Thus, four PGPR strains were selected from a series of bacterial strains isolated from maize roots at two locations in Malaysia. The PGPR strains were screened in vitro for their biochemical plant growth-promoting (PGP) abilities and plant growth promotion assays. These strains were identified as Klebsiella sp. Br1, Klebsiella pneumoniae Fr1, Bacillus pumilus S1r1 and Acinetobacter sp. S3r2 and a reference strain used was Bacillus subtilis UPMB10. All the PGPR strains were tested positive for N2 fixation, phosphate solubilisation and auxin production by in vitro tests. In a greenhouse experiment with reduced fertiliser-N input (a third of recommended fertiliser-N rate), the N2 fixation abilities of PGPR in association with maize were determined by 15N isotope dilution technique at two harvests, namely, prior to anthesis (D50) and ear harvest (D65). The results indicated that dry biomass of top, root and ear, total N content and bacterial colonisations in non-rhizosphere, rhizosphere and endosphere of maize roots were influenced by PGPR inoculation. In particular, the plants inoculated with B. pumilus S1r1 generally outperformed those with the other treatments. They produced the highest N2 fixing capacity of 30.5% (262 mg N2 fixed plant−1) and 25.5% (304 mg N2 fixed plant−1) of the total N requirement of maize top at D50 and D65, respectively. N remobilisation and plant senescence in maize were delayed by PGPR inoculation, which is an indicative of greater grain production. This is indicated by significant interactions between PGPR strains and time of harvests for parameters on N uptake and at. % 15Ne of tassel. The phenomenon is also supported by the lower N content in tassels of maize treated with PGPR, namely, B. pumilus S1r1, K. pneumoniae Fr1, B. subtilis UPMB10 and Acinetobacter sp. S3r2 at D65 harvest. This study provides evidence that PGPR inoculation, namely, B. pumilus S1r1 can biologically fix atmospheric N2 and provide an alternative technique, besides plant breeding, to delay N remobilisation in maize plant for higher ear yield (up to 30.9%) with reduced fertiliser-N input.Plant growth-promoting rhizobacteria (PGPR) may provide a biological alternative to fix atmospheric N However, only the latter demand is being widely addressed through maize cultivation as cash crop due to its higher return on investment . In 2012−1 . Thus, m−1 , the resAzospirillum [Klebsiella [Burkholderia [Bacillus [Pseudomonas [−1  in maize [Plant growth-promoting rhizobacteria (PGPR) isolated as free-living soil bacteria from plant rhizosphere can decrease chemical fertiliser-N use and increase plant growth and yield when associated with plant roots and other plant parts . Severalpirillum , Klebsieebsiella , Burkholholderia , BacilluBacillus  and Pseuudomonas  have beein maize , sugarcain maize  and oil in maize . Generalin maize . In addiin maize . It was in maize .2 fixed and their influence on plant-N remobilisation prior to anthesis and ear harvest, using the 15N isotope dilution technique.However, present information on indigenous PGPR associations with maize plant towards BNF and their influence on N remobilisation is still limited. Thus, a concerted effort is needed for an effective plant-N management. Therefore, the aim of this study was to select effective PGPR strains from a series of indigenous bacterial strains by biochemical characterisations and plant growth promotion assays. These selected strains and a reference strain, UPMB10 were identified using 16S rDNA gene analysis and further inoculated to maize plants grown under greenhouse conditions to estimate the amount of N2-fixing activity, phosphate solubilisation, indole-3-acetic acid (IAA) production and plant growth promotion assay. A parallel experiment on plant growth promotion assays indicated that PGPR inoculations with Fr1, S1r1 and S3r2 produced the highest maize plant top biomass and N uptake , Selangor  and Lentang Village in Sik, Kedah  using the modified method of Hoben and Somasegaran . Three fN uptake  Figs. Th2-fixing activity of the pure bacterial cultures was determined on N-free semi-solid malate medium (Nfb) [2-fixing activity. Phosphate solubilisation test was conducted according to the method of Pikovskaya [−1 of L-tryptophan and incubated on a rotary shaker at 200 rpm under room temperature, 28±2°C for 24 h. The cultures were centrifuged at 7000 rpm for 7 min and the supernatants were quantified spectrophotometrically at 535 nm with 2 mL of Salkowski’s reagent (2% of 0.5 M FeCl3 in 35% perchloric acid) after 25 min. The IAA concentrations were estimated from a standard IAA curve.Initial screening for Num (Nfb) . The plakovskaya , where tkovskaya . PGPR cu2, 2.5 U Taq DNA polymerase (Thermo Fisher Scientific), 0.16 μM of each primer 27F (5’-AGAGTTTGATCTTGGCTCAG-3’) and 1492R (5’-TACGGTTACCTTGTTACGACTT-3’) [™) at 5:1 (PCR product: dye) ratio and a marker  in 1% (w/v) agarose gel in 1X Tris-acetate EDTA (TAE) buffer for 1 h at 80 V. The agarose gel was stained with GelRed™ (Biotium Inc.) for 40 min and examined under UV light in a UV transilluminator (Bio-Rad Molecular Imager® Gel Doc™ XR+ System). Gel image was captured with Image Lab software  (®). The purified DNA products were sent for sequencing by First BASE Laboratories Sdn. Bhd. (www.base-asia.com) using BigDye® Terminator 3.1 cycle sequencing kit (Applied BioSystems). The quality of 16S rDNA sequences was checked manually by using Applied Biosystems Sequence Scanner Software (version 2.0). The sequences were analysed using the BLAST Sequence Similarity Search to identify the most closely related members in the NCBI GenBank DNA database (www.ncbi.nlm.nih.gov/geo). The partial 16S rDNA sequences of the PGPR strains were submitted to the NCBI database under their respective accession number as follows: Bacillus subtilis UPMB10 (KP641618), Klebsiella sp. Br1 (KP257586), Klebsiella pneumoniae Fr1 (KP641617), Bacillus pumilus S1r1 (KP295962) and Acinetobacter sp. S3r2 (KP295963).Total genomic DNA was extracted using Genomic DNA Mini Kit (Yeastern Biotech Co. Ltd.) according to the supplier’s instructions and used as DNA template in polymerase chain reaction (PCR) for amplification of the 16S rDNA gene. The DNA purity was quantified at 260 nm and 280 nm using NanoDrop Spectrophotometer , 1.6 to 2.2, to detect protein contamination in the DNA . PCR ampACTT-3’) , and ultatories) . DNA fraStreptomyces griseus FGQ9 (HQ202539) was used as an outgroup sequence.The partial 16S rDNA sequences of UPMB10, Br1, Fr1, S1r1 and S3r2 were aligned with the most closely related bacteria sequences obtained from the NCBI database using MUSCLE . The tre® PHM210); total N (semi-micro Kjeldahl method) [−1; exchangeable K (NH4OAc) [−1 and field capacity (pressure plate method) [2O5) and Muriate of Potash  at the recommended rates of 60 kg P2O5 ha−1 and 40 kg K2O ha−1 equivalents [15N-labelled urea  at 40 kg N ha−1 equivalent (a third of recommended fertiliser-N rate) [50 and D65) with four blocks and 0.75 m × 0.25 m planting distance between the polybags. The six treatments imposed were: (i) killed B. subtilis UPMB10 inoculum, autoclaved at 121°C for 20 min (Uninoculated control), (ii) B. subtilis UPMB10 inoculation (Reference control), (iii) Klebsiella sp. Br1 inoculation (Negative control), (iv) K. pneumoniae Fr1 inoculation, (v) B. pumilus S1r1 inoculation and (vi) Acinetobacter sp. S3r2 inoculation. Six maize seeds of similar size and shape, var. Hibrimas, were sowed and thinned to two per pot at 7th DAP (D7). The PGPR inocula were grown in TSB medium for 24 h  and cell suspensions were adjusted to OD600 between 1.4 and 2.0, which corresponded to the total plate counts of ca. 109 cfu mL−1, as determined on the TSA medium. Each polybag was inoculated with 20 mL of respective inocula (live or killed) on D0, D7 and D35. The polybags were watered and weighted daily to maintain at field capacity throughout the experiment to avoid any possible loss of applied 15N labelled fertiliser through denitrification, if the soil contained excess moisture. The total bacterial colonisations in soil, rhizosphere and root-endosphere were determined according to the method of Hoben and Somasegaran [65 harvest.The experimental soil  was collected from 0–15 cm soil depth at the University Agriculture Park, UPM . The soil was air-dried, ground, sieved (2 mm mesh) and analysed for its physico-chemical properties. The soil had pH 5.1  , 0.46%;  method) , 3.2%; a method) , 31.6 mg(NH4OAc) , 46.8 mg method) , 26.8%. ivalents , respect-N rate)  was diss50 and D65), each maize plant was separated into roots, young, ear and old leaves, stalk, tassel and ear (D65 only). The ear bearing leaf and its immediate lower leaf were labelled as ear leaves. The upward and downward leaves from the ear leaves were respectively labelled as young and old leaves. Every component was weighed, oven-dried  and ground (<1 mm) to determine the plant tissue N (except root) using the semi-micro Kjeldahl method [15N abundance using an emission spectrometer  at Malaysian Nuclear Agency in Bangi, Selangor. The 15N abundance in the sample was corrected for the natural 15N abundance present in the environment (0.3663 at. % 15Ne). Meanwhile, the N2 fixation in the whole maize plant (plant top basis) was calculated from the mean weighted atom excess (WAE) using the following formula [15Ne, TN = total N of a, b, c, d, e and f = tassel, young, ear and old leaves, stalk and ear, respectively, ear was only available in D65 harvest. The proportions of N derived from the atmosphere (% Ndfa) was calculated, as follows:At harvest  followed by Duncan’s Multiple Range Test (DMRT) at p<0.05 using Statistical Analysis System software (SAS version 9.0) . Pearson−1 compared to the