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Received Date: August 28, 2014; Accepted Date: September 11, 2014; Published Date: September 18, 2014
Citation: Natarajan SS, Khan FH, Luthria DL, Tucker ML, Song Q, et al. (2014) A Comparison of Protein and Phenolic Compounds in Seed from GMO and Non- GMO Soybean. J Data Mining Genomics Proteomics 5:161. doi: 10.4172/2153-0602.1000161
Copyright: 2014 Natarajan SS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
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Soybean; Transgenic; Proteomics; Two-dimensional gel electrophoresis; MALDI-TOF/TOF; Phenolic acids; Isoflavones
Soybean and soy derived products are consumed worldwide for benefits derived from their high protein and bioactive phenolic components, namely isoflavones and phenolic acids. Soybean proteins are used in human foods in a variety of forms. Consumption of soybeans reduced the risk of cancer, decreased risk factors for cardiovascular disease, and reduced chances of other chronic illnesses . Soybean seed contains 2 major storage proteins: β-conglycinin and glycinin. Other proteins such as β-amylase, cytochrome c, lectin, lipoxygenase, urease, Bowman-Birk Inhibitor (BBI) and trypsin are also present . Recently, Clemente et al.  reported that soybean trypsin and chymotrypsin inhibitor Bowman-Birk (BBI) were linked to the prevention and treatment of colorectal cancer. Soybean isoflavones also were reported to reduce the risk of breast, prostate, intestine, and stomach cancer [4,5].
Over the past few decades, genetically modified (GM) crops have played a significant role in increasing the productivity and nutritional value of crops, e.g., increasing tolerance to herbicides, improving resistance to pathogens and producing recombinant pharmaceutical molecules including human growth hormone and coagulation factor 1X [6-9].
Since 1996, GM crops have been commercially available in United States and many other countries have also approved the commercial use of GM crops. Clive  recently reported that approved GM crops are currently grown on approximately180 million hectares in more than 25 countries. Foods derived from GM crops are subjected to rigorous safety evaluation such as characterization of intended modification with specific tests for allergenicity and toxicity. In addition, potential unintended effects are evaluated on the basis of agronomic characteristics, compositional analysis, and evaluation of key nutrients .
A safety assessment of GMO crops is mandatory in US and other countries. Numerous international organizations have played vital roles in the formulation of universal safety/risk assessments of GMO crops (Codex Alimentarius Commission (CAC), 2003 and 2009; Food and Agriculture Organization of the United Nations (FAO), 2011; World Health Organization (WHO), 1993 and 2000; Organization of Economic Cooperation and Development (OECD), 1993). According to Millstone et al. , the safety assessment of new crops is based on the concept of “substantial equivalence”. If the chemical composition of a new crop is substantially similar to that of existing crop, it is not considered to pose a health risk . Unintended effects may occur due to unforeseen interactions with other proteins or biochemical, or effects caused by random insertion into the genome, which can alter normal plant processes. It is therefore important that each genetic modification be examined on a case-by-case basis for unintended effects . In 2010, the sequence of the soybean genome became available and greatly improved our ability to access unintended effects . However, a clear understanding of unintended effects in regard to protein and metabolites due to transgenic modifications is still lacking [16-19].
Holistic profiling approaches such as genomic, proteomics, transcriptomics, and metabolomics have broadened the spectrum of compounds that can be identified and analyzed in contrast to earlier targeted analytical approaches [20-23]. In the present study, we utilize a combination of a proteomic and a more classical targeted approach. Proteins were separated using two-dimensional gel electrophoresis (2D-PAGE) and individual proteins identified by mass spectrometry. In addition, two classes of phenolic compounds, phenolic acids and isoflavones, were analyzed using high performance liquid chromatography (HPLC) with diode array (DAD) and mass spectral detections (MSD).
Plant material and generation of transgenic plants
A soybean phage genomic library was screened for clones with similarity to a PG11 cDNA. A 17 kb genomic insert was sequenced and when the soybean genomic sequence became available the two sequences were compared, with only a few differences found between the two sequences. Polymerase chain reaction (PCR) was used to fuse a 1951 bpGmPG11a gene (Glyma12g01480) promoter to a ß-glucuronidase (GUS) reporter gene immediately downstream from the ATGGUS open reading frame. The GUS open reading frame included an intron from the castor bean catalase gene 18 bp down from the start of translation . The PG11a-GUS-NOS3 construct was cloned into the Bam H1- Eco R1 site of pTF101.1 in the opposite orientation to a herbicideresistance selectable marker gene (bialaphos resistance, bar) . The herbicide-resistance gene was constitutively expressed using a double CaMV 35S promoter . The Iowa State University Transformation Facility transferred this construct into A. tumefaciens (EHA101) and then transformed and regenerated transgenic soybean (Glycinel max Williams 82). Seven independent events displayed herbicide resistance and all seven tested positive for the GUS gene in a PCR genomic DNA assay. Five events displayed strong GUS staining in an abscission assay. Segregation analysis of second and third generation seed indicated that three events with strong GUS staining had a single copy of the transgene. Homozygous seed from third generation plants for each of the following three events were used for this study: ST-16-3-10, ST83- 28-1-15 and ST83-37-7-10. Seeds were collected from multiple plants for each event,mixed and several seeds were used for analysis
Protein extraction from transgenic soybean seeds
Protein was extracted from transgenic and isogenic control soybean seeds using a modified Trichloroacetic acid (TCA) / acetone method . Soybean seeds were first pulverized to fine powder in liquid nitrogen using a mortar and pestle. Fifty mg of seed powder was homogenized in 1 mL of precipitation solution containing 10% (w/v) TCA in acetone with 0.07% (v/v) β-mercaptoethanol. The protein was allowed to precipitate for 24 hrs at -20°C. The precipitated protein then twice underwent 1 hr rinses with cold acetone containing 0.07% (v/v) β-mercaptoethanol, with each rinse followed by centrifugation at 20,800 g for 20 min at 4°C. The supernatant from each rinse was discarded. The protein precipitate was dried using a vacuum centrifuge for 30 minutes, and then resolubilized in 1.0 mL of lysis buffer [7M urea, 2M thiourea, 4% (w/v) CHAPS, 0.5% (w/v) DTT]. The mixture was homogenized with repetitive pipetting, followed by the use of a sonicator for 10 minutes, and centrifugation at 20,800 g for 20 minutes at 4°C. The supernatant containing the solubilized protein was used for each of the 2D-PAGE separations
