteroidetes, Beta- and Gamma-proteobacteria differed significantly between the C:N ratios of 12.5:1 and 50:1. Acidobacteria are ubiquitous in soil and include metabolically and genetically diverse organisms; it is reported that due to their metabolic and genetic versatility that they potentially play an important role in agricultural systems ; howeverIt has been reported that Beta-proteobacteria and Bacteroidetes in particular exhibit copiotrophic attributes with their relative abundances being highest in nutrient-rich environments , 63, 64.Our results show that (i) amendment with artificial root exudate organic substrates of low C:N ratio increased the size of the microbial biomass and (ii) there was no P release from recalcitrant P pools (iii) where P was in sufficient supply there was a consequent increase in plant yield and plant P uptake. Where artificial root exudates with a C:N ratio of 50:1 were applied, the system quickly became N limited which decreased plant growth. We found no impact of C:N ratio on the diversity of the microbial community in terms of univariate measures; however, the overall structure of the community was impacted. We did not detect any movement of P from more recalcitrant pools into more-labile pools and although we did find P mobilised from moderately-available pools into readily available this was not related to the C:N ratio of the organic amendment. We conclude that plants had an advantage over the microbial biomass in accruing P when there was a surplus of easily-available P as opposed to the competitive advantage of microbes in maintaining larger P pools compared to plants under the limited and probably less readily-available P source of the NP treatment.


Myrica rubra Sieb. et Zucc.), a nude mouse tumor xenograft model was established. Treatments with C3G  significantly suppressed the growth of SGC-7901 tumor xenografts in a dose-dependent manner. Immunohistochemical staining showed a significant increase in p21 expression, indicating that the cell cycle of tumor xenografts was inhibited. qPCR screening showed that C3G treatment up-regulated the expression of the KLF6 gene, which is an important tumor suppressor gene inactivated in many human cancers. Western blot showed that C3G treatments markedly increased KLF6 and p21 protein levels, inhibited CDK4 and Cyclin D1 expression, but did not notably change the expression of p53. These results indicated that KLF6 up-regulates p21 in a p53-independent manner and significantly reduces tumor proliferation. This study provides important information for the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo.To investigate the antitumor effect of anthocyanins extracted from Chinese bayberry fruit ( Anthocyanins are the most abundant water-soluble pigment found in fruit, vegetables and beans. It has been well established that anthocyanins from different sources exhibit multiple functional properties including antioxidant , anticanGastric cancer, also called stomach cancer, is the fourth most frequently diagnosed cancer in humans and the third leading cause of cancer death worldwide . It rankMyrica rubra Sieb. et Zucc.) is a subtropical native Chinese fruit with high nutrient and health values. The red-colored Chinese bayberry pulp is a rich source of anthocyanins, especially cyanidin-3-glucoside (C3G) [Chinese bayberry (de (C3G) , which hde (C3G)  and in vde (C3G) . Previoude (C3G) , inhibitde (C3G)  and cellde (C3G) ,19. HoweKLF6) is a novel tumor suppressor gene and is involved in the pathogenesis of many cancers [KLF6 was observed in a number of other human cancers including prostate [KLF6 exhibits growth inhibition activity through several major cancer pathways such as p53-independent up-regulation of p21 [KLF6 gene is well known, it has not been reported whether natural nutrients such as anthocyanins could affect KLF6 expression.Krüppel-like transcription factor 6  fruits were harvested at commercial maturity from Xianju County, Zhejiang Province, China. The SGC-7901 gastric cancer cell line was obtained from the Department of Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University.Chinese bayberry (2O) was used in all experiments. All samples for HPLC (High Performance Liquid Chromatography) analyses were filtered through a 0.22 μm membrane before injection.C3G standards were purchased from Sigma-Aldrich Co. LLC . RIPA (Radio Immunoprecipitation Assay) lysis buffer was purchased from the Beyotime Institute of Biotechnology . Anti-KLF6, anti-caspase3, anti-p21, anti-p53, anti-Cyclin D1, anti-CDK4 antibodies were obtained from Proteintech . All the other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. . Double-distilled water (ddHw/v) three times. The supernatants from three extractions were combined and evaporated under reduced pressure at 36 °C to remove the methanol. A phenolic-rich extract (PRE) was obtained by solid-phase extraction (SPE) using a Sep-pak C18 cartridge . Sugars and organic acids were removed by eluting with ddH2O. Phenolics were eluted with 10% aqueous methanol after eluting with 5% aqueous methanol.Fifty grams of fresh Chinese bayberry fruit pulp was extracted ultrasonically with 80% aqueous methanol (1% formic acid) in a material-to-solvent ratio of 1:10  at a column temperature of 25 °C. The HPLC analyses were performed as per previously published procedures [v/v) formic acid in water (eluent A) and of acetonitrile: 0.1% formic acid  (eluent B). The gradient program was as follows: 0 to 40 min, 10% to 38% of B; 40 to 60 min, 38% to 48% of B; 60 to 70 min, 48% to 100% of B; 70 to 75 min, 100% to 10% of B; 75 to 80 min, 10% of B. Then 10 μL samples were injected and were detected from 200 to 600 nm. Anthocyanins were calculated as C3G equivalent at 515 nm.The purity of C3G was determined by HPLC  coupled with a Waters SunFire CLC-MS (Liquid Chromatograph-Mass Spectrometer) experiments were performed according to our previous publication . Briefly1H-NMR (Hydrogen-Nuclear Magnetic Resonance) (500.18 MHz) spectra were obtained on a Bruker Avance 500 instrument . Extracts (15 mg) were dissolved in 0.5 mL deuterated methanol (CD3OD) in a 5 mm ф tube at variable temperatures; δ (parts per million) and the coupling constants (J) in Hertz were presented as chemical shifts.The −1·bw−1·day−1), high-dose group  and positive control group  (three mice in each group). Mice were s.c. injected with 2 × 106 SGC-7901 cells on the right groin and were housed under a regular 12 h light/12 h dark cycle. After 18 days, the mice were sacrificed for the assay of tumorigenicity . All experiments were carried out in accordance with the ethical guidelines of the Animal Experimentation Committee in the College of Medicine, Zhejiang University. Our experiment ethic approval code is ZJU20160443.Balb/c nude mice (weighing 19–21 g) were used for building a model and were maintained at 23–25 °C and 50%–60% humidity in the Laboratory Animal Center of Zhejiang University . Balb/c-nu mice were randomly allocated to four groups: a model group (drinking water), low-dose group  paraformaldehyde/PBS  and embedded in paraffin for staining. The paraffin sections were deparaffinized in xylene, rehydrated in a 10 mM citrate buffer (pH 6.0), and heated in a microwave oven for 15 min to restore the antigens. To suppress endogenous peroxidase within the tissues, the samples were treated with 3% peroxide for 5 min and then with a blocking solution for 30 min. Slides were incubated with the primary antibody and secondary antibody in a humid chamber for 60 min. Tissue staining was visualized with a 3,3′-diaminobenzidine substrate chromogen solution and the images were taken by using a microscope set  at a 200× magnification.The tumor tissues were removed from each mouse and the samples were subsequently fixed in 4%  according to the manufacturer’s protocol. Equal amounts of total RNA (1.0 μg) were used to synthesize cDNA with the iScriptTM cDNA Synthesis kit . Quantitative real-time PCR was performed in triplicate using a SYBR Green Master I kit  and the LightCycler480 real-time PCR System . Gene-specific primers were used as mentioned in Tumor tissues were lysed using RIPA buffer containing 1% PMSF (Phenylmethanesulfonyl Fluoride), and protein inhibitor . The protein concentration was determined using the Pierce BCA Protein Assay Kit . The concentration was adjusted to 1 μg/μL using PBS and 5× loading buffer. Equal amounts (40 μg) of protein were separated on a 10% SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel and transferred onto PVDF (Polyvinylidene Fluoride) membranes. After blocking the membrane with 5% nonfat dried milk for 1.5 h, the protein abundance was detected with antibodies against KLF6 (1:1000 dilutions), p21 (1:1000 dilutions), p53 (1:1000 dilutions), Cyclin D1 (1:1000 dilutions), CDK4.actin was used as a loading control.