other strains (5–11 μg mL−1).The plant growth-promoting (PGP) abilities of the four selected strains and a reference strain, UPMB10 are shown in Bacillus subtilis (98% similarity), Klebsiella sp. (99% similarity), Klebsiella pneumoniae (99% similarity), Bacillus pumilus (99% similarity) and Acinetobacter sp. (98% similarity), respectively. A phenogram showing the genetic relationship among the PGPR strains and their most closely related bacteria obtained from NCBI database is presented in Gamma-proteobacteria: Klebsiella spp. (Br1 and Fr1) and Acinetobacter spp. (S3r2); (ii) Firmicutes: Bacillus spp. (UPMB10 and S1r1).Analysis of 16S rDNA sequences from the NCBI database suggested that UPMB10, Br1, Fr1, S1r1 and S3r2 were most closely related to 65) . In the root-endosphere, the PGPR inoculations particularly with Acinetobacter sp. S3r2 significantly stimulated up to 757 folds increase in total bacterial population compared to the uninoculated control (96 cfu cm−1 fresh root).PGPR inoculations increased rhizobacterial colonisation around the maize roots and their surrounding soils compared to uninoculated control at ear harvest (D65) . Rhizosp50) and ear harvest (D65), except in tassel (D65), young leaves (D65), old leaves (D50 and D65), stalk (D50) and root (D65) (B. pumilus S1r1 gave significantly highest biomass in tassel (D50), young leaves (D50), ear leaves (D65) and ear. Comparatively, the inoculation with K. pneumoniae Fr1 gave similar biomass in tassel (D50), young leaves (D50), ear leaves (D50 and D65), stalk (D65), ear (D65) and root (D50). Both inoculations with B. pumilus S1r1  and K. pneumoniae Fr1  gave significantly higher total plant biomass compared to uninoculated control  at D50 and D65 harvests. Plant stalk accounted for the highest proportion of dry biomass, making up to 40.6% and 38.9% of total plant biomass at D50 and D65, respectively. In D50, this was followed by tassel (15.2%), ear leaves (13.9%), young leaves (12.3%), older leaves (11.8%) and roots (6.1%). In D65, this was followed by ear (16.5%), young leaves (10.3%), ear leaves (9.1%), tassel (9.1%), old leaves (8.2%) and roots (7.8%). The part of maize plant that experienced a significantly lower biomass at D65 was the tassel (5.4–6.5 g to 5.4–5.7 g). On the contrary, the plant parts that experienced significant increment of biomass at D65 were the young leaves (4.2–5.7 g to 5.5–7.3 g), stalk (13.9–18.0 g to 21.7–26.1 g) and roots (1.9–2.7 g to 4.3–5.3 g).Generally, inoculation with PGPR significantly increased the dry biomass of whole plant and various plant parts of maize prior to anthesis (Dot (D65) . Among t50 and D65 harvests, except in the tassel (D65) , young, ear and old leaves, stalk and ear (D65) of maize. Generally, the significant differences in the N uptake of plant parts between D50 and D65 harvests were shown as a decrease in total N in the tassel and increased total N in the young leaves (S1r1 only), stalk and plant top.Inoculation with PGPR significantly increased the N uptake in all plant parts and plant tops of maize at Del (D65) . Meanwhi65 (119 mg plant−1) was significantly lower compared to D50 (160 mg plant−1), as shown among the inoculated treatments (except for Klebsiella sp. Br1). In young leaves, the mean N uptake was significantly higher at D65 (184 mg plant−1) compared to D50 (142 mg plant−1). However, this effect was evident only in inoculated treatment with B. pumilus S1r1. Similarly, the mean N uptake in the stalk and plant top significantly increased at D65  compared to D50 . The highest accumulation of N was found in plant stalk, containing 30.5% and 31.3% of the total plant top N at D50 and D65, respectively. The lowest accumulation of N was found in old leaves (D50) and plant tassel (D65) at 15.1% and 10.7% of the total plant top N, respectively. In general, majority of total N (70–81%) in the plant top of maize was accumulated before anthesis (D50).In the tassel, the mean N uptake at D15N atom excess (at. % 15Ne) in the plant top and in the different parts of maize than the uninoculated control at D50 and D65 harvests , old leaves and stalk. In general, the plant tops and all parts of maize  experienced significantly lower mean at. % 15Ne value at D65 than D50. However, the lower at. % 15Ne value in tassel at D65 among the inoculated maize plants was not statistically significant, except for the Klebsiella sp. Br1 treatment. Among the various plant parts, the lowest and highest concentrations of at. % 15Ne were found in the ear (0.651–0.885 at. % 15Ne) and old leaves (1.035–1.604 at. % 15Ne), respectively.Maize inoculated with PGPR generally had significantly lower weighted harvests . Among t2 fixed in the plant top and different plant parts of maize at D50 and D65 harvests, except for the plant stalk (B. pumilus S1r1 gave 30.5% Ndfa (262 mg N fixed plant−1) and 25.5% Ndfa (304 mg N fixed plant−1) at D50 and D65, respectively. Generally, the mean % Ndfa in the inoculated maize plant top at D65 (17.5%) was significantly reduced in comparison to D50 (21.1%), despite the significant increase in the mean amount of fixed N by 12.9%. Based on the fixed N content of the inoculated maize plants, the estimated mean N2 fixation rate was higher prior to anthesis (D50) at 3.5 mg N fixed plant−1 day−1 compared to after anthesis (D65) at 3.1 mg N fixed plant−1 day−1.Maize inoculated with PGPR gave marked increases in % Ndfa and amounts of Nnt stalk . In the B. pumilus S1r1 recorded significantly highest % Ndfa and fixed N content in tassel , young leaves , ear leaves , old leaves  and ear (26.4% Ndfa or 59 mg N fixed plant−1). Meanwhile, inoculation with K. pneumoniae Fr1 produced similar results in young leaves , ear leaves  and old leaves .Inoculation with 50 and D65, respectively. The lowest % Ndfa and amount of fixed N at D65 were found in tassel (3.3–10.2% Ndfa or 4–12 mg N fixed plant−1). The highest amount of fixed N was found in plant stalk at D50 (31–50 mg N fixed plant−1) and D65 (41–67 mg N fixed plant−1), albeit not the highest in % Ndfa (12.7–18.0% Ndfa). Interestingly, the maize plants inoculated with B. pumilus S1r1 had most of the fixed N concentrated in the ear leaves (62–75 mg N fixed plant−1), whereas other inoculated maize plants had most of their fixed N concentrated in the stalk. In tassel, % Ndfa and fixed N were significantly lower at D65 (6.1% Ndfa or 7 mg N fixed plant−1) compared to D50 (16.6% Ndfa or 28 mg N fixed plant−1). A similar trend was also observed in young leaves at D65, whereby % Ndfa (14.5%) and fixed N (29 mg N fixed plant−1) were significantly lower compared to D50 . In contrast, the ear leaves had significantly higher % Ndfa and fixed N in D65 than in D50. The amount of fixed N in the plant stalk significantly increased by 32.0% at D65 compared to D50, despite the fact that % Ndfa remained unchanged.Among the plant parts, the highest % Ndfa was found in young leaves (10.0–32.2% Ndfa) and ear leaves (16.0–40.8% Ndfa) at D65. There were significant interactions between PGPR strains and time of harvests for the parameters on total N uptake (tassel and plant top) and at. % 15Ne    Tables.Klebsiella sp. Br1, K. pneumoniae Fr1, B. pumilus S1r1 and Acinetobacter sp. S3r2) isolated from maize roots and a reference strain (B. subtilis UPMB10) from oil palm roots were used as inoculants for maize plants grown under greenhouse conditions. According to the 16S rDNA sequence analysis, these PGPR strains belonged to: (i) Gamma-proteobacteria: Klebsiella spp. (Br1 and Fr1) and Acinetobacter spp. (S3r2); and (ii) Firmicutes: Bacillus spp. (UPMB10 and S1r1). Similarly, Montañez et al. [gamma-proteobacteria subdivision and indicated a selective maize plant association with some of these bacterial genera such as Acinetobacter and Klebsiella. Other researchers have also observed that bacteria from Klebsiella genus were commonly sighted near the maize root system and soil environment [In the present study, four PGPR strains  condition, with strains such as Bacillus spp., Klebsiella spp., Azospirillum spp., Azotobacter spp. and Pantoea spp. [Bacterial genera such as by 30.7% . Similarr inputs . Many sioea spp. , 34. The2 through natural N2 fixation phenomenon. In addition, this N2 fixation screening method allows selection of strains with higher survival traits in N-deprived soil condition [2 fixation, these five strains have other PGP abilities, namely phosphate solubilisation and IAA production. The clear halo zones exhibited around the bacterial colonies in Pikovskaya media were in response to the solubilisation of insoluble inorganic phosphates by organic acids. Song et al. [Burkholderia cepacia, a PGPR isolated from cultivated soils in Korea, to its high level of organic acid productions namely gluconic acid. Other organisms such as Pseudomonas aeruginosa and B. megaterium have also been reported to have high phosphate solubilisation efficiency [The five selected PGPR strains showed positive reactions on Nfb media, a differentiating media for screening PGPR with potential BNF ability . The colondition . Besidesg et al.  attributficiency .