Protein (100 μg) was first separated by isoelectric focusing (IEF) on an IPGphor II apparatus using13 cm immobilized pH gradient (IPG) strips of pH ranges 4-7 and 6-11 (GE Healthcare, Piscataway, NJ).The dry IPG strips were hydrated 12 hrs in 250 μL rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 2% pharmalyte, 0.002% bromophenol blue) containing 100μg protein. The voltage settings for IEF were 500 V for 1 hr, 1000 V for 1 hr, 5000 V for 1 hr, and 8000 V to a total 24 kVhr. Following electrophoresis, the protein in the strips was reduced through incubation with equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, 1% DTT) and subsequently alkylated with the same buffer by substituting 2.5% iodoacetamide for DTT. Incubations for both reduction and alkylation were timed for 30 minute and took place on a shaker at room temperature. The second dimensional protein separation was achieved by polyacrylamide gel electrophoresis on a 12.5% polyacrylamide gel using a Hoefer SE 600 Ruby electrophoresis unit (GE Healthcare, Piscataway, NJ). The gels were then stained with colloidal CoomassieG-250. After de-staining with ddH20, gels were scanned using a GE ImageScanner III (GE Healthcare, Piscataway, NJ).
Protein visualization and image analysis
Protein expression analysis was conducted through the use of Progenesis SameSpots (TotalLab, Newcastle, England). Scanned images in Maya Embedded Language (MEL) file format were first uploaded and underwent a quality check for color saturation and ensured consistency in image resolution across all samples. The images were then aligned and spots were automatically detected. In addition to automated spot detection, a thorough visual inspection was used to eliminate nonspot fragments falsely reported by the software. Subsequent to the spot detection, the experimental design allowed the software to report differentially expressed spots across control and transgenic soybean gels. All of the differentially expressed spots with a p-value below 0.05 were chosen for subsequent analysis.
In-gel digestion of protein spots
Soybean protein spots differentially expressed across control and transgenic samples were excised with a 1.5 mm Spot Picker (The Gel Company, San Francisco, CA, USA). Protein digestion was performed using trypsin as described previously . For further removal of the gel stain, the gel plugs were hydrated with 25mM ammonium bicarbonate on a shaker for 10 minutes, and then dried with acetonitrile for 10 minutes. The hydration and dehydration cycle was performed twice. The gel plugs were then thoroughly dried under vacuum and incubated overnight at 37ºC with 20 μL of 10 μg/mL porcine trypsin (Promega, sequencing grade, Madison, WI) in 20 mM ammonium bicarbonate for protein digestion. The resulting peptides were eluted from the gel in 50% acetonitrile and 5% trifluoroacetic acid. The extract was vacuum-dried and the dried peptides dissolved in 50% acetonitrile and 0.1% trifluoroacetic acid.
Samples were spotted on a MALDI plate, co-crystallized with a 5 mg/ml concentration of α-cyanohydroxycinnamic acid (CHCA) matrix prepared in 70% acetonitrile containing 0.1% trifluoroacetic acid. Thirty fmol of a commercially prepared tryptic digest of bovine serum albumin (Michrom Bioresources, Inc. Auburn, CA, USA) was spotted onto the 13 calibration wells of the sample plate and 5 peptides with masses in the range of 927.493 m/z to 1881.905 m/z were used for the calibration. The mass spectrometer used was an AB SCIEX TOF/ TOF™ 5800 System (AB SCIEX, Framingham, MA, USA) operated in positive ion reflector mode to analyze tryptic peptides. Prior to analysis of unknowns, a plate model calibration was run to optimize mass accuracy and to update the instrument’s default calibration parameters. The instrument was operated in batch mode during peptide analysis, which entails first performing an MS survey scan on all spots of interest, followed by sequential MS/MS analysis of peaks detected in the MS scan. Acquisition of MS/MS data was controlled by an interpretation method that acquired MS/MS spectra on the strongest precursors first on up to 100 precursors detected in the MS scan. An exclusion mass list was prepared to prevent MS/MS analysis of common human keratin contaminant and minor porcine trypsin autolysis peaks. MS spectra for both standards and unknowns were acquired in positive ion reflector mode with 400 shots of a 349 nm Nd:YAG laser operating at 404 Hz. MS/MS spectra were also acquired in positive ion reflector mode with 250-1000 laser shots firing at a rate of 1010 Hz. Collision energy was set to 1kV and collision induced dissociation (CID) was enabled with air as the collision gas in the CID cell. When possible, known trypsin autolysis peaks at m/z 842.51 and 2,211.10 were used to internally calibrate the MS spectra.
Protein identification was performed using the Mascot search engine (http://www.matrixscience.com) against the NCBI nonredundant database with the taxonomy filter Viridiplantae (green plans). The parameters for database searches included: monoisotopic mass, trypsin as the digestive enzyme with allowance for 1 missed cleavage, peptide tolerance of 50 parts per million (ppm), MS/MS tolerance of 0.6 Da, allowance of 1+ peptide charge, fixed modification for carbamidomethylation of cysteine residues, and variable modifications for oxidation of methionine residues as well as N-terminal pyroglutamic acid resulting from glutamic acid or glutamine. Positive identification of proteins by MS/MS analysis required a single peptide having a significant ion score. Samples identified as uncharacterized/unknown identity were subjected to sequence alignment match via BLAST against UniProt knowledgebase, where sequence similarity of 85% and above was used as a minimum criteria for inclusion.
Extraction and analysis of isoflavones from soybeans
Ground soybean samples (250 mg) were placed in 15 ml polypropylene conical tube with 5 ml of hexane. The mixture was placed in an ultrasonic bath for 15 min. The mixture was centrifuged and the hexane layer, which contained oil, was removed and discarded. The residue was extracted twice with 5 ml of with the previously optimized solvent mixture (EtOH:H2O:DMSO, 75:20:5, v/v/v). The supernatant from the two extraction cycles were pooled together in a volumetric flask and the volume of the combined extract was adjusted to 10 ml with extraction solvent. Appropriate aliquots of extracts were filtered through a 0.45 μm PVDF syringe filter for isoflavone analysis by LC-MS. Analysis of isoflavones was carried out using out using an Agilent 1290 Infinity LC system coupled to a diode array and a mass spectrometry detector from Agilent Technologies (Palo Alto, CA, USA). Separation of isoflavones was achieved using a reversed phase C18 Luna column (Phenomenex, Lorance, CA, USA, 150×4.6 mm; particle size 5 μm), preceded by a guard column (Phenomenex, 4×3.0 mm). The structures for all isoflavones were confirmed by comparison of retention time, UV spectra and mass spectral analysis. Peak areas were integrated for quantitation. Comparison of extraction efficiencies was achieved by comparing HPLC peak areas.