(1:1000 dilutions), followed by incubation with peroxide-conjugated anti-rabbit immunoglobulin. β-Statistical analyses were carried out using SPSS version 19.0 . Data were analyzed by one-way ANOVA. Multiple comparison between the groups was performed using the LSD (Least Significant Difference) method. OriginPro 8.0 software packages  was used for plotting the experimental data. Values were expressed as the mean ± standard deviation.1H-NMR  molecule, furnishing the cyanidin aglycone, the main body structure of C3G. The ions at m/z 136.9 and 240.6 resulted from the ring-cross cleavage of cyanidin. The 1H-NMR spectrum showed the presence of one cyaniding nucleus and one hexose , which was 211.7 mm3 (A tumor xenograft model was established to study the inhibition effects of C3G on tumor growth in vivo. Through preliminary tests (data not shown), we chose 25 mg·kg30.8 mm3 A. At 18 11.7 mm3 A.The inhibitory effect of C3G on tumor growth was evaluated based on tumor xenograft size when tumor tissues were removed and weighed after sacrifice. The tumor weight and size of treatment groups were significantly lower than those of the control group B,C. InhiExperimental results showed no significant changes in body weight, liver index and spleen index between the control group and C3G treatment groups D–F, indiExpressions of caspase 3 and p21 in tumor tissues were determined by immunohistochemistry staining. The negative cellular nuclei were stained blue, while the positive nuclei were stained dark brown. Immunohistochemical studies showed clear positive p21 staining in cell nuclei in C3G treatment groups with a dose-dependent manner. In contrast, the positive caspase 3 staining, which appeared in the cytoplasm, showed no marked change among different treatments . These rKLF6, p21, Cyclin D1, CDK4 and p53, were further evaluated. Both mRNA levels, measured by quantitative real-time PCR, and protein levels, measured by Western blot, were analyzed to determine these effects. As indicated in KLF6 and p21 genes were up-regulated in a dose-dependent manner in C3G treatment groups. The relative mRNA expression of KLF6 in the low dose and high dose groups was about 1.61- and 2.06-fold higher than in the control group. Similarly, p21 was up-regulated 2.19- and 3.48-fold in the low dose group and high dose group, respectively. Both Cyclin D1 and CDK4 gene expressions in C3G treatment groups were inhibited significantly. In comparison with the control group  of transcription factors is characterized by three contiguous C2H2-type zinc finger motifs at the carboxy terminus which comprise the DNA-binding domain ,31. KLF6 protein . KLF6 isprostate , gastricprostate , ovarianprostate , breast prostate , liver [prostate ,26, and prostate ,23. KLF6n of p21  and inhin of p21 . This stCas/JNK interaction [p53 and other apoptosis-mediated gene expression [p53 expression in tumor tissues, either at the mRNA or protein level, illustrating that the mechanism of the antitumor effect of C3G in vitro and in vivo might be different. There have been multiple reports about antitumor effects of anthocyanins extracted from natural products. Baked purple-fleshed potato reduced the colon CSCs number via induction of apoptosis [N-Nitrosomethylurea–induced leukemia in vivo [In vitro effects of C3G are well studied and it is known that C3G can suppress tumor cell proliferation by various mechanisms such as apoptosis induction , cell cyeraction , elevatieraction , and inderaction . Mulberrpression . Howeverpoptosis . Anthocypoptosis . Delphinpoptosis . Pomegrapoptosis . A diet poptosis . Anthocy in vivo . A diet  in vivo . Further in vivo ,35,43,44 in vivo ,35,43,44KLF6 gene expression and KLF6 exhibited tumor growth–suppressing activity by cell cycle inhibition with a p53-independent up-regulation of p21 and disrupted the Cyclin D1 and CDK4 interaction. However, further progress could be made by studying the oral administration of C3G, its metabolism and digestion in mice to investigate the target points of C3G. Since the KLF6 gene belongs to the transcription factor Krüppel-like family, the antitumor function of other KLF family members could also be studied.In this study, by using high-purity C3G and a tumor xenograft model, it was demonstrated that C3G treatment could up-regulate KLF6 gene expression and the downstream effector p21 in a p53-independent manner. These results might provide important information concerning the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo and shed light on the potential application of food anthocyanins in cancer prevention.In conclusion, C3G treatment showed in vivo antitumor effects in dose-dependent manner, where significantly tumor growth inhibition was observed in nude mouse xenograft model. C3G treatments also showed no toxic effects or other side effects compared with the positive drug tegafur treatment. The tumor-suppressing effect was due to cell cycle inhibition but lower apoptosis induction through Immunohistochemical staining test. Further q-PCR and Western blot results showed that C3G treatment could up-regulate the


Box C/D snoRNAs are known to guide site-specific ribose methylation of ribosomal RNA. Here, we demonstrate a novel and unexpected role for box C/D snoRNAs in guiding 18S rRNA acetylation in yeast. Our results demonstrate, for the first time, that the acetylation of two cytosine residues in 18S rRNA catalyzed by Kre33 is guided by two orphan box C/D snoRNAs–snR4 and snR45 –not known to be involved in methylation in yeast. We identified Kre33 binding sites on these snoRNAs as well as on the 18S rRNA, and demonstrate that both snR4 and snR45 establish extended bipartite complementarity around the cytosines targeted for acetylation, similar to pseudouridylation pocket formation by the H/ACA snoRNPs. We show that base pairing between these snoRNAs and 18S rRNA requires the putative helicase activity of Kre33, which is also needed to aid early pre-rRNA processing. Compared to yeast, the number of orphan box C/D snoRNAs in higher eukaryotes is much larger and we hypothesize that several of these may be involved in base-modifications. Base-pairing of box C/D small nucleolar RNAs (snoRNAs) with the target RNA guides site-specific ribose methylation by fibrillarin (Nop1), a highly conserved methyltransferase. Some box C/D snoRNAs, like yeast U3, U14 and U8 in higher eukaryotes are not involved in methylation but are essential for pre-rRNA processing. Biochemical and computational analyses have led to the identification and characterization of most box C/D snoRNA targets. Several snoRNAs classified as ‘orphan’ snoRNAs lack a guide sequence or have complementarity to RNA that is not characterized. Considering the recent number of studies underscoring association of box C/D snoRNAs with genetic disorders including Prader–Willi Syndrome (PWS), cancer and obesity, it is feasible that apart from ribose methylation and RNA processing, these snoRNAs are involved in several other cellular processes. Here, we report a novel and unexpected function for two yeast orphan box C/D snoRNAs, snR4 and snR45, in site-specific base acetylation. We demonstrate that these box C/D snoRNAs guide cytosine acetylation by Kre33, a factor essential for assembly and processing of the 90S pre-ribosome. Kre33 is known to acetylate a subset of tRNAs with the help of the adaptor protein Tan1. However, here we show Kre33 dependent acetylation of 18S rRNA is guided by orphan snoRNAs. Our results expand the functional repertoire of box C/D snoRNPs and highlight yet another remarkable cellular modularity, where a single enzyme targets different substrates by means of either protein or RNA adaptors. O-ribose methylation, whereas H/ACA snoRNPs catalyze site-directed pseudouridylations of specific rRNA nucleotides ..19]. Ace4C-12 residue  . Kre33 m residue .Within the 18S rRNA, Kre33 binds predominantly to the 5′ domain of the 18S rRNA, specifically around helices 7, 8, 9 and 10, making direct contacts with U-residues surrounding positions U132, U192, U259, and U277 of 18S rRNA Figs  and S1C.Unexpectedly, we observed significant cross-linking to the orphan box C/D snoRNAs snR4 and snR45 Figs  and S1C.Recently, a vertebrate specific U13 box C/D box snoRNA was reported to be involved in 18S rRNA acetylation ,27. ConvS. cerevisiae are located at position 1280 in helix 34 and at position 1773 in helix 45  and nucleobase acetyltransferase (Kre33).We conclude that the acetyltransferase Kre33 is guided to its two substrate cytosines at positions 1280 and 1773 on the 18S rRNA by the box C/D snoRNAs snR4 and snR45, respectively. Box C/D snoRNAs are known to carry the 2′-in vivo DMS RNA structure probing data  in vivo  and GS2 (in the loop), are essential for acetylation. GS1 of snR4 extends from nucleotide number 1 to 5 and according to our model establishes base pairing with nucleotides 1286 to 1290 of 18S rRNA . GS2 conivo Figs  and S6G.Disruption of the base-pairing between GS1 