−1) which were comparable to those presented in other reports. Among other, Egamberdiyeva et al. [−1 and 0.5 μg mL−1 for Pseudomonas alcaligenes and Mycobacterium phlei, whereas Sachdev et al. [−1 from their collections of K. pneumoniae strains. In nature, IAA is synthesised by plants and PGPR from amino acid tryptophan, a common precursor in root exudates through transamination and decarboxylation biochemical reactions [50 strongly suggested the influence of phytohormone IAA on plant growth. However, these positive plant growth effects from IAA on plant top and root biomass were not apparent at ear harvest (D65), possibly due to the pot environment. IAA enhances plant growth mainly through extension of root system to reach larger soil volume for increased water and nutrient uptake. The common soil volume in all treatments could gradually limit this beneficial effects of IAA on root development, especially at the later stage of plant growth, ear harvest (D65) [The PGPR strains in this study produced considerable amounts of IAA  .8 cfu g−1) in the PGPR inoculated rhizospheres were deemed as sufficient to promote nitrogenase activity [Acinetobacter sp. S3r2 treatment.Rhizosphere usually has higher bacterial biomass and activity than the bulk soil due to the photosynthetically assimilated carbon sources such as carbohydrates, amino acids, amides, vitamins and organic acids in the root exudates . This phactivity , 41 in tactivity . The sigactivity . Furtheractivity  in whichB. subtilis UPMB10, Klebsiella sp. Br1, K. pneumoniae Fr1, B. pumilus S1r1 and Acinetobacter sp. S3r2) significantly increased the total N content and dry biomass of maize throughout the study (D50 and D65). Among the PGPR strains, inoculation with B. pumilus S1r1 and K. pneumoniae Fr1 recorded higher amounts of total N content and dry biomass in the respective whole plants, plant tops and different plant parts  of maize prior to anthesis (D50) and ear harvest (D65). These increments were strongly attributed to the inherent BNF abilities, as indicated by the significantly lower at. % 15Ne in the plant tops and different plant parts of the inoculated maize compared to the uninoculated control. Lower at. % 15Ne value indicates a marked increase in percentage of N within inoculated maize plant is derived from atmospheric-N [2 fixation rate of PGPR since the rate is calculated based on at. % 15Ne of reference plant, which will be indiscriminately influenced by the BNF of indigenous soil bacteria. Therefore, a suitable reference plant with similar plant type, root system and plant growth is crucial in a 15N isotope dilution study to minimise plant variations due to the influence of indigenous soil bacteria [15N isotope dilution technique is widely regarded as the most accurate and the only direct method available to quantify N status in plant and soil studies for short and long term experiments without any isotope effect or health risk under growth chamber, greenhouse and field conditions [Generally, PGPR inoculations  in the plant top, followed by K. pneumoniae Fr1. Inoculation with B. pumilus S1r1 contributed substantial amounts of fixed N to maize plant top of 262 mg N2 fixed plant−1 (30.5% Ndfa) and 304 mg N2 fixed plant−1 (25.5% Ndfa) at D50 and D65, respectively, which were equivalent to 14.0 kg N ha−1 and 16.2 kg N ha−1, respectively, based on an equivalent planting density of 53,333 plants ha−1. According to Schröder et al. [−1 day−1; thus, the amount of fixed N (0.25–0.28 kg N ha−1 day−1) from B. pumilus S1r1 inoculation could potentially complement ca. 50% of the total plant-N requirement. Other researchers have reported contributions of fixed N up to 26.7 kg N ha−1 or 70% of total plant-N in sugarcane inoculated with Azospirillum spp. [In the present study, maize inoculated with r et al. , the inilum spp. . The mailum spp. . These N15Ne in the plant tops and in the different plant parts of maize generally decreased upon ear harvest (D65). This phenomenon suggests a continuous contribution of unlabelled N from BNF and soil sources towards the dilution of at. % 15Ne in maize (inoculated and uninoculated control) until ear harvest. Nonetheless, the estimated rate of N2 fixed plant−1 day−1 in inoculated maize had declined after anthesis (from 3.5 to 3.1 mg N2 fixed plant−1 day−1), and this was possibly due to the occurrence of N feedback, as reported in Arabidopsis [Meanwhile, the at. % bidopsis . N feedbbidopsis . This subidopsis . Accordibidopsis , this ph15N isotope dilution technique can directly label the N in different plant parts to denote the two simultaneous N fluxes: (i) N remobilisation from the plant parts to grain and (ii) exogenous N uptake to the plant parts and grain [50). These stored N is continuously remobilised from structural compounds of senescing plant parts through proteolysis to developing plant parts in order to recycle the N and increase plant NUE [The nd grain , 53. Connd grain , 55, 56.nd grain . Accordind grain . In thislant NUE . Proteol50) and at ear harvest (D65) which could create a new approach to plant-N management.N remobilisation in plants could influence C production, as remobilised N is primarily sourced and initiated from senescing photosynthetic plant parts . Accordi50), the majority of N assimilated during the early growth of maize was derived from labelled fertiliser-urea source, instead of unlabelled soil and atmospheric sources, as indicated by the highest at. % 15Ne in the old leaves of maize (D50 and D65). This phenomenon demonstrated the role of old leaves as a major sink for N during the early maize growth [50) and the significant increments in N uptake and dry biomass of young leaves at D65. The old leaves continued to be the source of remobilised N for developing plant parts  after anthesis, as indicated in the significantly lower at. % 15Ne value at D65, while the N uptake and dry biomass parameters remained similarly low at D50. As ear leaves were optimal for photosynthate production at D50, the N usage for further organ growth has significantly reduced, as exhibited in the unchanged N uptake and dry biomass parameters of ear leaves between D65 and D50 harvests.Prior to anthesis  have delayed the dehiscence of tassels, thus exhibiting significant reductions in N uptakes at D65 harvest. In addition, the influence of PGPR on N remobilisation in maize was clearly demonstrated in the significant interactions found between the PGPR strains and the time of harvests in N uptake and at. % 15Ne parameters of tassel.At D50 and D65. Favourably, the significantly lower at. % 15Ne in ear leaves of D65 harvest suggested that the N content was diluted with “later” unlabelled-N uptakes from BNF and soil sources. The significant increments of fixed N (% Ndfa) in ear leaves of inoculated maize at D65 further emphasised the role of ear leaves as temporary transits to ears, which had the lowest at. % 15Ne at D65. Meanwhile, B. pumilus S1r1 displayed its superiority in BNF as indicated by the highest amount of fixed N found in the ear leaves instead of the plant stalk, where most fixed N of other PGPR strains was located at D65 harvest.During grain formation, the ear leaves functioned as supporting organs to continuously channel exogenous and remobilised N to ears . However15Ne in plant stalk remained mostly consistent throughout the plant growth, despite having the highest N uptake and dry biomass at D50 and D65 harvests. In addition to the significant increments of plant biomass and N uptake of stalk at D65, these phenomena demonstrated the role of plant stalk as an important N reservoir for maize [Amidst the fluctuations of N fluxes in the tassel, leaves  and ear of maize, the at. % or maize , 55. Accor maize , plant sor maize . In gene15N isotope dilution technique due to their lesser involvement in N fluxes, as they exhibited a low representation of plant biomass (<8%) compared to above ground plant parts [15Ne in the above and below ground plant parts are normally similar and either of these plant parts will give similar % Ndfa [B. pumilus S1r1 which fixed a significant amount of atmospheric N2, promoted vegetative growth and delayed plant senescence of maize, thereby produced a higher N content and yield of maize ear.Nonetheless, plant roots were not investigated in the present study using nt parts . Accordint parts , plant rr % Ndfa . This stBacillus pumilus S1r1, Klebsiella pneumoniae Fr1, Bacillus subtilis UPMB10 and Acinetobacter sp. S3r2 could significantly increase plant-N uptake, dry biomass and ear yield of maize. These increments are mainly attributed to the BNF ability of the strains, namely B. pumilus S1r1, which is able to fix up to 304 mg N2 fixed plant−1 at ear harvest (D65), and possibly other PGP abilities such as IAA production and phosphate solubilisation. The biomass production of plant tops and roots is correlated positively with IAA production by the PGPR strains. The maize plants inoculated with PGPR strains also exhibited delayed plant senescence, particularly in the tassel, which consequently improved the ear yield. This positively demonstrates that the remobilisation of N accumulated in maize top prior to anthesis from the leaves , tassel and stalk have served successively as N sinks and N sources toward the ear yield. Thus, this study indicates that PGPR inoculation can be considered as an alternative technique to improve grain yield besides the conventional plant breeding method for “delayed senescence” varieties, which is time consuming and tedious. Further studies are necessary to evaluate (i) the suitability and performance of B. pumilus S1r1 on maize under field conditions and (ii) the PGPR mechanisms involved in delaying plant senescence for higher N accumulation.This greenhouse study has demonstrated that inoculation with locally isolated PGPR strains, mainly S1 FigAsterisk,* on a bar indicates significant difference by Dunnett’s test (Uninoculated 1/3 N control) at p<0.05. Error bar indicates standard errors.(PDF)Click here for additional data file.S2 FigAsterisk,* on a bar indicates significant difference by Dunnett’s test (Uninoculated 1/3 N control) at p<0.05. Error bar indicates standard errors.(PDF)Click here for additional data file.S3 FigLanes: 1, Fr1 DNA; 2, S1r1 DNA; 3, S3r2 DNA; 4, Br1 DNA; 5, UPMB10 DNA; M, 1kb DNA ladder (Fermentas GeneRulerTM).(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.


Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in  Despite several million new systemic fungal infections annually worldwide, there are no commercial vaccines available. The development of effective vaccines requires a fundamental understanding of how protective immune responses are induced. Using experimental vaccine strains, we previously demonstrated that populations of T helper cells producing interleukin 17 (Th17 cells) and interferon gamma (Th1 cells) mediate vaccine resistance to systemic dimorphic fungi of North America. Here, we report how the immune system recognizes the fungal vaccines and induces the development of protective T cells. We delineate the role of pathogen recognition receptors (PRRs) and their common signaling pathway in host immune cells that recognize the fungal vaccine. While the signaling pathway studied is essential for the development of vaccine-induced T cells, the mechanism of action is novel and included T cell death after activation. The findings could be extended to virus-specific T cells suggesting that the mechanism is conserved among the microbial kingdom. Our work sheds new light on how protective T cells are induced and can be harnessed by vaccine strategies tailored against fungal and other microbial infections. Coccidioides posadasii [Histoplasma capsulatum [Blastomyces dermatitidis [+ T cells are the primary effector cells that control fungal infections in healthy hosts [The soaring rates of systemic fungal infections worldwide have spurred interest in developing vaccines ,4,5,6,7.osadasii , Histoplpsulatum  and Blasatitidis , which catitidis . CD4+ T hy hosts ,13 and Thy hosts . VaccineB. dermatitidis requires Dectin-2 recognition and signaling for the development of Th17 cells [H. capsulatum and C. posadasii require Dectin-1 and Dectin-2 for the induction of protective Th17 cell responses. Most TLRs (except for TLR3) and IL-1R family members trigger pathways via the adaptor protein myeloid differentiation primary-response gene 88 (MyD88) to activate NF-κB and mitogen-activated protein kinases (MAPK) [The development of effective vaccines requires a fundamental understanding of how protective immune responses are induced. We previously reported that the differentiation of Th17 cells and acquisition of vaccine immunity requires innate recognition and signaling through Card9 and MyD88 ,15. The s (MAPK) ,19.While TLRs and MyD88 have been implicated in the development of Th1 and Th2 cells ,21,22,23Toxoplasma gondii, LCMV and B. dermatitidis. In experimental T. gondii infection, T cell-intrinsic MyD88 is required for Th1 mediated resistance independent of IL-1R and IL-18R signaling, implying a role for TLRs [+ T cells require intrinsic MyD88 signals for differentiation and survival [B. dermatitidis infection [Both T cell-extrinsic and -intrinsic MyD88 signaling promotes adaptive immune responses. T cell-extrinsic signaling activates dendritic cells (DCs) and macrophages to produce pro-inflammatory cytokines and promote antigen presentation to initiate adaptive immunity during viral, bacterial and parasitic infections . Impairefor TLRs . During survival . Finallynfection .+ T cells during the contraction phase under non-inflammatory conditions. T cell-intrinsic MyD88 signals were largely dispensable for the development of anti-fungal CD4+ T cells. Moreover, TLR3, 7, and 9 served as the extrinsic upstream sensors and signaling receptors that initiate T cell survival signals under non-inflammatory conditions. Similar MyD88 requirements extrinsically governed the survival of virus-primed T cells, implying a general mechanism across microbial kingdoms.In the current study, we uncovered a novel mechanism by which MyD88 enables the development of vaccine-induced anti-fungal Th17 cells and resistance to infection. Instead of regulating the production of priming cytokines by APCs that shape T cell differentiation ,21,22,23Myd88-/- mice are highly susceptible to primary infections by B. dermatitidis, P. brasiliensis, A. fumigatus, C. neoformans, and C. albicans [Myd88-/- mice can acquire vaccine immunity against infection with the systemic dimorphic fungi B. dermatitidis and H. capsulatum. Myd88-/- mice were unable to control the live, attenuated #55 vaccine strain of B. dermatitidis and succumbed to dissemination and infiltration of the lungs by the yeast  mice that lack MyD88, as described in the methods. We adoptively transferred naïve Tg T-cells from these mice into wild-type recipients before subcutaneous vaccination. Thus, recipient host APCs are wild-type and transferred fungus-specific CD4+ T cells lack MyD88. As a positive control, we also transferred naïve wild-type 1807 into wild-type recipients before vaccination. At serial time intervals post-vaccination, we analyzed the behavior of the two transferred 1807 T cell populations. The number of activated (CD44+), Myd88-/- vs. Myd88+/+ 1807 cells was not reduced at day 7 post-vaccination (burst of T cell expansion)  , at day pansion)  and at dpansion) . Likewis reduced . Thus, M+ T cells. Here, we vaccinated Myd88∆T mice in which MyD88 expression is absent only in αβT cells [+ T cells upon recall. The numbers of activated (CD44+) and cytokine-producing T cells were similarly increased in vaccinated Myd88∆T and wild-type control mice, as compared to unvaccinated mice  and Th1 (IL-12p35) priming cytokines was comparable with bone marrow DCs from ent mice . Second,ent mice . Third, ype mice .+) T cells in Myd88-/- and wild type mice serially after vaccination. For this, we transferred 1807 cells into both recipients. We found that by 30 days post-vaccination the number of activated 1807 T cells was significantly reduced in the skin draining lymph nodes (sdLN) and in the lung upon recall  and enumerated in the lymph nodes and spleen. The number of transferred CD44+ CD4+ T cells was reduced in the lung, lymph nodes and spleen of Myd88-/- recipient vs. wild type recipient mice  1807 cells into ent mice . If MyD8Myd88-/- mice , purified CD4+ T cells from primary recipients and adoptively transferred them into mice that lack IL-1R; TLR2,3,4,7,9; TLR3,7,9; or TLR2,4 (as shown for MyD88 in +) 1807 T cells were sharply diminished in TLR2,3,4,7,9-/- and TLR3,7,9-/- mice, similar to vaccinated Myd88-/- mice -specific T cells in mice exposed to omycetes . We vacc-/- mice . Thus, tB. dermatitidis and C. posadasii. Vaccinated TLR2, 3, 4, 7, 9-/-, TLR3, 7, 9-/- and IL-1R-/- mice had increased lung CFU compared to vaccinated wild type controls and TLR2,4-/- mice  are the professional type I interferon producing DC subset, we sought to investigate whether IRF7 expression in pDC is compatible with the T cell survival phenotype in the knockout vs. wild type mice. Thus, we negatively enriched pDC from the spleen and analyzed IRF7 expression by real time RT-PCR. IRF7 expression in pDC was reduced in Myd88-/- vs. wild type mice but not in TLR3, 7, 9-/- and TLR2, 4-/- mice  cells [+ T cells, intrinsic MyD88 signals were indispensable for Tc17 cell responses, whereas Tc1 cells were less affected in the absence of these signals. MyD88 also has a T cell-intrinsic role for responses during LCMV, Vaccinia and Toxoplasma infections [+ Th17 and Th1 cells during pulmonary recall and resistance to fungal challenge.T cell intrinsic expression of MyD88 was largely dispensable for the generation of vaccine-induced Th17 and Th1 cells as assessed by the development of adoptively transferred 7) cells . In the fections ,43,44. IMyd88-/- and Myd88+/+ mice, we pinpointed the exact stage at which MyD88 regulates the development of Ag-specific T cells following vaccination. Extrinsic MyD88 regulated the contraction and was required for the survival of activated T cells during that phase, whereas it was dispensable for expansion, proliferation and differentiation of vaccine induced Th17 and Th1 cells. This conclusion is supported by the fact that absence of MyD88 in adoptive transfer recipients during the initial expansion and programming phase enabled the priming and differentiation of 1807 T cells, whereas absence during the maintenance phase led to accelerated contraction of 1807 cells. These results imply that extrinsic MyD88 provided a survival signal during non-inflammatory, resting conditions after adoptive transfer of effector cells into naïve recipient mice. The fact, that MyD88 regulates the survival of activated T cells under homeostatic conditions, in the absence of inflammation sets our findings apart from previously published studies in which LPS induced inflammation mediated by TLR4 signaling also promoted T cell survival [+ T cells [+ T cells for T cell activation, proliferation, and differentiation and do not require MyD88 for survival in our vaccine system [By using adoptive transfer of wild type 1807 T cells into survival . LPS-TLR T cells  that have system .Myd88-/-, TLR3, 7, 9-/- and wild type mice and the anti-apoptotic factors Bcl-2 and Bcl-xL by wild type 1807 T cells transferred into Myd88-/- and Myd88+/+ recipients over the course of the priming and contraction phase. Since enhanced T cell contraction does not occur when MyD88 is absent in CD11c+ cells, our data are compatible with the idea that non-myeloid stromal cells could play a role in MyD88 mediated survival of activated T cells.The mechanisms that guide transition from effector cells to memory cells are not well characterized. During the priming phase, T cell memory frequencies can be determined by the clonal burst size  and inflMyd88-/- vs. wild type recipients showed increased expression of active caspase 3, but not caspase 8. Since caspase 3 is an effector caspase that funnels signals from the extrinsic and intrinsic apoptosis pathways [-/- and Fas-/- mice with 1807 mice. Knocking out Fas or Bim in 1807 cells did not repair the exaggerated contraction in vaccinated Myd88-/- mice, indicating that MyD88 either does not mediate enhanced contraction through these two apoptotic pathways, or that the pathways are redundant.Since activated T cells underwent enhanced contraction in the absence of extrinsic MyD88, we investigated whether T cells showed enhanced apoptosis. We found that 1807 T cells from vaccinated pathways , we deleMyd88-/- mice. The engagement of TLR3, 7 and 9 distinguishes our findings from a previous report in which LPS induced TLR4 signaling promoted T cell survival under inflammatory conditions [T. gondii infection models [+ T cells with the dsRNA synthetic analog poly(I:C) and CpG DNA, respective ligands for TLR3 and TLR9, directly enhanced T cell survival without augmenting proliferation. Enhanced survival was associated with Bcl-xL up-regulation [Shigella dysenteriae type 1 upregulates TLR2 to activate mitogen-activated protein kinase (MAPK) and NF-κB to induce T cell expansion by promoting both proliferation and survival of CD4+ T cells. The proliferation, survival and effector function of CD4+ T cells through TLR2 co-stimulation show the ability of porin to directly call T cells into action [By using adoptive transfer of 1807 cells and enumerating endogenous calnexin-specific T cells in knockout recipients, we identified the receptors upstream of extrinsic MyD88 mediated T cell survival. In the absence of TLRs 3, 7 and 9, activated T cells underwent enhanced contraction similar to that in nditions . Impairen models ,29,56. Tgulation . Porin oo action .