Extraction and analysis of phenolic acids from soybeans
Ground soybean samples (250 mg) were hydrolyzed in 2N NaOH containing 10 mM EDTA and 1% ascorbic acid for 30 min in an ultrasonic bath at 56°C as described previously . After hydrolysis, the samples were cooled to an ambient temperature and the pH of the extract was adjusted to 2.5 with 6N HCl. Phenolic acids were isolated from the acidified extract with ethyl acetate (5 ml×2). The mixture was vortexed for 30 sec and centrifuged on a bench top centrifuge (Damon IEC HN-SII, Ramsey, Minnesota, USA) at 5000 rpm for 10 min. The upper organic ethyl acetate layer containing hydrolyzed phenolic acids was carefully transferred into a separate tube and evaporated under nitrogen gas. The dried residue was re-dissolved in 2 ml of 80% aqueous methanol filtered through a 0.45 μm PVDF syringe filters into HPLC vials for analysis. Four replicates of hydrolysis, extraction, and analysis were carried out with each sample. The structures for identified phenolic acids were confirmed by comparison of retention time, UV and mass spectral analysis as reported earlier . Peak areas were integrated for quantitation. Comparison of phenolic acids was achieved by total peak area under the peak as detected by the UV-diode array detector.
Transgenic soybean seeds consist of single gene insertions containing both the bar herbicide gene and the β-glucuronidase (GUS) reporter gene controlled by the double CaMV 35S promoter and PG11a gene promoter, respectively. Although the 35S promoter is generally used as a constitutive promoter in plants, its expression level varies in different tissues. One study of soybean demonstrated that the CaMV 35S promoter was expressed strongly in leaves but very weakly in seed . Similarly, we demonstrated using GUS staining that the PG11a promoter is expressed throughout the soybean seed with the highest expression in the vascular cylinder of the root radical (Figure 1). Seed morphology and coat color were similar between the 3 transgenic events and control soybean seeds tested in our current investigation
Figure 1: Histochemical GUS staining of non-transgenic and transgenic soybean seeds. Soybean seeds: event 1 (ST83-16-3-10), event 2 (ST83-28-1-15), event 3 (ST83-37-7-10), and control. Seeds were imbibed with water for 3 hours and then stained for GUS activated overnight at 37°C. Seeds were then split down the middle and pictures taken of the outside and inside of each half.
|Savi #||Spot #||Control||Event1||Event2||Event3||Bonferroni-corrected p-value|
** significant at 1% probability level
Table 1: Average protein levels in the seeds of control and transgenic soybeans and the Bonferroni-corrected p-value
Analysis of proteins
A modified TCA/acetone method was used to extract soybean proteins . Three biological replicates of protein extracts from three transgenic events and control lines were separated by 2D-PAGE. Analysis was initially carried out with immobilized pH gradient (IPG) strips using a pH range of 3 to 10 (data not shown). However, over this broad pH range, storage proteins were poorly separated and the total numbers of resolved protein spots were insufficient. Improved separation and spot resolution were achieved using pH gradients between 4 to 7 and 6 to 11. Therefore, 100 μg of each protein extract was used for two separate 2D gels, one with a pH range from 4 to 7 and another with a pH range of 6 to 11. Representative 2D-PAGE gels for soybean seed protein are shown in (Figure 2) (Table 1). Comparison of 2D gel images for each of the transgenic events to that of control images identified 30 proteins spots that differed among treatments. Each of the 30 differentially expressed spots were excised manually from the gels and digested with trypsin. The tryptic digests were purified and analyzed by MALDI/TOF/TOF mass spectrometry. (Table 2) includes MASCOT information for each excised protein referenced by its spot number including: predicted protein identity, theoretical isoelectric point (pI), molecular weight (Mr), the original species that the protein was identified in, number of peptides matched, MOWSE score, and gene ID or accession number of the best match.
Figure 2: 2-D proteomic map of transgenic soybean. Proteins (200 μg) were separated in gradients of pH 4–7 for the first dimension, and resolved in the second dimension using 12.5% SDS-PAGE. The resulting gels were stained with Colloidal Coomassie Blue G-250. Numbered protein spots were excised and identified by MALDI/TOF/TOF. Differentially expressed proteins spots were numbered in the gel and protein information were listed in (Table 1).
|Spot #||Protein||PI #||MOWSE Score||# of PM||Mr (Da)|
|1||Beta-conglycinin, alpha chain||gi|121281||669||7||121281|
|2||Beta-conglycinin, alpha chain-like [Glycine max]||gi|356535993||67||1||68392|
|3||Alpha' subunit of beta-conglycinin [Glycine max]||gi|9967361||277||2||65160|
|4||Alpha' subunit of beta-conglycinin [Glycine max]||gi|9967361||566||6||65160|
|5||Alpha' subunit of beta-conglycinin [Glycine max]||gi|9967361||533||6||65160|
|6||Beta-conglycinin alpha subunit [Glycine max]||gi|335353923||943||11||70451|
|7||*Beta-conglycinin beta subunit [Glycine max]||F7J077||1364||18||50442|
|8||*Beta-conglycinin beta subunit [Glycine max]||F7J077||529||5||50442|
|9||*Beta-conglycinin beta subunit [Glycine max]||F7J077||1462||20||50442|
|10||Beta-conglycinin beta subunit [Glycine max]||gi|341603995||299||3||50010|
|11||Beta-conglycinin beta subunit [Glycine max]||gi|63852207||1523||19||48358|
|12||Beta-conglycinin beta subunit [Glycine max]||gi|341603995||906||11||50010|
|13||Beta-conglycinin beta subunit [Glycine max]||gi|63852207||881||13||48358|
|14||Beta-conglycinin beta subunit [Glycine max]||gi|341603995||432||3||50010|
|15||Beta-conglycinin beta subunit [Glycine max]||gi|341603995||911||12||50010|
|16||Beta-conglycinin beta subunit [Glycine max]||gi|341603995||172||1||50010|
|17||Beta-conglycinin beta subunit [Glycine max]||gi|341603995||908||11||50010|
|18||Glycinin G1 precursor [Glycine max]||gi|121276||100||1||56299|
|19||Glycinin A3B4 [Glycine max]||gi|225440||521||6||27447|
|20||Glycinin G1 precursor [Glycine max]||gi|121276||464||5||56299|
|21||Glycinin G1 precursor [Glycine max]||gi|121276||405||5||56299|
|22||Isoflavone reductase homolog 2 [Glycine max]||gi|351726399||811||9||33919|
|23||Isoflavone reductase homolog 2 [Glycine max]||gi|351726399||826||11||33919|
|24||Cysteine synthase [Glycine max]||gi|351727525||858||6||34362|
|25||*Pv42p [Phaseolus vulgaris]||Q41108||119||1||41312|
|26||Quinone oxidoreductase-like protein At1g23740||gi|356571378||180||1||34660|
|27||*Alcohol dehydrogenase 1 [Glycine max]||Q8LJR2||501||4||40007|
|28||Nucleoside diphosphate kinase [Glycine max]||gi|26245395||252||3||16402|
|29||Peptidyl-prolyl cis-trans isomerase 1 / Cyclophilin 1 [Glycine max]||gi|75304514||359||4||18395|
|30||Seed maturation protein PM30 [Glycine max]||gi|351727184||228||2||15145|
|* Refers to identity based on BLASTp on resulting sequence from MASCOT|
Table 2: Identification of differentially expressed protein between control and transgenic soybean seed variteies
Variation of storage proteins