of snR4 and snR45 with 18S rRNA resulted in complete loss of acetylation at residue C1280 and C1773, respectively . SimilarApart from guide sequences, different conserved regions of both snR4 and snR45 .In addition to its acetyltransferase domain, Kre33 contains an N-terminal DEAD-box like helicase module . Changin0, A1 and A2 cleavages, leading to an accumulation of aberrant 23S and 22S rRNA species, a pattern that was previously observed upon hypomorphic expression of Kre33 . Sedimentation profiles of snR41, snR57, snR51 and snR55 in the helicase mutant appeared very similar to those of snR4 and snR45 , suggestO-methylation at these sites to be significantly reduced in the kre33-K289A mutant , but in view of its length and irregularity, this seems a very poor guide for 2′-O-methylation.Apart from guide sequences that target snR4 and snR45 for Kre33 mediated acetylation, these snoRNAs contain canonical C/D and C′/D′ boxes with a complete set of core C/D box proteins; Nop1, Nop56, Nop58 and, being the primary binder, Snu13  [4]. We Our Kre33 CRAC analysis revealed three 18S rRNA binding sites: the 5′ domain, helix 34 and helix 45. Our data suggest that Kre33 predominantly interacts with the 5′ domain of 18S rRNA and our results support the hypothesis that its interaction with h34 and h45 is aided by snR4 and snR45, respectively. The Kre33 binding sites on 18S rRNA are spatially adjacent to that of snR4 and snR45 and a chimeric RNA that covers both GS2 of snR4 and its base-pairing site on h34 provides direct evidence.Chaetomium thermophilum and S. cerevisiae, Kre33 has been modeled into the head domain formed by the 5′ domain of 18S rRNA and suggested to bind there as a homodimer [Interestingly, Kre33 has been previously characterized as a component of the SSU processome and shown to physically interact with several components of the U3 snoRNP containing 90S particle including Rrp9, Enp1 and Nop14 ,34. In romodimer ,34. Our Recent biochemical analyses of the early assembly of the SSU processome have shown that Kre33 joins the SSU processome relatively late once the 3′ major h34) and minor (h45) domain of 18S rRNA have been transcribed . Interes and minoThe snoRNA-rRNA hybrids found in our CRAC-libraries yielded support for Kre33 action on snR4, snR40 and snR55 and indicated that Kre33 could regulate the interactions of 18S rRNA sequences with other snoRNAs, such as snR77, snR52, and possibly U14. CLASH on Kre33 should enable us to generate a more complete overview of snoRNA-rRNA interactions mediated by Kre33. Overall, we conclude that the defect of 18S rRNA processing in the helicase mutant is concomitant with or due to defective release of a subset of snoRNAs.4C-12 acetylation of serine and leucine tRNAs [O-ribose methyltransferase Nop1/Fibrillarin, has been shown to target modification of histone H2A on glutamine residues both in yeast and human [Apart from 18S rRNA acetylation in eukaryotes, Kre33 catalyzes acnd human . With thAll yeast strains and plasmids used in the present study are listed in 600nm of 1 followed by 1:10 serial dilutions. From the diluted cultures, 5 μl were spotted onto YPD plates and incubated at 37°C, 30°C or 16°C.For growth analysis, yeast cells were grown over night in YPD medium and diluted to an OD600nm of 0.1 and were grown to an OD600nm of 0.8 in 1 L YPD. Cells were collected by centrifugation and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 volume of ice-cold TNM150 , 5 mM β-mercaptoethanol) + protease inhibitors (Roche protease inhibitors cocktail tablets) and disrupted by shaking with 2.5 volumes of Zirconia beads (5x1 min with 1min resting on ice between each round). 3 volumes of ice-cool TMN-150 was then added to lysate which was clarified using centrifugation at maximum speed for 20 minutes at 4°C. The lysates were incubated for 2 h, at 4°C with end-over-end rotation with 125μL of IgG Sepharose beads. that had been equilibrated with TNM150 buffer (2 times washing with 3 mL of TNM150). The beads were washed 5 times with 1 mL of TNM150 (w/o protease inhibitors and were collected and resuspended in 250 μL of TNM150 (w/o protease inhibitors). For analysis of the protein, beads were directly boiled in protein loading buffer  and separated by SDS-PAGE and analyzed by Western blotting using anti-TAP antibody (Roche). RNA was isolated using phenol-chloroform extraction and the snoRNAs were characterized by Northern blotting as described previously [After an overnight growth in YPD medium, TAP or HTP tagged strains were diluted to an ODeviously . Percentwww.ncbi.nlm.nih.gov/geo (accession no. GSE87480). For CRAC data analysis, data from two biological replicates were analyzed as described[CRAC was performed exactly as recently described . Cells wdescribed; yieldindescribed. As a cowww.jalview.org) and secondary structures generated with Varna (http://varna.lri.fr/). The base-pair interactions proposed to form the pseudoknot were identified by analyzing the alignments with SPuNC (http://www.ibi.vu.nl/programs/spuncwww/).snoRNAs homologous to snR4 and snR45 were retrieved and aligned as described previously [E. coli homolog, TmcA [The 3D structure prediction was carried out with amino acid sequence of yeast Kre33 using recent protocol . Kre33 sog, TmcA . USCF Chog, TmcA .Mung bean nuclease protection assay and RP-HPLC analysis for acetylation were performed exactly as described before .Sucrose gradient centrifugation for the pre-ribosome analysis and co-localization studies was performed as described previously .600nm of 0.2 and grown at 30°C to an OD600nm of 0.8 to 1.5. In the hood, two 15 mL aliquots of yeast culture in 50 mL polypropylene tubes were treated with 300 μL of 95% ethanol with 1:4, v/v DMS (Sigma) or without as a negative control and mixed vigorously for 15 seconds. Both aliquots were then incubated with shaking for 2 minutes at 30°C. The reaction was stopped by placing the tube on ice and adding 5 mL of 0.6 M β-mercaptoethanol and 5 mL of isoamyl alcohol. After addition of the stop solutions the tubes were vortexed for 15 seconds and then centrifuged at 3000xg at 4°C for 5 minutes. The cell pellets were washed with another 5 mL of 0.6 M β-mercaptoethanol.A flask with 100 mL YPD media was inoculated with an overnight culture to a starting ODTotal RNA was isolated from the DMS treated cell pellet using GTC  as described . The sitRibose methylation was analyzed by deoxynucleoside triphosphate (dNTP) concentration-dependent primer extension, exactly as described elsewhere . Um898 iS1 FigCRAC analysis revealed specific binding of Kre33 to leucine and serine tRNAs (A), 18S rRNA (B) and snoRNAs snR4 and snR45 (C). A) Overview of CRAC-results for Kre33 binding to serine and leucine tRNA species. The left y-axis shows the total number of times each nucleotide within an RNA fragment was mapped to the RNA sequence (x-axis); the right y-axis shows the number of reads carrying a substitution (red) or deletion (orange). Around the acetylated cytosine (*) a specific peak is observed in Kre33 cross-linked tRNAs. For other tRNA species or for the same species in control CRAC-experiments these substitution-peaks were not found. B) Read-alignments for Kre33 on the 18S rRNA show specific binding to the 5′ domain (pink), around helices 34 (orange) and 45 (blue). Putative crosslink sites (yellow circles) are seen as a gap in these sequences. Acetylated residues C1280 and C1773 (purple) fall within cross-linked segments. Note that Kre33 binding could be incompatible with that of modifying snoRNPs snR44, snR49, snR51, snR55, snR40, and snR83/Ruf3. C) As (B) but then for Kre33 binding to snR4 and snR45. Models of snoRNAs interacting with target rRNA sequence (blue) with the acetylated cytosine residue (red) are as in (PDF)Click here for additional data file.S2 FigA) Alignment of snoRNAs homologous to snR45. Sequences were retrieved and aligned as described previously [(PDF)Click here for additional data file.S3 FigTorulaspora delbrueckii snR45 could be modeled with a D′ box using a canonical sequence  instead of the CUGU found in S. cerevisiae and closely related species.Phylogenetic analysis of snR4 with alignment (A) and 2D-models (B) as described for (PDF)Click here for additional data file.S4 Fig4C1280  and ac4C1773  isolated from a strain lacking snR4 (Δsnr4). Loss of snR4 leads to complete loss of ac4C1280 without affecting ac4C1773. B) Overlaid RP-HPLC chromatogram of tRNAs from strains lacking snR4 , snR45  and Tan1  strain. Loss of snR4 and snR45 does not affect tRNA acetylation.A) Overlaid RP-HPLC chromatograms of the nucleosides derived from fragments isolated using mung bean nuclease assay, containing ac(PDF)Click here for additional data file.S5 Fig2D structure models (top panel) based on phylogenetic comparison and DMS-modification for snR4 (A) and snR45 (B) showing Kre33 cross-linked sites , along with the binding regions (transparent) and cross-linking sites  of Nop1 (purple), Nop56 (green), and Nop58 (blue) determined previously [(PDF)Click here for additional data file.S6 FigSNR4 and SNR45 expressing the indicated mutant versions of snR4 (E) and snR45 (F). (G) Northern blot developed with snoRNA-specific probes showing stable expression of snr4 and snr45 mutants. 