+ T cells, and that these signals likely originate with upstream TLR 3, 7 and 9, which likewise preserve the memory pool of T cells by stemming exaggerated contraction. Our findings may guide the rational design of vaccines against fungal and other microbial infections.Collectively, we show that MyD88 signals extrinsically regulate anti-fungal vaccine immune responses by preventing apoptosis of activated CD4-/- B6.129S7-Il1r1tm1Imx/J mice (stock # 003245) [a/Cy (stock #00406) mice carrying the Thy 1.1 allele were obtained from Jackson Laboratories, Bar Harbor, ME. Blastomyces-specific TCR Tg 1807 mice were generated in our lab and were backcrossed to congenic Thy1.1+ mice as described elsewhere [Myd88-/- x 1807 mice that carry the congenic marker Thy1.1 were generated by crossing Thy 1.1+ 1807 mice with Myd88-/- mice twice. TLR24-/-, TLR379-/- and TLR23479-/- [Myd88-/- [Myd88∆T mice in which MyD88 expression is selectively deleted in all αβT cells [+Myd88fl/fl [-Myd88fl/fl were housed in specific-pathogen free conditions at the University of California at San Francisco (UCSF).Inbred wild type C57BL/6, IL-1R1 003245)  and conglsewhere . Myd88-23479-/-  mice weryd88-/-  and Myd8βT cells  were a g88fl/fl  and CD11To enrich epitope-specific T cells in mice we generated an anti-Cnx-specific MHC class II tetramer  and used6 to 107 live or heat killed BAD1 null B. dermatitidis yeast strain #55 [7 live H. capsulatum strain G217B or 5 x 104 live attenuated vaccine strain (∆T) that lacks the chitinases 2,3 and D-arabinotol-2-dehydrogenase as described previously [3 or 2 x 104 wild-type yeast of B. dermatitidis strain 26199, 2 x 105H. capsulatum G217B, 2 x 105 FKS or 100 spores of the virulent C. posadasii isolate C735 [Mice were vaccinated as described  twice, train #55 , 107 liveviously . Mice weate C735 . To asseate C735 .+ T cells after vaccination we purified and transferred 106 CD4+ T cells from naïve 1807 Tg cells into C57BL/6 wild-type mice before vaccination. On the same day, recipients were vaccinated, and challenged four weeks post-vaccination.To assess the T helper cytokine phenotype of Calnexin-specific CD4+ wild type mice were infected with 105 pfu of LCMV Armstrong. 8 days later, CD4+ T cells were purified from the spleen and adoptively transferred into naïve Myd88-/-, IL-1R-/-, TLR23479-/-, TLR379-/- and wild type Thy1.2+ mice. After 4 weeks of rest the number of GP66-specific CD4+ T cells were assessed by tetramer that were generously provided by the NIH tetramer core facility.Congenic Thy1.17 heat-killed strain #55 yeast at a single dorsal site. On day 10 post-vaccination inflamed s.c. tissue was excised from the site of vaccination, placed in ice-cold collagenase buffer and minced into fine pieces. To digest the tissue, 5 ml of dissociation buffer (0.025 mg/ml Liberase (Roche Diagnostics) and 50 μg/ml DNAse I (Sigma-Aldrich) in collagenase buffer) was added and samples were incubated at 37°C and 5% CO2 for 30 minutes. To further release single cells, the tissue was mashed with the back of a 10-ml syringe plunger through a 70-μm cell strainer with an additional 5 ml of dissociation buffer and incubated for another 30 minutes. The dissolved tissue was washed with ice-cold PBS containing 5 mM EDTA and 1% BSA and strained again. Filtered cells were spun at 1500 rpm, the supernatant was carefully aspirated and cells were resuspended in complete media for stimulation.Mice were vaccinated once s.c. with 106 cells/ml) were stimulated for 4 to 6 hours with anti-CD3  and anti-CD28  in the presence of Golgi-Stop (BD Biosciences). Stimulation with fungal ligands yielded comparable cytokine production by transgenic T-cells compared to CD3/CD28 stimulation as shown previously [+ T cells per organ was calculated by multiplying the percent of cytokine-producing cells by the number of CD4+ cells. Intracellular Caspase 3 Alexa647 , Caspase 8 PE , Bcl2 Alexa 647 (633510 Biolegend) and Bcl-xL PE  were stained from ex vivo derived lymph node cells and splenocytes.Lung cells were harvested at day 4 post-infection. Cells  when data were normally distributed [P value < 0.05 is considered statistically significant.Differences in the number and percentage of activated, proliferating or cytokine-producing T cells were analyzed using the Wilcoxon rank test for nonparametric data or the tributed . A P valThe studies performed were governed by protocols M00969 and AN101733 as approved by the IACUC committees of the University of Wisconsin-Madison Medical School and University of California at San Francisco (UCSF), respectively. Animal studies were compliant with all applicable provisions established by the Animal Welfare Act and the Public Health Services (PHS) Policy on the Humane Care and Use of Laboratory Animals.S1 Fig(A) Purified CD4+ T cells from naïve Bim-/-, Fas-/- and wild type 1807 mice were transferred into Myd88-/- and wild type recipient mice prior to vaccination. (B) IL-7 and IL-15 transcripts harvested from the skin draining lymph nodes and spleen of naïve Myd88-/-, TLR3, 7, 9-/- and TLR2, 3, 4, 7, 9-/- mice vs. wild type controls. (C) Relative expression of IFN-α, IFN-β, IFNAR1, IFNAR2, IRF7 and IRF8 of Myd88-/- mice vs. wild type mice. (D) Relative IRF7 expression in splenocytes from Myd88-/-, TLR2,3,4,7,9-/-, TLR2,4-/- and TLR3,7,9-/- vs. wild type mice. (E) IRF7 expression in negatively enriched pDC from Myd88-/-, TLR2,4-/- and TLR3,7,9-/- vs. wild type mice. * P < 0.05 vs. corresponding wild type recipient mice. (F) CD11c+ cells do not mediate MyD88 regulated T cell contraction. Effector 1807 T cells were primed in wild type recipient mice vaccinated with heat inactivated B. dermatitidis vaccine yeast as outlined in +-Myd88fl/fl and CD11cCre—Myd88fl/fl mice and rested for four weeks. The numbers of Thy1.1+ 1807 T cells from the skin draining lymph nodes and the spleen were enumerated by FACS. Data are the mean ± SEM (n = 5–7 mice/group). Data are the average of two independent experiments.(TIF)Click here for additional data file.S2 Fig+ T cells from naïve 1807 mice were transferred into Myd88-/-, IL-1R-/-, TLR2, 3, 4, 7, 9-/-, TLR3, 7, 9-/- and TLR2, 4-/- and wild type mice prior to vaccination. After vaccination and challenge (at day 4 post-infection), activated and cytokine-producing T cells in the lung were enumerated by FACS. (A) Data are expressed as the mean ± SD of 4–6 mice/group. Data are from single experiments representative of three independent experiments. * P < 0.05 vs. wild type control mice. (B) The dot plots show the sum of concatenated events from 4–6 mice/group and the values indicate the mean number of 1807 CD4+ T cells. Data are expressed as the mean ± SD of 4–6 mice/group from a single experiment representative of three independent experiments. * P < 0.05 vs. wild type control mice. (C) The frequencies of cytokine producing 1807 T cells in IL-1R-/- and wild type mice that were vaccinated or not from P < 0.05 vs. wild type control mice. (D) Resistance to C. posadasii infection. TLR2, 3, 4, 7, 9-/-, TLR3, 7, 9-/- and wild type mice were vaccinated with 5 x 104 live attenuated (∆T) strain or not. Seven weeks later, mice were challenged with 102 spores of C. posadasii strain C735 and the number of CFU determined at two weeks post-infection.Purified CD4(TIF)Click here for additional data file.


The ongoing avian H7N9 influenza outbreaks in China have caused significant human fatal cases and the virus is becoming established in poultry. Mutations with potential to increase mammalian adaptation have occurred in the polymerase basic protein 2 (PB2) and other viral genes. Here we found that dual 627K and 701N mutations could readily occur during transmission of the virus among ferrets via direct physical contact, and these mutations conferred higher polymerase activity and improved viral replication in mammalian cells, and enhanced virulence in mice. Special attention needs to be paid to patients with such mutations, as these may serve as an indicator of higher virus replication and increased pathogenicity. Since the initial outbreak in Eastern China in 20133With the development of the H7N9 virus in the field, some amino acid mutations have occurred which are believed to be associated with host preference and specificity in mammalian species3567910Here we used a ferret transmission model and deep-sequencing of the viruses shed by ferrets experimentally exposed to the prototype H7N9 virus, A/Anhui/1/2013 6 median tissue culture infective dose (TCID50) of the wild type AH1 or SH5 viruses shed viruses through the nasal cavity for 6 days post-inoculation (dpi) with peak titers, which occurred at 2 dpi, ranged from 6.0 ∼ 6.3 log TCID50/ml for AH1 and from 5.3 ∼ 6.3 log TCID50/ml for SH5 (50/ml) were maintained in SH5-PC3 till 8 dpe , except for the mutant with PB2-627E+701D where the level of activity was not affected by temperature (p < 0.01).To investigate whether mutations at positions 627 and 701 in the PB2 protein could alter the activity of the RNA-dependent RNA polymerase, the expression levels of a luciferase reporter gene in minigeome-based assays in 293T cells were tested. A ribonucleoprotein (RNP) complex containing the PB2 627E and 701D (ED) residues consistently showed the lowest level of activity. The activity level increased from the PB2-627E+701N (EN) mutant to the wild type PB2-627K+701D (KD), with the mutant with 627K and 701N (KN) having the highest level of activity . At all perature . For mutp < 0.05), with the average peak replication discrepancy occurring at 48 hpi , with rgAH1-KN showing marginally higher replication at 24–36 hpi  of 0.001, and supernatant was sampled at various time points. At 33 °C rgAH1-KN exhibited significantly higher growth properties when compared to rgAH1-EN and the wild type rgAH1-KD virus at 36–72 hours post-inoculation  and rgAH1-KN viruses were ≥106.3, 104.5, and 103.3 TCID50  were inoculated into groups of five mice. At inoculation doses of 10er cases . At a doted mice  and 4a. talities  and 4b-d3 TCID50 , while tectively . Overallectively  and 4.50 than at 3 log TCID50  in C57BL/6 mice was detected in both the upper and lower respiratory tracts  and occasionally in extrapulmonary organs . Virus rg TCID50  and 6. Tat 1 dpi  and 6.Influenza A viruses can readily acquire mutations through virus replication in host cells, owing to the low fidelity of their RNA dependent polymerase13134th December 2014, all available H7N9 virus sequences from the poultry and environmental samples were found to exclusively carry PB2-627E while the majority  of the human isolates had a lysine mutation at this position (E627K), and a tree sparrow virus identified in Shanghai possessed both E and KAs of 4In this study, by using a ferret transmission model, we demonstrated that both the E627K and D701N mutations could occur within one passage in physical contact ferrets, and these two mutations could co-emerge in the same virus particle. The dual 627K and 701N mutations in the PB2 protein of AH1 viruses was associated with increased polymerase activity, better replication in human cells and enhanced virulence in mice. However, as the E627K and D701N mutations were not found in airborne exposed ferrets, this suggests they may not increase the airborne transmissibility of the viruses.Structural studies of the C-terminal domain of PB2 show that residue 701 is located in the nuclear localization signal (NLS) and importin binding domain, and it