Soybean seeds contain two major classes of storage proteins, the 7S and 11S proteins, which are normally referred to as β-conglycinin and glycinin, respectively. The β-conglycinins are trimeric glycoproteins consisting of three types of non-identical, but homologous polypeptide subunits: α, α′, and β. They form seven different combinations with the molecular weight of 180 kDa . The second class of storage protein, glycinin, is hexameric with molecular weights of 360 kDa and consists of acidic (A) and basic (B) polypeptides. Glycinin is encoded by five non-allelic genes, Gy1, Gy2, Gy3, Gy4, and Gy5. It codes for five precursor protein molecules, G1, G2, G3, G4, and G5, respectively . Based on physical properties, these five subunits are classified into two distinct major groups; group I consists of G1 (A1aBx), G2 (A2B1a), and G3 (A1aB1b) proteins and group II contains G4 (A5A4B3) and G5 (A3B4) subunits. Beillinson et al.  identified additional two genes, glycinin pseudogene (gy6) and functional gene (Gy7).
In this investigation a total of 30 differentially expressed protein spots were detected over pH ranges 4-7 and 6-11. Among the 30 spots, a total of 21 spots were identified as storage proteins. More specifically, 6 protein spots (spot#1-6) were identified as α subunits of β-conglycinin, 11 protein spots (spot#7-17) were identified as of β-subunits of β-conglycinin, and 4 spots (spot#18-21) were identified as glycinin G1 subunits. Based on the results, these protein spots are distributed over a wide range of pI and molecular weights and varied among the 3 transgenic events. Transgenic events 1, 2, and 3 had between 1.0 to 1.5 fold increase/decrease in protein abundance for the α-subunit and β-subunit of β-conglycinin. The significance of the protein level of each storage protein spot among the control and three events were tested using ANOVA procedure of SAS , as multiple comparisons were involved, a Bonferroni correction at 1% threshold was used to determine the significance of each protein spot. If a significant difference was observed for any spot, a comparison of the control vs. each of the three events was conducted using Dunnett’s test.
Table 1- includes results and statistics for the relative abundance of each protein spot for each of the 3 events compared with the nontransgenic control. There was a broad range of variability of both acidic and basic storage proteins components among the transgenic events. Gomes et al.  investigated alpha subunit of β-conglycinin spot variation in four conventional soybean genotypes namely BRS 257, 258, 267 and Embrapa 48, using 2D-PAGE analysis and also by ID-PAGE. They showed variation of total number of protein spots in BRS 257, BRS258, Embrapa 48 and BRS 267 is 102, 124, 113, and 99 respectively. They are also reported 46 differentially expressed proteins (storage, allergenic, maturation, agglutinin, and trypsin inhibitors) in 2D gels among 4 non-GM soybeans. Similarly, significant differences of both β-conglycinin and glycinin storage proteins using proteomics was reported in 14 Canadian commercial soybean varieties by Zarkada et al.  and in 90 Brazillian soybean cultivars by . Houston et al.  quantified soybean allergens in 20 non-GM soybeans and observed 10-fold variation of glycinin G3 when comparing 2 different varieties. Multiple authors have reported variation in protein profiles in different cultivars of soybeans grown under different environmental conditions using different proteomics techniques [37,39, 40]. In addition, Hughes and Murphy  reported that glycinin content varies from 7.5% for soybean grown in a uniform environment to 10% for soybeans grown at different locations. The previous publications from our laboratory also showed variation of storage proteins, allergen, and anti-nutritional proteins among several wild and cultivated soybean genotypes [42,43]. In another study, Fehr et al.  reported environmental effects caused significant variation of glycinin and β-conglycinin with the exception of A3 subunit of glycinin observed in 14 genotypes grown in 8 locations for three years. In addition, Helm et al.  also reported variation of above proteins due to environment and genotype and the magnitude of the differences between the 2 cultivars for 11S/7S ratio varied among locations within years. Piper and Boote  reported that maturity group, location, and environmental variation affect characteristics of soybean seeds, which suggested temperature has a significant effect on protein expression . Koo et al.  reported 19 differentially expressed protein spots of P34, storage, and seed maturation related proteins identified by immunoblot and MALDI-TOF/TOF analysis between PI 567476 and Clark accessions. The authors suggested that β-conglycinin subunits are products of a multigene family, and the variation in the distribution of protein spots could be due to post-translational modifications such as a sequence of glycosylation, deglycosylation, and proteolysis . Several other transgenic studies in soybeans and other crops have been also reported in the literature, which concluded that the occurred variation is within the natural variation of conventional cultivars [13,22,50,51].
Variation of other proteins
Other differentially expressed proteins found in this investigation were involved in primary metabolic processes and synthesis of nucleotides and other secondary compounds including: 2 spots of isoflavone reductase (spot# 22, 23); one spot of cysteine synthase (spot#24), one spot of Pv42P (Phaseolus vulgaris) (spot# 25), one spot of quinine oxidoreductase-like protein (spot#26), one spot of alcohol dehydrogenase (spot# 27) ; one spot of nucleoside diphosphate kinase (spot# 28); one spot of peptidyl-prolylcis-trans Isomerase 1 (spot# 29); one spot of seed maturation proteins (spot# 30). Except for the spots 22 and 26, no significant difference of the protein level was observed among the control, and the three events at the 1% probability level. For the spots 22 and 26, only the difference between Control and event 2 was significant, the significance of Control vs. Event 3 and Control vs. Event 4 were not significant. The authors concluded that no significant changes were detected between transgenic and non-transgenic lines and the differences occurred in 2 spots fell in the range of natural variation
Variation in isoflavones and phenolic acids content
Isoflavonoids are the principal group of phenolic compounds present in soybeans. Lee et al.  extracted soybean protein and investigated the effects of year, location, and genotype on soybean isoflavones. The authors concluded that environmental and genotype effects were the most important sources of isoflavones content variation in soybeans. In another study, Wei et al.  investigated the differences in isoflavones content in locally grown soybeans and genetically modified imported soybeans. They reported that the isoflavone content of imported genetically modified soybeans were similar to the regular Taiwanese grown soybeans. Taie et al.  reported that varying levels of organic fertilization produced significant influence on the phenolic content and antioxidant activity of soybeans. Similarly, influence of postharvest and storage conditions on phenolic content was reviewed by Amarowicz et al. . The authors revealed that the variation in polyphenol content was often negligible as compared to the differences in content between various plant varieties.