5S rRNA was used as loading control. H) Sedimentation profile of snR4 (snr4-b) and snR45 (snr45-b) with an altered GS2 (cf. 2D models of snR4 (A) and snR45 (B) along with zoomed-in views (C and D) for mutated regions. The dotted red box in (B) indicate a mutation that interfered with stable expression of the snoRNA. See for further details the legend for  GS2 cf.  to test (PDF)Click here for additional data file.S7 FigKre33 was depleted using a strain containing HA tagged Kre33 under galactose promoter (pGAL1::3HA-kre33) that was grown in YPGSR  to mid-log phase, washed in pre-warmed water and transferred to YPD for up to 12 h. We compared t0 (time point at which cells were transferred to YPD) and t6 (6hours after transfer to YPD). A) Western blot showing that glucose mediated depletion of Kre33 (top panel) was extremely effective and after 6 hours Kre33 was depleted to a level beyond the Western blot detection limit. The bot was developed using mouse anti-HA (SIGMA) and goat anti-mouse-HRP (Santa Cruz Biotechnology) antibodies. Glucose 6 phosphate dehydrogenase (G6PDH) was used as a loading control (lower panel). The blot was developed using rabbit anti G6PDH and donkey-anti rabbit (Santa Cruz Biotechnology) antibodies. B) Sedimentation profiles of snR4 and snR45 at t0 and t6.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Drosophila eIF5A can functionally replace yeast eIF5A and is required for actin-rich cable assembly during embryonic dorsal closure (DC). Furthermore, Diaphanous, the formin involved in actin dynamics during DC, is regulated by and mediates eIF5A effects. Finally, eIF5A controls cell migration and regulates Diaphanous levels also in mammalian cells. Our results uncover an evolutionary conserved role of eIF5A regulating cytoskeleton-dependent processes through translation of formins in eukaryotes.Elongation factor eIF5A is required for the translation of consecutive prolines, and was shown in yeast to translate polyproline-containing Bni1, an actin-nucleating formin required for polarized growth during mating. Here we show that  Interestingly, FH1 domains contain long stretches of consecutive prolines, which decrease translation efficiency due to prolines being poor acceptors and donors during peptide bond formation thus causing ribosome stalling2. Therefore, the FH1 domain may have an impact in how formin protein levels are regulated and hence on how the cell responds to cytoskeletal demands.Cytoskeletal remodeling in response to signal transduction pathways is critical to many cellular processes. Actin-nucleating proteins of the formin and Arp2/3 protein families mediate the first step of filament assembly, contributing to cell shape, polarity, adhesion, and migration among others. Formins in particular allow the initiation and growth of linear filaments through their conserved actin polymerization domains FH1 and FH25. Remarkably, eIF5A is the only known protein to contain the polyamine-derived amino acid hypusine, a post-translational modification that is essential for its role in translation  and promotes cell migration, invasion and cancer metastasis14.The eukaryotic translation initiation factor 5A (eIF5A), and its homolog in prokaryotes EF-P, has recently been described to alleviate ribosome stalling during translation of proline stretches (polyPro motifs)Saccharomyces cerevisiae (Sc) TIF51A gene, one of the two genes encoding Sc eIF5A, causes a sterile phenotype due to reduced synthesis of the Bni1 formin. Translation of the polyPro motifs located in the FH1 domain of Bni1 required eIF5A; consequently, eIF5A conditional mutants in yeast haploid cells were unable to grow a mating projection, termed shmoo, towards a pheromone gradient during mating15. Despite the functional connection between eIF5A and translation of polyPro-containing proteins in yeast, it is unclear whether eIF5A can regulate cytoskeletal remodeling through the translation of formins in other systems.Recently, we showed that the depletion of the essential Drosophila eIF5A (Dm eIF5A) in dorsal closure (DC), the last major morphogenetic rearrangement during embryogenesis. DC involves the formation of an actomyosin contractile supracellular cable together with the migration, spreading and fusion of lateral epidermal cellular sheets17. We have found that Dm eIF5A is the functional homolog of Sc eIF5A and its depletion in Drosophila causes defects in actomyosin cable formation in the embryonic epidermis during DC. Our results also revealed that eIF5A regulates the protein levels of Diaphanous (Dia), and that this formin is required during DC downstream of eIF5A. Lastly, depletion of active eIF5A decreases the levels of Dia and migration in mouse neural stem cells (NSCs). Together, our results indicate an evolutionary conserved function of eIF5A in the translation of actin-nucleating formins with important consequences on the regulation of cytoskeleton-mediated processes from yeast to mammals.In this study, we identified a role of Drosophila embryogenesis, the dorsal epidermis appears discontinuous due to germ band retraction and is transiently covered by an extraembryonic tissue, the amnioserosa (AS). The hole is subsequently sealed by a complex process called DC, which involves the formation of an actomyosin contractile cable at the leading edges of the dorsal-most epidermal (DME) cells, the emission of cellular protrusions from opposite sides of the epidermis that interdigitate and zip the dorsal opening at its corners, and the apical constriction and apoptosis of AS cells that facilitates the occlusion of the hole. As a result, the two lateral epidermal sheets converge towards the dorsal midline while the AS reduces its surface area until it disappears inside the embryo17. The cabut (cbt) gene encodes a transcription factor with high similarity to the vertebrate TIEG (TGF-β-inducible early-response genes) proteins and is involved in several key events during DC including actin dynamics19. However, its direct target genes during this process are unknown. Using genome-wide ChIP-on-chip analysis for Cbt binding sites in extracts from 9–14 h embryos we identified eIF5A as a Cbt putative target gene . Reduced eIF5A function affects cell growth and causes autophagy in several Drosophila tissues and increases polysome size in S2 cells20, suggesting a potential role in translation elongation. However, the role of the only Dm eIF5A gene in DC has never been investigated. To clarify this issue, we reduced the expression of Dm eIF5A specifically in the embryonic epidermis using a UAS-iReIF5A RNAi line and the arm-GAL4 or 69B-GAL4 epidermal drivers  embryos displayed DC defects with holes in the dorsal and anterior epidermal regions  or two  amino acid substitutions, respectively, and are unable to grow at restrictive temperature (37 °C)23. Yeast mutants transformed with plasmids containing either Dm eIF5A or Sc eIF5A exhibited restored growth at the restrictive temperature  wild-type levels at 37 °C  based on their FH2 domain and additional conserved regions32. Among them, only the diaphanous (dia) gene has been analyzed in DC . Similar results have been obtained in DAAM mutants . Therefore, we tested whether Dia could be the translational target of Dm eIF5A during DC. Dia protein levels in arm-GAL4/UAS-iReiF5A or 69B-GAL4/UAS-iReiF5A embryos were apparently lower than in control embryos (arm-GAL4/+ and 69B-GAL4/+) by immunoblot detection (data not shown). The differences were, however, very small, most likely because the strategy used deletes eIF5A in only a small fraction of cells. Therefore, we decided to analyze Dia expression at the single cell level in embryos in which eIF5A RNAi is driven in a mosaic pattern by the en-GAL4 line  showed that Dia levels were reduced in eIF5A mutant cells compared to nearby wild-type cells  with respect to that without Dia . Similarly, partial suppression was observed in yeast for the shmooing defects of eIF5A mutants when increasing Bni1 formin levels15, which could indicate that either higher levels of this formin would be necessary for a complete rescue of the phenotype or that additional proteins involved in DC might also be targets of eIF5A. In summary, phenotypic analysis and immunofluorescence visualization indicate that Dia is a functional target of Dm eIF5A during DC and suggest that defects in actomyosin cable assembly in eIF5A mutant embryos are likely the result of low Dia levels.Since 36. A requirement for eIF5A in cytoskeletal-mediated processes such as cell migration has also been reported37, however a link between