might disrupt the salt bridge between 701D and 753R, thereby facilitating the unfolding of the NLS and its exposure to and binding of the importin molecules16171816171719The avian H7N9 influenza virus that emerged in 2013 has caused three outbreak waves in humans but relatively few viral sequences are available, given the large numbers of affected birds and humans. This paucity of information on the possible adaptations of H7N9 viruses within infected birds or patients makes the prediction of clinical outcomes difficult. Our study, together with previous reports910All experiments using mice and ferrets were conducted in compliance with approvals from the Committee on the Use of Live Animals in Teaching and Research (CULATR) of The University of Hong Kong (HKU CULATR 3264-14) and the Institutional Ethical Review Board (IERB) of Shantou University Medical College (Ref. No. SUMC2013-111 and SUMC2015-017). The methods were carried out in accordance with the approved guidelines of the International Council for Laboratory Animal Science and the University Policies “Animal Ethics and Welfare” and “Use of Animals in Research”.All manipulations involving H7N9 viruses were performed in biosafety level 3 (BSL3) containment facilities by trained staff with all necessary personal protective equipment, following the institutional Guidelines on Safety Policy & Management. Staff allowed to handle viruses and animals in the BSL-3 facilities must have completed BSL-3 training and got a license to conduct animal experiments. Active surveillance of body temperature and reporting of symptoms of influenza-like illness were required for researchers involved in this project. Individually ventilated cages and powered air purifying respirators were used for the animal experiments. Chemical and biosafety approvals were obtained from the Li Ka Shing Faculty of Medicine, HKU. The Faculty’s safety policy, management and practice are subject to constant review by the Faculty Safety Committee and its Safety Officers. Matters of dual use research are considered under the ethical approvals and safety approval procedures.2 at 37 °C. Two human H7N9 virus isolates50) on MDCK cells.Human embryonic kidney 293T and human type II alveolar epithelial A549 cells ATCC CCL-185) were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , glutamine , penicillin (100 units/ml), and streptomycin . Madin-Darby canine kidney  cells were maintained in minimum essential medium , supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin . All cells were incubated in a humidified atmosphere of 5% CO85 were oPfuUltra® II Fusion HS DNA Polymerase (Stratagene) and gene-specific primers as described21Renilla luciferase (Rluc). Reporter plasmid pFluc was constructed by cloning the firefly luciferase (Fluc) gene from the pGL3 vector (Promega) into the RNA polymerase I promoter/terminator cassette. The untranslated regions (UTRs) of the influenza A/WSN/33 NP segment was introduced into the flanking regions of the Fluc gene to produce artificial influenza NP-like RNA segment. All plasmids were confirmed to have the exact sequences as designated.Viral RNAs of the A/Anhui/1/2013 (H7N9) influenza virus were extracted and reverse transcribed with the Uni-12 primer50 was calculated using the Karber method910in vivo mouse experiment.Recombinant viruses rgAH1-KD (with wild type PB2-627K and 701D), rgAH1-KN (rgAH1-PB2-627K and 701N), rgAH1-ED (rgAH1-PB2-627E and 701D) and rgAH1-EN (rgAH1-PB2-627E and 701N) were generated by co-transfection of the eight reverse-genetically reconstructed plasmids, each carrying a viral gene segment, into 293T/MDCK co-cultured monolayers50 values were determined on MDCK cells.A549 cells were inoculated with each rescued virus at an M.O.I. of 0.001, and incubated at 33 °C or 37 °C. At time points 0, 12, 24, 36, 48, 60, 72, 84 and 96 hpi, supernatants were collected and TCIDEqual amounts of the reporter plasmid pFluc and internal control plasmid pRluc (0.5 μg each) were co-transfected with expression plasmids encoding PB2, polymerase basic protein 1 (PB1), polymerase acidic protein (PA), and nucleoprotein (NP) genes into 293T cells using the PolyFect (Qiagen) reagent according to the manufacturer’s instructions. Mock transfections were performed with pFluc and pRluc only. Transfected cells were incubated at 33, 35, 37 or 39 °C, respectively. After 24 hours of incubation, supernatants were discarded and the cell extracts were prepared in 120 μl of lysis buffer. Light units were measured using Dual-luciferase Assay Kits (Promega) on a GloMax® 96 Microplate Luminometer (Promega). All results shown are the averages from triplicate experiments with standard deviations (SD).Mustela putorius furo, Wuxi Sangosho Co. Ltd.), held in separate cages, were intranasally inoculated with 106 TCID50 of AH1 or SH5 viruses in 500 μl of MEM. At 24 hpi, three naïve ferrets  were introduced to each of the three main cages with the directly inoculated ferrets. Another three were placed in adjacent cages at a 10 cm distance from the main cage, to serve as airborne exposed animals50 assays. When viruses shed by the contact ferrets  reached their peak titers, RNAs from the nasal swabs were extracted and subjected to deep sequencing on a Mi-Seq desktop sequencer (Illumina), giving a coverage of 6,000 ∼ 25,000×. The PB2 gene sequences at the 627 and 701 positions were analyzed for their genetic heterogeneity. At 14 dpi, sera were collected from each animal for detection of seroconversion and antibody titers by hemagglutination inhibition (HI) tests.Three six-month-old influenza-free male ferrets (1 ∼ 106 TCID50) in a 50 μl volume, or mock inoculated with 50 μl PBS. Body weights and clinical signs of infections were recorded daily. Mice that lost more than 30% of their original weight were euthanized for humane reason and recorded as a fatal infection. At 14 dpi, blood was collected from each of the surviving mice, and serum was separated for determination of antibody titers using HI tests. Mouse median lethal dose (MLD50) and 50% infectious dose (MID50) were determined using the Karber formula3 or 105 TCID50 of the indicated viruses and three mice in each group were euthanized at 1, 3, 5, and 7 dpi. Brain, nasal turbinate, trachea, lung, heart, spleen, kidney, liver, intestinal tract, eyeball, conjunctiva and spinal cord were collected and virus titers were determined by TCID50 assays.Groups of five 8- to 10-week-old specific-pathogen-free (SPF) female C57BL/6 mice  were anesthetized with 0.2 ml of 0.75% pentobarbital sodium and inoculated intranasally with recombinant viruses at the indicated dose  staining and immunohistochemical staining of NP antigens in the lung tissues were performed by using a mouse anti-NP monoclonal antibody, kindly provided by Professor Ningshao Xia, the National Institute of Diagnostics and Vaccine Development for Infectious Diseases, Xiamen University, and a goat anti-mouse IgG–biotin conjugated secondary antibody t test. A p-value < 0.05 was considered statistically significant.Differences between experimental groups were evaluated using the Student’s How to cite this article: Zhu, W. et al. Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models. Sci. Rep.5, 14170; doi: 10.1038/srep14170 (2015).


PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers,  These cells have the capacity to differentiate into neurons as well as glia cells , 2. NPCs targets , 4. Compin vitro –8. in vitro assays to analyze the properties of NPCs [NPCs are commonly isolated from different regions of murine and human brain by microdissection and subsequently cultured as free-floating aggregates using both classic and stirred suspension 3D culture system methods , 10. It  of NPCs . This sp of NPCs . in vitro it is crucial that the culture methods display the desired regional and subtype specificity compared to the affected neurons of the patient. Consequently, disease modeling in 3D tissue culture systems has recently been successfully applied in Alzheimer's disease [A common approach of human NPCs derivation from iPSC involves neural induction by inhibition of SMAD signaling by means of two inhibitors (SB431542 and Noggin or LDN193189), followed by expansion of NPCs and subsequent terminal differentiation into neurons using the 2D culture system , 14. Yet disease , 17 and The human brain is made up of various subtypes of neurons but also by a substantial amount of glia cells (more than 50%) . One sub in vitro conditions, is the extended time frame (42–84 days) for achieving functional neuronal maturation [Another aspect of neuronal differentiation, which may be a challenge underturation , 33. Thituration , 35. Oneturation , 37. Traturation . Consequ PAX6 and NESTIN, which are NPC markers. Furthermore, we directly established a link between the expression of BLBP, PAX6, and astrocyte differentiation efficiency [ GFAP and AQP4 expression.In the present study, we describe a potentially efficient 3D method of astrocyte enrichment from human iPSC-derived NPCs. The method progresses through an initial phase of NPC formation with increasing expression officiency . Our metμM SB431542  and 100 ng/mL Noggin . NPCs were expanded in neural maintenance medium and maintained on plates coated with poly-L-ornithine (Sigma-Aldrich) and laminin  (POL/L). Terminal differentiation on POL/L coated plates was initiated using neural differentiation medium as described in the Media. Total time of terminal differentiation was 21 days.The three human iPSC lines used in this study were generated by using the Sendai virus (CytoTune-iPS 2.0 Sendai Reprogramming Kit) . Three cell lines were investigated in this study: clone N S5 and N S8 derived from a healthy 34-year-old male donor and clone RT S11 derived from a 33-year-old healthy female donor. NPCs cell lines were induced from each of these human iPSCs by dual inhibition of SMAD signaling with 10 6 cells/mL were plated on the low attachment dish  in neural maintenance medium and maintained for 7 days as neurosphere cultures. Neurospheres were visualized in an inverted microscope under phase contrast mode. Three representative pictures of neurospheres were taken from 3 independent cultures with an inverted microscope under phase contrast mode . Subsequently, the neurospheres were dissociated into single cells using Accutase and next seeded on plates coated with POL/L. Finally, the cells were differentiated for 21 days in neural differentiation medium.Monolayer NPCs from all three lines  were dissociated into single cells with Accutase (Sigma-Aldrich). A total of 1 × 10NMM consisted of a 50/50 mixture of DMEM/F-12 with GlutaMAX (Life Technologies) and neurobasal medium (Life Technologies). This basic media was supplemented with N2 (Life Technologies), B27 (Life Technologies), 2 mM L-Glutamine (Sigma-Aldrich), 10 ng/mL basic fibroblast growth factor (bFGF) (Life Technologies), 10 ng/mL epidermal growth factor (EGF) (Life Technologies), 1x nonessential amino acid (Sigma-Aldrich), and 50 U/mL penicillin/streptomycin (Life Technologies).