In the present study, we compared the isoflavone content from four soybean samples and the results are presented in (Table 3A). Identification of isoflavones was achieved by comparison of retention times, ultraviolet, and mass spectral data with authentic commercial standards or results published in the literature. The six isoflavones were identified as daidzein, glycetin, glycetein, daidzin, genistin, and genistein. Four additional peaks were tentatively identified as malonyl and acetyl conjugates of the three isoflavones at retention times 20.4, 20.9, 24.6, and 26.9 mins respectively. These compounds were present in all four soybean samples. Similar compounds have been identified and reported previously . Quantification in the present study was achieved by comparing peak areas under the curve for the identified isoflavones. The relative standard deviation for four replicate analyses for most samples was less than 5%. There were minor differences in individual isoflavones between control and transgenic samples. The total quantity of isoflavones extracted from four different samples varied between 10-20%. Based on the statistical data no systemetic variation in different isoflavones content was observed between different events and control sample. For example, two isoflavonoids, genistein and daidzein, showed no significant differences between event 1 and control but showed difference among events 1, 2, 3 and control at the 1% probability level. We concluded that no consistent trend in genetic variation was observed in our study with isoflavones, but the results indicated that the variation is due to environmental effects since the seeds used in this investigation were harvested in different times. Berman et al.  reported that the second generations of transgenic soybeans were compositionally equivalent to the conventional soybeans. However comparative large variations in isoflavone content that ranged from 551-7584 μg/g with an average of 2973 μg/g were reported in soybeans grown in Northern and Southern China from maturity groups 0-6 . Similar high variations in isoflavones content in different cultivars and growing conditions in Kansas grown soybeans were also reported .
|Isoflavone||Control||Event 1||Event 2||Event 3||Bonferoni corrected p-value||Trait significance|
|Average (HPLC area)||St. Dev||%RSD||Average (HPLC area)||St. Dev||%RSD||Average (HPLC area)||St. Dev||%RSD||Average (HPLC area)||St. Dev||%RSD|
|Glycetin (Rt 12.8 min; n=4)||131.500||6.165||4.688||144.875||5.652||3.901||124.175||4.388||3.534||122.025||3.242||2.657||0.002|
|Diadzein (Rt 30.2 min; n=4)||50.525||2.644||5.232||50.800||3.477||6.844||83.200||2.810||3.377||82.925||4.172||5.032||< 0.001||2,3 (1,C)*|
|Glycetein (Rt 31.6 min; n=4)||15.275||2.689||17.602||18.925||1.374||7.263||23.100||1.608||6.962||20.700||1.610||7.780||0.009|
|Genistein (Rt 41.0 min; n=4)||64.425||3.075||4.773||65.750||1.686||2.565||117.650||2.983||2.535||78.500||4.511||5.747||< 0.001||2,3 (1,C)*|
|Phenolic acid||Control||Event 1||Event 2||Event 3||Bonferoni corrected p-value||Trait significance|
|Vanilic acid (Rt 27.8 min; n=4)||Average (mg/g)||0.062||0.065||0.063||0.068||0.002|
|Syringic acid (Rt 31.4 min; n=4)||Average (mg/g)||0.242||0.276||0.266||0.275||0.236|
|p- Coumaric acid (Rt 38.5 min; n=4)||Average (mg/g)||0.070||0.082||0.108||0.087||< 0.001||2,(3,1),C*|
|Ferulic acid (Rt 42.2 min; n=4)||Average (mg/g)||0.083||0.084||0.077||0.091||0.012|
|Sinapic acid (Rt 43.7 min; n=4)||Average (mg/g)||0.089||0.093||0.098||0.101||0.014|
Table 3: LC-MS analysis of phenolic compounds from control and genetically modified soybean. (A) HPLC area of four isoflavones was used for comparison and four separate extractions and analysis were carried out for each sample. (B) Quantification of five phenolic acids was achieved using authentic commercial standards and four separate extractions and analysis were carried out for each sample.
It is well documented in the literature that the phenolic acid content in seeds of several crops is influenced by cultivars, growing conditions, and the methodologies used for analysis [28, 60-62]. Table 3B includes information for the identity and quantification of phenolic acids from the four soybean samples. Identification of phenolic acids was achieved by comparison of retention times, ultraviolet and mass spectral data with authentic commercial standards. The five prominent phenolic acids were identified as vanillic, syringic, p-coumaric, ferulic, and sinapic acids. In addition, there were four minor peaks detected at retention times 14.4, 47.2, 50.6, and 56.4 mins respectively. These compounds were present in minor amounts in all four soybean samples. The total quanity of phenolic acids extracted from four different samples ranged between 0.55 mg/g to 0.62 mg/g. The relative standard deviation of four replicate analyses was below 5%. Similar phenolic acids profiles have been reported in soybean, flaxseed, and olives . However, our results are different from the data published by Taie et al. , where authors observed chlorogenic acid in addition to the other phenolic acids in soybeans grown under organic growing conditions. Based on the statistical data, only one out of five acids, p-coumaric acid, showed significant difference among transgenic events 2, 3, 1 and control at the 1% probability level with no consistent trend. The variations in the phenolic phytochemicals content observed in the present study are within the natural range of variations observed in various conventional cultivars grown and processed under different environmental conditions or analyzed by different methods.
We separated soybean proteins using 2D-PAGE and identified 30 proteins that appeared to be differentially expressed in at least one of 3 transgenic events in comparison to non-transgenic control seeds. Each of these proteins (spots) was excised from the gels and their identity determined with MALDI-TOF/TOF tandem mass spectrometry. Phenolic acids and isoflavones were extracted and analyzed by LCMS analysis. The results indicated that minor variations in proteins, isoflavones, and phenolic acids profiles exist between control and transgenic soybean seeds. The variations observed in the present study are generally within the natural range of variations observed in conventional cultivars grown and processed under different environmental conditions. However, additional long term studies with different cultivars grown over multiple years with more transgenic insertions with their isogenic lines are needed to precisely evaluate the impact of genetic transformation. Detailed metabolomic profile analysis looking at multiple classes of phytochemicals in both control and transgenic lines are needed to confirm the current results
We thank Dr. Richard Sicher for critical review of this manuscript. Funding for this research was provided by ARS project 1245-21220-232-00D. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.