mDia and eIF5A has not been established. We found that NSCs isolated from the subependymal zone of the adult mouse brain and grown as neurospheres express detectable levels of eIF5A1 but not eIF5A2 ; in addition, we also found detectable levels of mDia mRNA . Therefore, we decided to test whether mDia protein could be regulated by eIF5A in NSCs. Hypusination is a post-translational modification selectively found in eIF5A that is essential for its function6. A 3-day treatment of neurospheres with the cell-permeable inhibitor of deoxyhypusine synthase GC7 at 5 or 10 µM did not affect cell viability (not shown), but resulted in a 3-fold reduction in the levels of hypusinated eIF5A that could be completely reversed by washing the drug out for 3 additional days  catalyzes actin polymerization and regulates microtubule dynamics, playing an important role in cell polarization, adhesion and migration downstream of GTPase RhoAays Fig. . Individays Fig. . More inays Fig.  without 1, therefore the unique modulation of eIF5A activity by polyamine-dependent hypusination opens exciting possibilities for the pharmacological targeting of cytoskeletal remodeling in different biological systems.Our results together indicate that eIF5A is required for Dia translation in both vertebrate and invertebrate cells. We demonstrate that structural and sequence conservation of eIF5A across evolution correlates with its conserved role in cytoskeletal dynamics through the regulation of formin translation. Actin nucleation is a tightly regulated process69B-GAL4 and arm-GAL4 driver lines, UAS-iReIF5A, UAS-iRDAAM, UAS-iRcapu, UAS-iRform3, UAS-iRfhos, and UAS-iRfrl RNAi lines, and UAS-Dia-GFP line were obtained from the Bloomington Drosophila Stock Center (BDSC). The stock harboring both UAS-Dia-GFP and UAS-iReIF5A constructs was generated by standard genetic techniques. Flies were grown on standard media at 25 °C. All crosses were performed at 25 °C except those using UAS-iRcapu, UAS-iRform3, UAS-iRfhos, and UAS-iRfrl RNAi lines which were performed at 29 °C.Fly stocks 19 and examined using Nomarski DIC optics. Lethality assays were performed at 25 and 29 °C, except when using UAS-iReIF5A and UAS-iReIF5A; UAS-Dia-GFP that were performed only at 25 °C.For cuticle analyses, embryos were collected and processed as reported38. Primary antibodies used were: rabbit anti-Dia  and rabbit anti-eIF5A . Secondary antibodies coupled to fluorochromes were purchased from Invitrogen and used at a 1:200 dilution. For phalloidin staining, embryos were fixed with 4% paraformaldehyde in PBS and devitellinization was done with in 1:1 heptane-80% ethanol. After washing in 100% ethanol, embryos were incubated twice in PBS with Triton-X100 (PBS-Tx)−1% BSA for 10 min each time, twice in PBS-Tx-2% BSA for 10 min each time and 1 h in Alexa Fluor 568 phalloidin reagent . Phalloidin incubation was followed by 15 min incubations of PBS-Tx with decreasing concentrations of BSA (2-0%). Specimens were mounted in Mowiol and images were acquired either on a LeicaTCS-NT confocal laser-scanning microscope or on an Olympus FV1000MPE confocal microscope. Several Z sections of each embryo were taken and processed with ImageJ. Imaginal discs images were acquired in a Leica DMI2500 optical fluorescence microscope. For quantification of fluorescence intensity in en>GFP> iReIF5A embryos, images of anti-Dia stainings were converted to grayscale. The Mean Gray Values of ten regions of interest of similar areas were measured per segment with ImageJ. The fluorescence intensity was calculated as the average of the Mean Gray Values obtained from three eIF5A mutant and three control segments.For immunohistochemistry, embryos were fixed with 3.7% formaldehyde diluted in phosphate buffer saline (PBS) and 1:1 heptane-methanol for devitellinization. Third instar larval wing imaginal discs were dissected, fixed and stained as describedSaccharomyces cerevisiae haploid strains wild-type BY4741 (MATa his3∆0 leu2∆0 met15∆0 ura3∆0) and temperature-sensitive eIF5A mutants tif51A-1 and tif51A-3 derivatives of the BY4741 strain23 were grown in liquid YPD or SC complete medium when carrying plasmids at 25 °C until the exponential phase and then treated as indicated. The Sc and Dm eIF5A genes were cloned in the yeast centromeric heterologous expression plasmid p416TEF (URA3 marker), under the control of the constitutive TEF promoter and using the CYC1 terminator . For this, we used a GAP repair strategy40. The ORF of the yeast TIF51A gene  was PCR amplified using primers GAPR 5′-eIF5A-Sc (5′-GTTTTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATGTCTGACGAAGAACA-3′) and GAPR 3′-eIF5A-Sc (5′-ACTAATTACATGACTCGAGGTCGACGGTATCGATAAGCTTGATTTAATCGGTTCTAGCAG-3′) and yeast genomic DNA as template. The Dm eIF5A unique gene was amplified with primers GAPR 5′-eIF5A-Dm (5′-GTTTTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATGGCTGAGTTGGACGA-3′) and GAPR 3′-eIF5A-Dm (5′-ACTAATTACATGACTCGAGGTCGACGGTATCGATAAGCTTGATCTATTTGTCCAGAGCAG-3′) using the GH16179 cDNA clone from the Drosophila Genomics Resource Center (DGRC) EST collection as template. PCR cassettes were co-transformed with EcoRI linearized p416TEF plasmid41 in BY4741 yeast cells and cells with GAP repaired plasmids were selected in SC-ura media. Plasmids were recovered from yeast cells and directly transformed in E. coli competent cells, then plasmids were extracted from bacteria and the correct cloning was confirmed by DNA sequencing.per sample from at least two independent experiments.Strains were grown in experimental temperatures and treated with 10 μg/ml of α-factor (Sigma) for 2 h. After incubation for 4 h at the indicated temperatures, cells were visualized by microscopy and images were taken . Approximately 200 cells were analyzed 42. Viability was analyzed using a commercial MTS assay (Promega). Deoxyhypusine synthase inhibitor GC7  was dissolved in sterile distilled water. For migration assays, neurospheres were individually selected according to their size and plated on Matrigel®-coated coverslips. Migration area was measured with ImageJ software and normalized to the initial neurosphere area.Subependymal NSCs were isolated from 2 month-old C57BL/6J mice and cultured as describedNeurospheres were fixed with 2% paraformaldehyde in 0.1M phosphate buffer, incubated with mouse anti-mDIA1  or rabbit anti-hypusine  overnight and then with Alexa Fluor® 488 donkey anti-mouse  or Cy3 donkey anti-rabbit  secondary antibodies for 1 h. DAPI (1 μg/ml) was used to counterstain nuclei. Pictures were taken using a confocal microscope (Olympus FV10) and all images were processed equally with Photoshop.43) as described44. Denatured protein extracts from neurospheres were separated by 15% (for hypusine detection) and 8% (for mDia1) SDS-PAGE and transferred to a nitrocellulose membrane. Antibodies used were: mouse anti-mDIA1 , mouse anti-GAPDH , mouse anti-α-tubulin , rabbit anti-hypusine, FabHpu24 , HRP goat anti-mouse  and HRP goat anti-rabbit . Band intensities of immunoblots were detected and quantified with the ImageQuant LAS 4000 mini image analyzer .Western blot analyses with yeast extracts were performed with anti-HA 12C5A  and anti-Hxk2  obtained from cell samples of the same experiment. BNI1-HA translation efficiency is expressed relative to HXK2 translation efficiency and represented as a fraction against 25 °C for each strain. For NSCs, RNA was isolated using RNeasy Mini Kit (Qiagen), cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara) and qRT-PCR was performed using TaqMan® probes (Life Technologies). For qRT-PCR in yeast, BNI1-3/ BNI1-4, HXK2-1/HXK2-2, and ACT1-1/ACT1-2 primers were used, which are described in ref. RNA extraction, reverse transcription and quantitative PCR (qRT-PCR) analyses in yeast were used to quantify