μM 2-mercaptoethanol (Life Technologies), and 50 U/mL penicillin/streptomycin.NDM was composed of a 50/50 mixture of DMEM/F-12 with GlutaMAX and neurobasal medium. This basic media was supplemented with N2, B27, 2 mM L-Glutamine, 1x nonessential amino acid, 0.2 mM ascorbic acid (Sigma-Aldrich), 25  Alexa Fluor 647 mouse anti-NESTIN and PE mouse anti-human Pax6 antibodies . Flow cytometry analysis was performed using a “Flow Cytometer Cytomics FC 500” . A red solid state laser 635 nm and an argon laser 488 nm were used to detect NESTIN and PAX6 expression in NPCs. Subsequently, we set the specific gate to calculate the proportion of low PAX6 expressing NPCs from 2D and 3D culturing system. Data was analyzed using FlowJo software (version 7.6.5) from 3 independent cultures.In order to perform flow cytometry analysis, NPCs harvested from 2D monolayer and 3D neurosphere were dissociated into single-cell suspension with Accutase. Dissociated cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and subsequently permeabilized with 0,2% Triton X-100 for 20 minutes. Cells were stained for 1 hour at RT withμg/mL diluted in PBS to detect nuclei counterstaining. Unspecific binding of the secondary antibodies was excluded by using only secondary antibody controls. Cells were analyzed under fluorescent microscope equipped with 3D imaging module  and controlled by AxioVision 4.8.1 Microscope software . The number of proliferative cells (KI67+) was quantified. The numbers of neurons (TUBB3+) and astrocytes (GFAP+ and AQP4+) were quantified. Five pictures per coverslip were acquired from 3 independent cultures (20x magnification). On average, approximately 300 cells were counted for each picture.To analyze neural markers cells were fixed in 4% PFA for 20 minutes at RT, washed in phosphate buffered saline (PBS), and permeabilized with 0.2% Triton X-100 diluted in PBS for 20 minutes. Afterwards, cells were blocked in 3% BSA + 0.2% Triton X-100 in PBS for 1 hour at RT. NPCs were incubated overnight at 4°C with the following primary antibodies: rabbit anti-PAX6 , mouse anti-NESTIN , rabbit anti-Musashi-1 , mouse anti-SOX2 , and mouse anti-KI67 . Neurospheres were incubated overnight at 4°C with rabbit anti-PAX6  and mouse anti-NESTIN . Differentiated cells were incubated overnight at 4°C with mouse anti-MAP2 , rabbit anti-beta-III tubulin , mouse anti-beta-III tubulin , rabbit anti-AQP4 , and rabbit anti-GFAP . The primary antibodies were detected using the following fluorescence labeled secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG (H + L), Alexa Fluor 594 donkey anti-mouse IgG (H + L), Alexa Fluor 488 donkey anti-mouse IgG (H + L), and Alexa Fluor 594 donkey anti-rabbit IgG (H + L) . Finally, the cells were incubated for 20 min at RT with 4′,6-diamidino-2-phenylindole (DAPI) at a final concentration of 0.2  GAPDH was used for data normalization. Samples were collected from 3 independent cultures. Data was analyzed based on 2−ΔΔCt method [Total RNA was isolated from cultured cells using the RNeasyPlus Mini kit . Two micrograms of total RNA was transcribed to cDNA using the SuperScript VILO cDNA Synthesis kit (Life Technologies) in accordance with the manufacturer's instructions. QPCR reactions were performed using LightCycler 480 SYBR Green I (Roche) on a LightCycler 480 real-time PCR machine (Roche). In this study, the house-keeping genet method . All use post hoc multiple comparison test was applied to compare the neurosphere diameter across all studied cell lines at days 4 and 7. Distribution of relative abundance of mRNA from the different genes was examined by using D'Agostino and Pearson omnibus normality test. Student's t-test was performed to compare the difference between groups. Pearson's correlation tests were performed to establish the correlation between the following: (1) the relative mRNA abundance of PAX6 in 3D neurosphere NPCs and neurosphere diameter at day 7; (2) the relative mRNA abundance of BLBP and PAX6 in NPCs and GFAP in differentiated cells over three studied cell lines. Distribution of proportion of KI67+, low PAX6 expression, TUBB3+, AQP4+, and GFAP+ cells was tested using Kolmogorov-Smirnov normality test. One-way ANOVA with a Tukey post hoc multiple comparison test and Student's t-test was performed to compare the difference between groups. A p value below 0.05 was considered significant. Results are reported as the Mean ± SEM (standard error of the mean). Data was analyzed using OriginPro Statistical software (version 9).Distribution of neurosphere diameter was tested using Kolmogorov-Smirnov normality test. A one-way analysis of variance (ANOVA) with a Tukey in vitro, we set up a direct comparison of 2D and 3D NPC expansion methods (In order to investigate whether 3D culturing methods can enhance astrocyte differentiation from NPCs methods .μm) was larger than N S5 (74.26 ± 5.45 μm) and RT S11 (65.05 ± 6.18 μm) at day 4. Even though we observed significant differences amongst cell lines, all of them were proliferative as neurospheres and increased in diameter during culturing. At day 7 the diameter of neurospheres was significantly increased in all lines  compared to day 4. One cell line (N S8) showed a significant larger diameter at day 7 compared to N S5 and RT S11 (p < 0.01). All lines analyzed  expressed NESTIN and PAX6 (p < 0.01). This result demonstrates that NPCs maintain the capacity to proliferate in 2D and 3D culturing system. Furthermore, 3D suspension culture induces the formation of neurospheres with the expression of NPC markers.We assessed the morphology of day 4 and 7 neurospheres by phase contrast imaging and their expression of early neural lineage markers (PAX6 and NESTIN) by immunocytochemistry. Neurosphere size was evaluated at days 4 and 7. We observed varying sizes of neurospheres within the different cell lines analyzed . As showand PAX6 , confirmICC was performed to examine the expression level of NPC markers  in 2D and 3D NPCs. Both were dissociated and plated as single cells on day 7. As expected, 2D and 3D NPCs were positive for NESTIN and PAX6 expression . FurtherSOX2, PAX6, and NESTIN). The abundance of PAX6 and NESTIN was significantly increased (p < 0.01) in 3D cultured NPCs in comparison with 2D cultured NPCs in all investigated cell lines  correlation with mRNA abundance of PAX6 in 3D cultured NPCs  is increased.Flow cytometry was implemented to study the expression level of PAX6 and NESTIN in 2D and 3D NPCs. The intensity of the PAX6 signal in the FACS analyses revealed two apparently divergent populations in 2D NPCs whilst 3D NPCs showed only one distinct population. The appearance of two individual populations was most prominent with the 2D NS8 cells . Subsequll lines . Howeverred NPCs . This inp < 0.01) ( GFAP compared to 2D derived cells (p < 0.01). Furthermore, qPCR was performed to verify the ICC quantification results. Significantly higher expression of AQP4 was observed in cells differentiated from 3D culturing method (p < 0.01). Together these data indicate that neurosphere aggregation could promote astrocyte differentiation. Similar expression patterns of MAP2 and TUBB3 and the population of TUBB3 positive neurons were detected in neurons derived from NPCs using both culture systems  to the expression of astrocyte marker (GFAP) in the astrocytes derived from all studied NPCs lines. Therefore, BLBP and PAX6 expression during NPC stages can be used as an indicator for potential differentiation into the astrocyte lineage as verified by GFAP expression. Furthermore, we performed qPCR analysis to prove that the increase of PAX6 expression and the promotion of astrocyte differentiation were due to the loss of cells with low PAX6 expression during neurosphere formation. These low PAX6 expressing cells are considered early IP cells. Our qPCR results demonstrated a significant decrease of expression of the IP cell marker eomesodermin (TBR2) in cells differentiated from 3D cultured NPCs compared to 2D cultured NPCs (p < 0.01). Herein it can be concluded that the 3D culture method might promote the astrocytes differentiation and provoke an inhibition of the IP cells generation.The 2D and 3D NPCs were dissociated and differentiated on POL/L plates for 21 days. ICC analysis showed that the majority of cells derived from NPCs cultured in the two different systems were positive for neuronal markers , and microtubule-associated protein 2 (MAP2); Figures  < 0.01) . We alsoed cells . Subsequg method   and neurospheres (3D). Human NPCs derived from iPSCs have traditionally been maintained and proliferated in 2D culture system, demonstrating an efficient differentiation into the neuronal lineage , 14. Howo models . Thus, ncreening , 15, 17.creening , 48. We  PAX6 and NESTIN expression levels were significantly higher in 3D NPCs. This indicates that neurosphere aggregation promotes PAX6 and NESTIN expression. Furthermore, a more homogenous PAX6 expression was detected in 3D NPC populations suggesting that an uneven distributed PAX6 expression pattern in 2D NPCs was avoided by neurospheres aggregation. PAX6 is an established NPC marker expressed in RG cells which plays an essential role in maintaining the NPC population, neuronal and glial differentiation [ PAX6 induces production of larger neurospheres [ PAX6 in 3D NPCs was positively correlated to subsequent neurosphere diameters at day 7.In the present study, we further compared the NPC identity and differentiation ability of NPCs cultured in 2D and 3D culturing systems. We chose PAX6, NESTIN, SOX2, and Musashi-1 (NPC markers) to evaluate the neural identity of our 2D and 3D NPCs. PAX6 is uniformly expressed in early neural ectoderm cells of human fetuses and in NPCs differentiated from human ESCs . Pan-neuntiation . Previountiation . As a coospheres . Consist in vitro has been described earlier [ GFAP and AQP4 expression compared to the 2D condition. Hence, we draw a conclusion that 3D NPCs culturing methods stimulate astrocyte differentiation. This observation is in agreement with previously described findings, which show efficient 3D differentiation of ESC-derived NPCs to glial cells of the peripheral nervous system, in particular Schwann cells [ GFAP expression in astrocytes was significantly positively correlated to the astrocyte progenitor marker BLBP expression in NPCs, which is in accordance with observations of other groups [ GFAP expression, resulting in inhibition of astrocyte maturation [ GFAP expression in astrocytes was strongly and positively associated with PAX6 expression pattern during the NPC stage. These correlation studies underline the importance of BLBP and PAX6 expression in NPCs for differentiation towards an astrocyte fate.Herein, we demonstrated that human iPSC-derived NPCs cultured under 2D and 3D conditions differentiated into cells positive for mature neuronal markers (MAP2 and TUBB3) and the astrocyte markers (GFAP and AQP4). The overall process of directing differentiation of human iPSCs-derived NPCs to neurons and astrocytes earlier . As a nonn cells . Moreover groups , 26, 27.turation , 60. Sim TBR2 in the differentiated cells derived from 3D NPCs when compared to 2D NPCs. This indicated that neurosphere formation is associated with the loss of early IP cells (low PAX6 expression cells), resulting in the decrease of TBR2 expression in differentiated cells. During neurogenesis, IP cells asymmetrically divide into neurons and IP daughter cells [Single-cell mRNA profiling indicates that neurospheres are composed of RG cells (high PAX6 expression) other than early IP cells or IP cells (low PAX6 expression) . Similarer cells . Moreoveer cells , 25. HowPAX6 and NESTIN). Subsequently, GFAP and AQP4 expression is increased during terminal astrocyte differentiation in the cells differentiated from 3D cultured NPCs. These results revealed an attractive method to improve the astrocyte differentiation ability from iPSC-derived NPCs in a shorter time frame and with higher efficiency compared to derivations from 2D cultured NPCs.In summary, we established a reliable NPC culture system in 3D that efficiently enriches GFAP and AQP4 positive astrocytes during terminal differentiation. Propagation of NPCs using the 3D culture system promotes the expression of RG cell markers (