domain: chemistry


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Starting in 2020, the 5.2 million voters in Los Angeles county will begin seeing a brand new device when they head to the polls later this year to vote in California's primary and general elections. It's called the Voting Solutions for All People, or VSAP, and it aims to make voting for L.A. residents easier, secure, and transparent.
The Ballot-Marking Devices, which will make their debut on March 3 for the California primary, were designed by the Registar-Recorder/County Clerk in response to the previous, aging systems and are meant to make it easier for voters to to customize their voting experience to fit their needs.
Voters can access 13 languages and adjust the touch screen to a comfortable angle as well as change the display settings such as text size and contrast or go through the ballot using the audio headset and control pad. The iPad-like device is attached to a plastic table with privacy dividers to allow voters privacy while they cast their ballot.
Because election security has become such a serious issue for voters, the new ballot marking device will not be connected to the internet or any other kind of network. It can also guide voters through the voting process and give them an easy-to-understand step-by-step instruction on how people can vote for their preferred candidates. The county has even made the code that runs the machines available as an open-source, allowing it to be used or modified by other voting jurisdictions.
Voters can also research their votes at home and make their selections by using the Interactive Sample Ballot, prior to arriving to the vote center. After voters make their selection, they can scan their "Poll Pass" on the device, which will transfer their selections to the ballot.

domain: news


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IndiaDisplaying results 1 - 3 of 3
Seizing the opportunities of a circular economy in textiles
28 Jun 2021
By Henrique Pacini, Economist, UNCTAD
Zambian cotton entrepreneur goes beyond the fibre
28 May 2021
UNCTAD supports efforts to add value to cotton by-products in eastern and southern Africa to make the crop more profitable for farmers.