The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs  in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission.The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through IMPORTANCE In this study, we found that our P[19] strain and other P[II] RVs recognize mucin cores and the type 1 HBGA precursors as the minimal functional units and that additional saccharides adjacent to these units can alter binding outcomes and thereby possibly host ranges. These data may help to explain why some P[II] RVs, such as P[6] and P[19], commonly infect animals but rarely humans, while others, such as the P[4] and P[8] RVs, mainly infect humans and are predominant over other P genotypes. Elucidation of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis. Rotaviruses (RVs) are the principal cause of severe diarrhea in children, responsible for approximately 200,000 deaths in children younger than 5 years old worldwide in 2011 13. It has4–8–14–17–The recent findings that almost all major RV genotypes in genogroups P[II] to P[IV] infect humans and recognize histo-blood group antigens (HBGAs) –27 have –28–in vitro binding assays. For example, the VP8*s of the human P[8] and P[4] RVs recognize the Lewis b (Leb) and H-type 1 HBGAs, while the P[6] RVs bind the H type 1 only  in five P genogroups (P[I] to P[V]) based on sequences of the viral surface spike proteins VP4 and VP8* , 33 Fig. While tIn this study, we characterized a P[19] RV that commonly infects animals (pigs) but rarely humans . A uniquDQ887060) was expressed in E. coli BL21 and purified by glutathione S-transferase (GST) tag affinity purification, as described previously , 18 mM 15NH4Cl, and 22 mM glucose) or 15N,13C-minimal growth medium supplied with 22 mM [13C]glucose (The VP8* sequences (amino acids 46 to 231) from a human RV P[19] . The recombinant GST-VP8* proteins were applied to individual glycan arrays at a protein concentration of 200 μg/ml, and the bound GST-VP8* proteins were detected using a fluorescence-labeled anti-GST monoclonal antibody. Relative fluorescent units (RFU) of each glycan were calculated to rank the reactivity in the interaction with P[19] VP8*. A further microarray analysis was performed at the carbohydrate microarray facility of the Glycosciences Laboratory at the Imperial College, London, United Kingdom, using a focused array of sequence-defined glycan probes arrayed as lipid-linked probes  and biotinylated anti-rabbit IgG (Sigma). Binding was detected with Alexa Fluor 647-labeled streptavidin . The probes have been described previously   experiments and used to calculate solution dissociation constants (dKs) for binding of the two ligands to P[19] VP8*. The dKs were defined as [VP8*][ligand]/[VP8*-ligand], where [VP8*-ligand] refers to the concentration of the complex of VP8* and the glycan ligand and [VP8*] and [ligand] are the concentrations of free protein and ligand. NMR data were collected at 20°C with samples of ∼300 μl of Tris NMR buffer  in 5-mm Shigemi NMR tubes on a 600-MHz Bruker Avance III spectrometer. Samples of 0.25 mM 15N-labeled VP8* were titrated with each ligand at 0, 0.1, 0.3, 0.5, 1.0, 1.4, 1.9, 2.7, 4.3, and 6 mM, and 2D 1H-15N-HSQC spectra were acquired after each titration step. Spectra were processed with NMRPipe software (http://www.cgl.ucsf.edu/home/sparky). The weighted chemical shift change (CSC) upon titration was determined for 10 strongly affected backbone amide peaks that were not overlapped according to the following formula (N)2 + (ΔδN/6.5)2]}1/2, ΔδHN = δHNfree − δHNbound, and ΔδN = δNfree − δNbound, where δNfree and δNbound are the amide 15N chemical shifts in the absence and presence of ligand and δHNfree and δHNbound are the backbone amide hydrogen chemical shifts, respectively. The concentration of free ligand, [L-free], was determined by the equation [L-free] = [L-total] − (1/f) × [P-total], where f is the fraction of VP8* in the bound state is CSC/CSC-max, and [L-total] and [P-total] are the total concentrations of ligand and protein at each titration. The CSC-max and dKs were determined by fitting the CSC/CSC-max at each ligand titration versus the [L-free] to the equation f = [L-free]/(dK + [L-free]), with the variables CSC-max and dK. The [L-free] values were calculated with the updated CSC-max, and this process was repeated until there were no further changes. The error bars for dK were determined based on the average and standard deviation of the 10 peaks that had their CSC/CSC-max versus [L-free] plotted and fit to dK in the same way.Titrations of P[19] VP8* with mucin core 2 (kindly provided by James Paulson) and LNFPI (V-lab) were followed by two-dimensional heteronuclear single quantum correlation . Backbone assignments were 91% complete for residues 63 to 225, and only 5 to 10 HN cross-peaks could not be assigned. However, none of the unassigned peaks shifted in the titration studies.In order to assign the backbone chemical shifts, 2D and 3D NMR data were collected for ∼300 μl of 1.1 mM  spectra : HNCACB,1H-13C HSQC, heteronuclear multiple-bond correlation (HMBC), HSQC-total correlation spectroscopy (HSQC-TOCSY), 1H-1H correlation spectroscopy (COSY), TOCSY, and nuclear Overhauser effect spectroscopy (NOESY), and 1D 1H NMR experiments recorded at 20°C and referenced to internal trimethylsilylpropanoic acid (TSP) (2O), both at pH 8.0. Chemical shift assignments for LNFPI were in agreement with published values  . The NMRd values , althougIo − Isat)/Io, where Io and Isat are the intensities of the signals in the reference and saturated spectra, respectively, and the normalized STD amplification percentage was determined by dividing by the largest STD amplification factor for each ligand. The protons with the largest STD amplification percentage along with data from the 2D STD TOCSY spectrum, which were used to resolve overlap from the 1D spectra, were used to assign the binding epitopes.All saturation transfer difference (STD) NMR spectra were acquired in Shigemi Tubes  with a Bruker 600-MHz Avance III spectrometer at 10°C using a conventional HCN probe and the TopSpin 3.1 pulse sequence STDDIFFGG19.3. The NMR sample for mucin core 2 in complex with P[19] VP8* was prepared with 200 μM protein and 6.0 mM ligand in a final volume of 300 μl of Tris NMR buffer. The LNFPI STD sample was prepared with 130 μM P[19] VP8* and 6.6 mM LNFPI in a final volume of 300 μl of phosphate NMR buffer. This results in total protein-ligand ratios of 1:35 and 1:50 for the mucin core 2 and LNFPI samples, respectively. The protein resonances were saturated at 0 ppm with a cascade of 40 Gaussian-shaped pulses with duration of 50 ms, resulting in a total saturation time of 2 s. The off-resonance saturation was applied to 30 ppm, and 1,024 experiments were acquired for each with a 5-s recycle delay. A spin-lock filter with strength of 10 kHz and duration of 100 to 200 ms was applied to suppress the broad protein resonance signals, and Watergate 3-9-19 was used to suppress the residual water signal. The STD spectra were obtained by subtracting the on-resonance spectrum from the off-resonance reference spectrum. STD amplification factors were determined by the equation (http://swissmodel.expasy.org/). Mutant VP8* proteins with single amino acid substitutions were constructed from template wild-type VP8* constructs using the QuikChange site-directed mutagenesis kit .A homology model of the P[19] VP8* protein was built based on the X-ray crystal structure of the DS1 strain of RV (PDB identifier [ID] 2AEN) as the template by SWISS-MODEL automated protein structure homology-modeling server (A P[19] RV (G9P[19]) was adapted from a positive stool sample obtained from Taiwan's Centers for Disease Control (CDC) collected on 17 December 2014 by multiple blind passages on MA104 cells. The identity of the strain was confirmed by reverse transcription-PCR (RT-PCR) and sequencing following detection of viral replication in the cell culture by ELISA using a Rotaclone kit . The cell culture-adapted P[19] RVs were then used for inhibition assays with human milk, neonate saliva, and porcine mucin FPLC fractions as well as the oligosaccharides of LNFPI and mucin core 2 using procedures described previously . In briein vitro binding assays of P[19] VP8* to human milk, human saliva, and porcine mucin for the detection of potential host receptor ligands. The P[19] VP8* proteins bound the high-molecular-weight gel filtration fractions of human milk and porcine mucins  against a library containing 610 mammalian glycans. Binding signals arose for a group of glycans containing the type 1 HBGAs and/or mucin cores 2, 4, and 6 . FurtherIn vitro assays of binding of P[19] VP8* to mucin core glycans (mucin cores 1 to 8) also confirmed the glycan array results, with the positive binding signals observed only in those containing the disaccharide GlcNAcβ1-6GalNAc, while further additions of residues GlcNAc β1-3 (mucin core 4) or Gal β1-3 (mucin core 2) linking to GalNAc enhanced the binding  abolished binding . In vitr binding .Gal) is also involved in the interaction with P[19] VP8*, in addition to the GlcNAc and the terminal (H) fucose , demonstrated that the extended type 1 chain of an internal Gal  fucose  and b. S) fucose  and d.To examine the biologic significance of reactive glycan ligands for P[19] RVs, viral replication inhibition assays were performed on a human P[19] RV. P[19] RV titers were significantly reduced following incubation of the viruses with saliva, porcine mucin fractions (gel filtration), and human milk samples that previously bound P[19] VP8*  and b. I1H-15N heteronuclear single quantum coherence (HSQC) spectra. All backbone chemical shift files were available from the BioMagResBank database (accession number 25441). Although LNFPI caused slightly more amino acid perturbations, the residues with HSQC chemical shift perturbations for mucin core 2 and LNFPI largely overlapped  were smaller than those for binding to LNFPI (dK = 2.6 mM)  , indicatMore than 90% of