Three compounds were identified as potential candidates for further development. The listed compounds were also assayed for their antimalarial efficacy against chloroquine (CQ) sensitive strain (3D7) of Plasmodium falciparum. Assay of twenty seven hydroxyethylamine derivatives revealed four  as strongly active, which were further evaluated against CQ resistant strain (7GB) of P. falciparum. Compound 5e possessing the piperidinopiperidine moiety exhibited promising antimalarial activity with an IC50 of 1.16 ± 0.04 μM. Further, compounds 5e, 6j, 6o and 6s exhibited low cytotoxic effect on MCF-7 cell line. Compound 6s possessing C2 symmetry was identified as the least cytotoxic with significant antimalarial activity (IC50: 1.30 ± 0.03 μM). The combined presence of hydroxyethylamine and cyclic amines (piperazines and piperidines) was observed as crucial for the activity. The current studies suggest that hydroxyethylamine based molecules act as potent antimalarial agent and may be helpful in drug development.A novel class of phthalimides functionalized with privileged scaffolds was designed, synthesized and evaluated as potential inhibitors of plasmepsin 2 (K Plasmodium falciparum alone owns the majority of lethal cases worldwide. The malarial problems in the endemic regions have reached a critical stage owing to the arrival of extensive resistance to the current antimalarial drugs and insecticide-resistant mosquitoes. The increasing resistance of P. falciparum to the available drugs, e.g., chloroquine (CQ)  sample/ [abs] control × 100.Phthalimides were serially diluted in DMSO in a final concentration of 100, 25 and 6.25 μg mLell line . Cell liIn vitro hemolytic activity of all synthesized compounds was performed as per reported protocol . Th. ThC2 sds 5a-f,  were deprocedure . N-phthag points . N-phthapoxide R,S phenylaterature .P. falciparum has proven to be an excellent enzyme system for testing the inhibitory potential of new synthetic compounds. It can be prepared by recombinant methods as the inactive zymogen, proplasmepsin 2, and it converts smoothly to the mature and catalytically active enzyme, plasmepsin 2, by simple incubation at pH 4.5. Similarly, plasmepsin 4 from P. falciparum can be prepared and activated. Catalytic activity of the plasmepsins can be studied by following the change in absorbance at 300 nm as cleavage occur between a Phe and a nitroPhe in the synthetic substrate, Lys-Pro-Ile-Glu-Phe-NO2Phe-Arg-Leu [i, for the compounds and these values are reported in 6u and 6r have Ki values in the micromolar to submicromolar range. For compounds 6p and 6t tested against plasmepsin 4, Ki values were higher, in the micromolar range. These values are adequate to direct further drug optimization studies.Plasmepsins, a family of aspartic proteases of malaria parasite, are known to participate in a wide variety of cellular processes essential for parasite survival. Plasmepsin 2 from -Arg-Leu . The com6r has a Leu-like side chain at the R position, which is expected to be the P2 position in the Schechter and Berger nomenclature [6u has an Ile-like side chain in the same position. Compounds 6n and 6s, with a benzyl-like side chain, apparently do not fit well into the pocket where the R group would fit, which would be the S2 pocket in the enzyme active site. The benzyl group next to the–OH would fit into the S1 pocket of the active site of enzyme. Furthermore, the compounds 8a-8u have the phthaloyl group on the N-terminal side of the structure and this should be accommodated in the S3 site of the plasmepsins. The phthaloyl group is much larger than the t-butyl group present on 5a-5f and this can account for why the compounds 5a-5f does not show inhibition when they are tested against plasmepsin 2 and plasmepsin 4.Derivative nclature  when bin6p in sec-butyl group in the R position  and a 4-bromobenzyl group in the R1 position. This pairing produces a Ki value of 27.4 μM for plasmepsin 4. Apparently, the Ile residue in the P2 position is of optimal size, as smaller or larger substituents do not work as well and hydrophobicity is required to interact with the S2 pocket of the enzyme. Consideration of the substitution in the R1 position shows that the bromobenzyl group is optimal; the fluorobenzyl group does not provide as strong interaction as bromobenzyl.Compound 6t may be compared to the other compounds in i value of 3.3 μM. These results provide clues to the structure activity relationship for new inhibitors of the plasmepsins.Compound 5a-6u were screened for their ability to inhibit the growth of parasite activity, which was determined by the standardized SYBR green based assay [50 values for the inhibition of parasite growth by compounds (5a-6u) are listed in 6q (IC50: 8.54 ± 0.04 μM). It was observed that four of the compounds, 5e (IC50: 1.16 ± 0.04 μM), 6j (IC50: 1.33 ± 0.04 μM), 6o (IC50: 1.25 ± 0.08 μM), and 6s (IC50: 1.30 ± 0.03 μM) inhibited the parasite growth in a concentration-dependent manner and showed the maximum inhibition as compared with other compounds. Further, these most active members were also evaluated for their antiplasmodial activity against CQ resistant strain (7GB) of P. falciparum and a significant inhibition of the parasite growth was observed.We have studied the antimalarial effect of all the listed molecules in asynchronized culture conditions in order to identify a putative lead structure for pharmacological applications. Compounds, ed assay . The IC55e on the parasite developmental stages , we treated tightly synchronized ring-stage parasite cultures with 5e at 5 μM concentration and counted the parasites at different developmental stages  in Giemsa-stained smears at three time points . The control (untreated) parasite progressed through the first cell cycle (0–48 h), increased in size and density, developed into trophozoites and schizonts and subsequently formed viable merozoites. These merozoites were able to invade fresh RBCs and form new ring-stage parasites.  showed low cytotoxic effect. Compound 5f showed maximal toxicity among all tested compounds followed by compound 5a, 5c, 6k, 6m and 6q with IC50 values above 8 μM. The compounds (5e and 6s) were found to be less toxic. Compound, 6s possessing C2 symmetry was identified as least cytotoxic as compared to other molecules.Assaying all new compounds for cytotoxic effect, 13 compounds were found to display low cytotoxic effects on MCF7 cell line. Interestingly, four potent molecules with significant antimalarial activity  and their binding sites were analyzed using CASTp, Q-Site Finder and also verified with literature survey. The top predicted sites were taken for grid formation in docking studies. The structures of compounds  were prepared and all the possible tautomers were generated. These compounds were found to follow Lipinski's rule of 5 and show no reactive functional groups according to the ADME calculations  followed by the estimation of binding free energy estimates  were identified as top hits for plasmepsin 2 and 6r, 6u, 6t for plasmepsin 4  or aromatic (6o and 6j), the potency is increased. Compounds with smaller aliphatic groups viz., methyl and ethyl directly attached to fourth position of piperazine are less effective against parasitemia suppression. At the P2 position compounds that exhibit an extended aromatic system (R = benzyl group) enhanced the lethal effect on the parasite followed by sec-butyl and isobutyl groups. The above observation was further reinforced when 6l and 6p, possessing 4-(p-fluorobenzyl)piperazine and 4-(p-bromobenzyl)piperazine, respectively, displayed higher potency  than their positional isomers 6i (IC50: 5.27 ± 0.06 μM) and 6m (IC50: 6.96 ± 0.10 μM), respectively as shown in Functionalised hydroxyethylamine based molecules were synthesised and screened for ersified . It is eP. falciparum. Almost all the molecules were active as anticipated owing to the presence of hydroxyethylamine and piperazine or piperidine moieties. The lower Ki values of all the screened compounds showed their efficacy as inhibitors of plasmepsin 2 (6u) and plasmepsin 4 (6t). The experiments demonstrated that the treatment of 5e inhibits the parasite to transit from early to late schizont stage, which was evidenced by absence of ring stage. Among all the compounds tested, 5e (an intermediate) was proved to be the most potent compound. However, compound 5e did not inhibit the activity of plasmepsins (2 and 4), which advocates the possibility of other drug targets like non-digestive vacuole plasmepsins which, in fact, might be the crucial drug targets according to the current understanding of plasmepsin biology. As there is lack of potent inhibitors of non-DV plasmepsins, we can’t rule out 5e and similar compounds as inhibitor of non-DV plasmepsin, which has been proved as good recipe for research in the area of plasmepsin medicinal chemistry.In conclusion, we have reported the antimalarial activity of hydroxyethylamine based phthalimides. Significantly, the obtained functionalized small molecules were found to provide a new avenue for the discovery of antimalarial agents against CQ resistant strain of S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S17 Fig(TIF)Click here for additional data file.S18 Fig(TIF)Click here for additional data file.S19 Fig(TIF)Click here for additional data file.S20 Fig(TIF)Click here for additional data file.S21 Fig(TIF)Click here for additional data file.S22 Fig(TIF)Click here for additional data file.S23 Fig(TIF)Click here for additional data file.S24 Fig(TIF)Click here for additional data file.S25 Fig(TIF)Click here for additional data file.S26 Fig(TIF)Click here for additional data file.S27 Fig(TIF)Click here for additional data file.S28 Fig(TIF)Click here for additional data file.S29 Fig(TIF)Click here for additional data file.S30 Fig(TIF)Click here for additional data file.S31 Fig(TIF)Click here for additional data file.S32 Fig(TIF)Click here for additional data file.S33 Fig(TIF)Click here for additional data file.S34 Fig(TIF)Click here for additional data file.S35 Fig(TIF)Click here for additional data file.S36 Fig(TIF)Click here for additional data file.S37 Fig(TIF)Click here for additional data file.S38 Fig(TIF)Click here for additional data file.S39 Fig(TIF)Click here for additional data file.S40 Fig(TIF)Click here for additional data file.S41 Fig(TIF)Click here for additional data file.S42 Fig(TIF)Click here for additional data file.S43 Fig(TIF)Click here for additional data file.S44 Fig(TIF)Click here for additional data file.S45 Fig(TIF)Click here for additional data file.S46 Fig(TIF)Click here for additional data file.S47 Fig(TIF)Click here for additional data file.S48 Fig(TIF)Click here for additional data file.S49 Fig(TIF)Click here for additional data file.S50 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOC)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.