domain: agronomy


Early Morning Robo Calls Not From Us Says Monmouth County Republicans
Recipients of an early morning robo-call are being told the Monmouth County Republicans had nothing to do with it.
Assemblyman Declan O'Scanlon says registered Republicans in the thirteenth legislative district were called around four in the morning with a message seemingly endorsing him, Assemblywoman Amy Handlin, and Senator Joe Kyrillos in Tuesday's primary race; but O'Scanlon believes his opponents were behind the call.
"It was done to antagonize people and it was pretty effective...It was not even a mistaken call by us it was a purposeful call by our opponents to make people mad at us." says O'Scanlon.
The message which said “Hi, we are calling on behalf of Joe Kyrillos, Declan O’Scanlon and Amy Handlin to remind you you can still vote until 8 p.m., another four hours,” the call urged. “Remember to get out and vote. Thank you.”
O'Scanlon says they don't have hard proof the call was placed by opponents of the Republicans for Conservative Leadership, who have Leigh Anne Bellows running for Senator Kyrillos' seat, Stephen Boracchia and Edna Walsh running for assembly seats, and Daniel Peters running for Sheriff.
The 13th district assemblyman believes it wasn't an accident by his opponents.
"The return number that shows up on caller ID's they mimicked the Monmouth County Republican Headquarters number, yet the call just talked about the legislative candidates."
O'Scanlon notes the Monmouth County Republicans have reported the incident to the Prosecutor's Office.
"This is very serious infraction to do something like this. The last time it happened it was a forty two thousand dollar fine." says O'Scanlon.
Numerous calls to members of the Republicans for Conservative Leadership were not returned.


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Oops! I kiiiind of forgot about this last little Beer Diary Travel Edition post. My bad. It’s not even because it was a lackluster, unmemorable experience or something. In fact, it was by far the beer highlight of our trip. For those of you who […]
Tag: Arizona Craft Beer
Yay! Part 2. Exciting stuff. This is the part where I get to tell you about a new (to me) microbrewery in Phoenix, AZ, Sun Up Brewing. Mike’s dad brought us to Sun Up one cold, miserable day during our vacation. You know, the day […]
And might I add that it was a complete coincidence that my husband’s vacation time/our plans to visit family in Phoenix serendipitously occurred during Arizona beer week. Lucky us.

domain: travel


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LOS ANGELES (AFP) - A US man convicted of murdering two Chinese students during a botched robbery was on Monday jailed for life without the possibility of parole for his role in the killings.
Javier Bolden, 22, was found guilty last month of shooting dead Wu Ying and her boyfriend Qu Ming as the couple sat in a car outside the downtown Los Angeles campus of the University of Southern California.
An accomplice, Bryan Barnes, had already pleaded guilty to the crime and was given life without parole in February.
Sentencing Bolden on Monday, Judge Stephen A. Marcus said the murders had "stained the reputation of Los Angeles".
Marcus said Bolden had shown "no remorse" during the trial, and at one stage held up a copy of the Los Angeles Times newspaper which showed the killer smiling in court.
"You are not repentant," Marcus said, describing it as "truly one of the saddest cases that I've had to preside over."
The father of Qu Ming on Monday described Bolden and Barnes as "two extremely evil specimens of human trash".
Qu Wanzhi, who had requested the death penalty for his son's killer, also criticised the sentence as too lenient.
"This judgment is ... unfair to our children and of little comfort to their souls in heaven," Qu said, adding that the sentence was "humiliating to the people of China as well".
During the trial, prosecutors told how the 23-year-old graduate students were "helpless victims" inside a parked BMW and that Bolden and Barnes "counted down the seconds" as they approached the car before opening fire.
Barnes and Bolden were arrested in May 2012 after being traced by the dead students' stolen cell phones.


Is a unique new generation multi-functional ceramic ester engine oil additive with a superior performance under the most extreme conditions. The synergy of micro ceramic particles with high-en esters and PAO’s create a high degree of bonding to metal which results in a solid and unequalled lubricating film on the most critical and wear-sensitive parts of the engine. This results in high performance lubrication, ensures a rapid heat transfer and prevents metal to metal contact
For all gasoline, diesel and LPG engines with or without turbochargers. Can be mixed with all commercially available engine oils. Does not clog filters and is not harmful for catalytic converters. Not suitable for motor cycles using a wet clutch. One bottle treats 4 to 6 litres of motor oil. Recommended use: every 50.000 km.
|Densità a 15 °C||kg/l||0.847|
|Viscosità a 100 °C||mm²/s||13.7|
|Punto di infiammabilità COC||°C||>200°C|
|Punto di scorrimento||°C||-39 °C|


Rent a PlayStation, Xbox, or Nintendo in Grand Rapids, MI
If you want to buy or rent an Xbox, PlayStation, or Nintendo gaming console in Grand Rapids, then you need to visit your nearest Michigan Rent-A-Center to browse our selection of the latest video game systems. We consider our selection and payment options are the best out there. Not only can check out all the latest video game systems from the top names in video game consoles, but you can decide to rent-to-own, pay 4-6 Months Same As Cash, rent, or buy — all in the same store. We'll even help you pick the payment plan that's right for you, whether you want to pay bi-weekly, bi-monthly, weekly, or monthly!
Worry-Free Game Console Rentals in Grand Rapids
Are you concerned about your gaming system dying on you right in the midst of a difficult level or online multiplayer game? Do you always have good luck smiting bosses but bad luck with keeping your electronics alive? You may be the right person to rent a Nintendo or Xbox system with our Worry-Free Guarantee, which protects you if your video game system fails during the span of your rental agreement.**
Why Should You Get a Rent-to-Own Video Game System in Grand Rapids, MI?
One of our most popular options is rent-to-own for gaming customers that shop at Rent-A-Center, so you might be curious if rent-to-own is the best option for you when getting a new video game system. Making this decision can depend on the kind of video game player you are. If you're a legacy gamer who collects old-school Nintendo cartridges and Atari games and loves busting out those older games even if their graphics aren't as good as the most up-to-date games, rent-to-own gives you the ability to rent now and make the newest gaming console yours at the end of your rental. Alternatively, if you're the kind of person who won't get a game if it's more than a day old, you'd probably be a good candidate to choose a rental. And if you're like a lot of people, coming somewhere between the two... then come on in to your local Grand Rapids Rent-A-Center, and our expert team members can help you pick the best option!
Enjoy Gaming System Rentals with Payment Flexibility in Grand Rapids
At Rent-A-Center, there are various ways to pay for your PlayStation, Nintendo, or Xbox game console rental. Put away that checkbook and your car keys — with our gaming system rentals, you can pay for it online, on Rent-A-Center's app, or by giving us a call. You don't even need to drive across Grand Rapids! But if you decide to stop by to make a payment at our Grand Rapids location, that works as well. Come see us to find the newest gaming systems today!