chemical shift perturbations occurred for a group of amino acids scattered in a location near the previously identified SA- and A-HBGA binding interfaces , 21, 44 Sequence alignment showed that all P[19] strains had identical amino acid compositions on the deduced ligand binding site (data not shown). In addition, alignment of the group A RV VP8* sequences showed that the amino acids of the deduced ligand binding interface of the P[19] RVs are conserved among all other three P[II] RVs  , suggestAdditional binding studies for other P[II] RVs revealed similar but genotype-specific binding properties among the three major human RVs  and the P[19] RVs. Like the P[19] RVs, all of the other three P[II] RVs recognized the type 1 HBGA disaccharide precursor with low affinities, while VP8* proteins of P[4] and P[8], but not of P[6], also recognized mucin cores 2, 4, and 6  and b. Sin vitro binding assays of P[10] as well as other three randomly selected P[I] RVs . Nearly identical saliva binding  of human infections worldwide . Interes46–b) antigens containing the Lewis epitope  to these type 1 HBGA glycans completely blocked P[19] binding . This ty epitope . A stron epitope . In the  epitope , which cThe differences in Lewis epitope involvement for P[II] RV binding may also play a role in human versus animal host ranges, as some animal species, such as mice and pigs, lack Lewis antigens .The P[6] RV did not bind the mucin core glycans and bound poorly to the human adult saliva , but it The finding of nearly identical saliva and glycan binding profiles between P[10] and P[19] suggests a potential evolutionary connection between P[10] and P[19] RVs, which may be selected by a common host factor such as the mucin cores and/or type 1 HBGAs. The conserved amino acids of the deduced binding interface, particularly the exclusively conserved residue W175, between P[10]/P[12] and P[19] and other P[II] RVs  further supports this hypothesis. Interestingly, P[10] and P[19] have very similar host ranges, both pigs and humans, while the majority of other P[I] RVs exclusively infect animals. This observation in our view raises concern regarding the strategy to develop live, attenuated animal RV reassortant vaccines for humans, because these animal RVs, such as the bovine (P[5]) and sheep (P[15]) RVs , 53, mayOur study raises additional issues that need to be addressed in the future. For example, based on the binding properties of P[19] and other P[II] RVs, we noted a potential binding specificity switch from simple mucin core glycans to more complex type 1 HBGAs, leading to host range changes between animal (P[I]) and human (P[II]) RVs and among P[II] RVs in different human populations. However, direct evidence, including supporting structural data, for such host range switching and variations remains lacking. While mucin core 2 is commonly found in a variety of cells and tissues, mucin core 4 and 6 sequences were found only in a very limited type of mucin, that is, in human intestinal mucins . Thus, f


Influenza activity in the United States began to increase in early November 2017 and rose sharply from December through February 3, 2018; elevated influenza activity is expected to continue for several more weeks. Influenza A viruses have been most commonly identified, with influenza A(H3N2) viruses predominating, but influenza A(H1N1)pdm09 and influenza B viruses were also reported. This report summarizes U.S. influenza activityU.S. World Health Organization (WHO) and National Respiratory and Enteric Virus Surveillance System laboratories, which include both public health and clinical laboratories throughout the 50 U.S. states, Puerto Rico, and the District of Columbia, contribute to virologic surveillance for influenza. During October 1, 2017–February 3, 2018, clinical laboratories tested 666,493 specimens for influenza virus, 124,316 (18.7%) of which tested positive . During Public health laboratories tested 51,014 specimens collected during October 1, 2017–February 3, 2018. Among these, 27,669 tested positive for influenza virus, including 23,257 (84.1%) for influenza A and 4,412 (15.9%) for influenza B viruses . Among tData on age were available for 23,578 influenza-positive patients whose specimens were tested by public health laboratories. Overall, 1,863 (7.9%) were aged 0–4 years, 5,208 (22.1%) were aged 5–24 years, 7,576 (32.1%) were aged 25–64 years, and 8,931 (37.9%) were aged ≥65 years. Influenza A(H3N2) viruses were predominant among all age groups, accounting for 68%–72% of viruses identified among persons aged 0–4 years, 5–24 years, and 25–64 years and 84% of viruses reported among persons aged ≥65 years. The largest proportion of reported influenza B virus infections occurred in persons aged 5–24 years; influenza B viruses accounted for 21.9% of the viruses reported in this age group.Six human infections with novel influenza A viruses were reported to CDC during October 1, 2017–February 3, 2018. All of these were variantThe A(H3N2)v virus detected in Iowa had a hemagglutinin (HA) gene segment derived from a seasonal human H3N2 virus that was likely introduced into swine by reverse zoonosis  in 2010. This virus was closely related to H3N2 viruses known to circulate in the U.S. swine population , using previously described genomic enrichment practices  viruses propagated in tissue culture have lacked sufficient hemagglutination titers for antigenic characterization using HI assays. Therefore, a subset of influenza A(H3N2) viruses are selected for antigenic characterization using the virus neutralization focus reduction assay to assess the ability of various antisera to neutralize infectivity of the test viruses. CDC has antigenically or genetically characterized 1,365 influenza viruses collected and submitted by laboratories in the United States since October 1, 2017, including 276 influenza A(H1N1)pdm09 viruses, 695 influenza A(H3N2) viruses, and 394 influenza B viruses.Phylogenetic analysis of the HA gene segments from 276 A(H1N1)pdm09 viruses collected since October 1, 2017, showed that all belonged to subclade 6B.1 . Of the A total of 695 influenza A(H3N2) viruses were sequenced, and phylogenetic analysis of the HA gene segments illustrated that multiple clades/subclades were cocirculating . CirculaAmong influenza B viruses, phylogenetic analysis of 338 influenza B/Yamagata-lineage viruses showed that all the HA gene segments belonged to clade Y3 . A totalAmong the 56 influenza B/Victoria-lineage viruses sequenced and phylogenetically analyzed, the HA gene segment of all viruses belonged to genetic clade V1A, the same genetic clade as the vaccine reference virus, B/Brisbane/60/2008. However, the HA gene segment of 28 viruses (50.0%) had a six-nucleotide deletion (encoding amino acids 162 and 163), and viruses like these, abbreviated as V1A-2Del, were previously reported pdm09 viruses tested for oseltamivir and peramivir susceptibility, four (1.1%) were resistant to both drugs and contain H275Y, the established NA marker of resistance to oseltamivir. A total of 265 of those influenza A (H1N1)pdm09 viruses also were tested for zanamivir susceptibility, and all were susceptible. All 903 influenza A(H3N2) viruses tested for oseltamivir and zanamivir susceptibility were susceptible to both of these medications. A total of 638 of those A(H3N2) viruses also were tested for peramivir susceptibility, and all were susceptible. All 387 influenza B viruses tested for oseltamivir, peramivir, and zanamivir susceptibility were sensitive to all three recommended antiviral medications. High levels of resistance to the adamantanes (amantadine and rimantadine) persist among influenza A(H1N1)pdm09 and A(H3N2) viruses. Adamantane drugs are not recommended for use against influenza at this time.During October 1, 2017–February 3, 2018, the weekly percentage of outpatient visits to heath care providers participating in the U.S. Outpatient Influenza-like Illness Surveillance Network (ILINet) for influenza-like illness††† within their jurisdiction . During the 2017–18 season, the peak number of jurisdictions reporting widespread activity in a single week was 50 (93%); this occurred for the 3 consecutive weeks . During the previous five influenza seasons, the peak number of jurisdictions reporting widespread activity in a single week during each season has ranged from 41 (76%) in the 2015–16 season to 48 (89%) during the 2012–13 season.Influenza activity levels reported by state and territorial epidemiologists indicate the geographic spread of influenza activityCDC monitors hospitalizations associated with laboratory-confirmed influenza infections in adults and children through the Influenza Hospitalization Surveillance Network (FluSurv-NET),The cumulative influenza hospitalization rates per 100,000 population during October 1, 2017–February 3, 2018, for persons aged 0–4 years, 5–17 years, 18–49 years, 50–64 years, and ≥65 years were 40.0, 10.3, 18.3, 63.1, and 263.6, respectively. Among all hospitalizations, 14,770 (86.4%) were associated with influenza A virus infection, 2,251 (13.2%) with influenza B virus infection, 43 (0.3%) with influenza A virus and influenza B virus coinfection, and 37 (0.2%) with influenza virus infection for which the type was not determined. Among the 3,841 patients for whom influenza A subtype information was available, 3,308 (86.1%) were infected with influenza A(H3N2) viruses and 533 (13.9%) with influenza A(H1N1)pdm09 viruses. Among hospitalized persons aged 0–64 years for whom influenza A subtype information was available, 23.6% were infected with influenza A(H1N1)pdm09 viruses, compared with only 7.0% of those aged ≥65 years.Information on underlying medical conditions was available for 2,147 (12.6%) hospitalized patients with laboratory-confirmed influenza as of February 3, 2018. Among 1,955 hospitalized adults with information on underlying medical condition available, 1,325 (67.8%) had at least one underlying medical condition that placed them at high risk for influenza-associated complications. The most commonly reported medical conditions were cardiovascular disease (35.5%), metabolic disorders (33.0%), obesity (25.2%), and chronic lung disease (23.6%). Among 192 hospitalized children with information on underlying medical conditions available, 97 (50.5%) had at least one underlying medical condition, the most commonly reported being asthma (22.8%), neurologic disorders (14.4%), and obesity (10.1%). Among 151 hospitalized women aged 15–44 years with information on pregnancy status, 36 (23.8%) were pregnant.CDC tracks pneumonia and influenza (P&I)–attributed deaths through the National Center for Health Statistics (NCHS) Mortality Reporting System. The percentages of deaths attributed to P&I are released 2 weeks after the week of death to allow for collection of sufficient data to produce a stable P&I mortality percentage. From October 1, 2017, to January 20, 2018, the weekly percentage of deaths attributed to P&I has ranged from 5.8% to 10.1% and has exceeded the epidemic thresholdAs of February 3, 2018, (week 5), 63 laboratory-confirmed influenza-associated pediatric deaths occurring during the 2017–18 season were reported to CDC. Fifteen deaths were associated with an influenza A(H1N1)pdm09 virus infection, 16 were associated with an influenza A(H3N2) virus infection, 14 were associated with infection with an influenza A virus for which no subtyping was performed, and 18 were associated with an influenza B virus infection. Since influenza-associated pediatric mortality became a nationally notifiable condition in 2004, the number of influenza-associated pediatric deaths per season has ranged from 37 to 171, excluding the 2009 pandemic, when there were 358 pediatric deaths during April 15, 2009–October 2, 2010. The mean age of the reported pediatric deaths reported this season was 7.4 years (range 2 months to 17 years); 40 (63%) of the children died after admission to the hospital. Among the 56 children with a known medical history, 30 (54%) had at least one underlying medical condition recognized by ACIP as placing them at increased risk for influenza-related complications. Among the 54 children who were eligible for influenza vaccination (≥6 months of age at date of onset) and for whom vaccination status was known, 14 (26%) had received at least 1 dose of influenza vaccine before onset of illness .Influenza illness this season has been substantial, with some of the highest levels of ILI and hospitalization rates in recent years and elevated activity occurring in most of the country simultaneously. Influenza A(H3N2) is the predominant influenza virus circulating this season. Past A(H3N2) virus–predominant seasons such as the 2012–13 and 2014–15 seasons had increased numbers of influenza related infections, hospitalizations, and deaths compared with A(H1N1)pdm09 virus-predominant seasons, and the 2017–18 season is on track to reach or exceed estimates from those seasons.The percentage of outpatient visits to doctors’ offices, urgent care centers, and emergency departments that were for ILI rose sharply in late 2017 to 7.7% in early February. This is the highest level of ILI activity since the pandemic in 2009 which peaked at 7.7%. During the previous five influenza seasons, the peak weekly percentages of outpatient visits for ILI ranged from 3.6% to 6.1% and remained above baseline levels for an average of 16 weeks (range = 11–20 weeks). The weekly percentage of outpatient visits for ILI this season has been above the national baseline for 11 weeks, suggesting that influenza activity is likely to continue for several more weeks.The cumulative hospitalization rate attributed to laboratory-confirmed influenza for the week ending February 3, 2018,  exceeded the rate for the same week in 2014–15 , an A(H3N2) virus–predominant season categorized as high severity, and is the highest rate observed for this week since the system expanded to include adults during the 2005–06 season. Persons aged ≥65 years account for the majority of cases (59%); however, hospitalization rates for all adult age groups  are higher than those observed during the same week in 2014–15. These hospitalization rates are not adjusted for testing practices, which can vary from season to season; therefore, caution should be used when comparing hospitalization rates across seasons.P&I-related deaths also rose sharply in the first weeks of 2018, accounting for 10.1% of all deaths recorded on death certificates during the week ending January 20, 2018. It is anticipated that the number of P&I-related deaths will continue to increase for several more weeks and might exceed the peaks in past recent A(H3N2) virus–predominant seasons (11.1% in 2012–13 and 10.8% in 2014–15). Through the week ending January 20, P&I-related mortality has been above the epidemic threshold for 5 consecutive weeks. During the past five seasons, the average number of weeks this indicator was above threshold was 11 (range of 7–15 weeks).Sixty-three laboratory-confirmed influenza-associated pediatric deaths have been reported to CDC as of February 3, 2018; 46% of these children were otherwise healthy. Among those children who were eligible for vaccination and for whom vaccination status was known, only 14 (26%) had received any influenza vaccine this season before the onset of illness . In a previous analysis of pediatric deaths with a similar percentage of eligible children vaccinated (26%), influenza vaccination was associated with a 65% reduction in risk for laboratory-confirmed influenza-associated pediatric death  against influenza A and influenza B virus infection associated with medically attended acute respiratory illness in the United States was 36% . VE was estimated to be 25% (95% CI = 13%–36%) against illness caused by influenza A(H3N2) virus, 67% (95% CI = 54%–76%) against A(H1N1)pdm09 virus and 42% (95% CI = 25%–56%) against influenza B virus , inhaled zanamivir  and intravenous peramivir . Resistance to these medications is not a concern at this time because only four influenza viruses  collected in the United States since October 1, 2017, were identified as not being sensitive to oseltamivir and peramivir.https://emergency.cdc.gov/han/han00409.asp.Treatment with neuraminidase inhibitors has been shown to reduce illness duration and severe outcomes of influenza based on evidence from randomized controlled trials, meta-analyses of randomized controlled trials, and observational studies (https://www.cdc.gov/flu/weekly). Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A infections in humans is available online (https://www.cdc.gov/flu).Influenza surveillance reports for the United States are posted online weekly  viruses predominating, but influenza A(H1N1)pdm09 and influenza B viruses were also detected. Influenza illness this season has been substantial, with some of the highest levels of influenza-like illness and hospitalization rates in recent years, and elevated activity occurring in most of the country simultaneously. Elevated influenza activity is expected to continue for several more weeks.With several more weeks of elevated influenza activity expected, the increasing proportion of influenza A(H1N1)pdm09 and influenza B viruses, and the potential to prevent significant illness through influenza vaccination, CDC continues to recommend influenza vaccination at this time. In influenza seasons with increased severity, influenza antiviral medications are an increasingly important adjunct to vaccination in the treatment of influenza. Early treatment with neuraminidase inhibitor antiviral medications is recommended for patients with severe, complicated, or progressive influenza illness and those at higher risk for influenza complications, including adults aged ≥65 years who develop influenza symptoms.