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A Taste of Home, Wherever You’re From
Los Ranchos Bakery has big city vibe with neighborhood charm
Chef du Jour
Chef Brent Moore at Pueblo Harvest
New things in store for the restaurant
All Good News All the Time
Whether you’re more familiar with the French Riviera or the French Quarter, there are plenty of places in Albuquerque to get a taste of authentic French cuisine. Read Hosho McCreesh’s review of Le Quiche Parisienne in this issue, and check out these other restaurants in the city that will cater to your wanderlust and make you feel, if only for the evening, that you’re dining in the City of Lights. Bon appetit, mes amis.

domain: food


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In April 2008, the former director of the Deutsches Schauspielhaus in Hamburg, Friedrich Schirmer, endowed a fellowship designed to support a young dramatist from the German-speaking theater scene in memory of Marie Zimmermann, the dramatist and festival organizer who died in 2007. Akademie Schloss Solitude is responsible for organizing and awarding the fellowship.
Since 2019, the Marie Zimmermann Fellowship for Dramaturgy has been funded by the Ministry of Science, Research and the Arts Baden-Wuerttemberg. This fellowship is unique in the context of artist-in-residence programs, and is aimed at young, particularly talented dramatists from the German-speaking area. It includes prize money of 5,000 euros and a two-month stay at Akademie Schloss Solitude in a free, furnished living/working studio.

domain: artistic


text:
Im 6'1, 195lbs size 13 boot and i ride...
Im 6'1, 195lbs size 13 boot and i ride mostly groomers but im trying to learn the park this season. Im a intermediate rider who normally ride a 158, what size board should i go with? I have spoken to some DF gear experts who Ski (no snowboarders were available) they recommended a 156. The Dark series only comes in a 155, will a 155 be too small for me? Im not interested in speed just carving and learning the park.
going down one cm is no big deal but check the width on the board to make sure your boots won't drag. i"m 6'2" and my park board is a 154 and my pow baord is a 160. hope this helps.
My current 158W waist width is 25.8cm, which fits my size 13 perfectly. The dark Series 155W im looking at has a 25.1cm. Is the .7cm difference going to matter as far as toe drag? Or should i just take my chances with the dark series 158W?
go with the 158W. The 155W is only 25.1 CM at the waist. You will have a lot of toe drag

domain: hobby


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Congratulations to Carlos Whitlow and GP! They are the 2015 Grand Prizewinners of the Allstate Gospel Superfest Battle of the Bands....
Bishop Elect Marvin Winans receives the 2015 Founders Award from Gospel Superfest during Chicago event.
Look for Carlos and his dynamic ensemble at the 2016 Allstate Gospel Superfest.... Details forthcoming....
Hollywood film and TV stars shine in Chicago for the 16th Annual Allstate Gospel Superfest.
House of Hope Arena - Chicago, IL
Allstate Gospel Superfest Chicago 2014!
Allstate Gospel Superfest Chicago 2015
Joshua's Troop Clap Your Hands from Robert Cartwright, Jr. on Vimeo.
Dorinda He Brought Me AGSF 2013 from Robert Cartwright, Jr. on Vimeo.
Speechless Anita Wilson from Robert Cartwright, Jr. on Vimeo.


text:
K#RWA Fatality Brutal Fruity Gum
Slim all white portion with taste of strawberries, raspberries and hints of banana
About K#RWA Fatality Brutal Fruity GumK#RWA Fatality Brutal Fruity Gum combines the delicious flavours of strawberries, raspberries and banana. The taste comes complimented with a very high nicotine content of 50mg/g (36mg/pouch). K#RWA Fatality Brutal Fruity Gum from the "Fatality" series by K#RWA delivers super strong nicotine pouches that are not recommended for beginners or unexperienced users.
CONTENT AND INFORMATION
Declaration of content
Cellulose (E 460), Sodium carbonate (E 500), Aromas, Nicotine, Sweeteners (E 960), Propylene glycol (E 1520), Sodium alginate (E 401), Stabilizer (E 504)


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I was curious if any of you out there have experienced or believe that the music industry has a glass ceiling or not regarding woman and pay. For those of you that are not very familiar with what a glass ceiling is, I will explain in lamens terms. A glass ceiling is a visual expression to help you “see” how the men in the workforce are on a higher level standing on the floor which is made out of glass looking down at the women who are not allowed upstairs in to the same room and are also being paid a less wage for doing the same amount of work or even the exact same type of work. It has been researched that women can earn as much as five dollars per hour less than a man although it could be preforming the exact same job.
Now having a better understanding of what a glass ceiling is, the question still remains. Do you think there is still a glass ceiling issue within the music field of DJ, radio, etc.? Do you feel there is an obvious difference in hourly or salary pay between men and women. If the answer is yes, why do you believe this to be so. Conversely, if the answer is no, why do you believe that to be the case. Have you personally experienced this or seen this in your life? What do you think can be done to ensure the equality of women can be implemented?
On a different note, I am wondering if certain company benefits are equally allowed to females? Take for instance drug and alcohol rehab help. Most companies have a rainy day fund set aside for such occurrences. Some even have a well organized (EAP) employee assistance program. So the question remains, do employers provide the sam opportunities (substance abuse treatment) to women as they do men. Or do they expect the women to heal through attending a local 12 step program or even more severe, do they fire them altogether?
You might have the opinion that the glass ceiling does not exist, or even the opinion that there is no problem with having a glass ceiling. No matter what your personal feelings are about the matter, I would be very curious to see what all the different thoughts and opinions are regarding this topic. Feel free to comment or personally email me as this topic may be controversial for some of you to post your thoughts publicly.

domain: sociology


Surly is a leading manufacturer of bicycle frames, and was one of the first Fat Bike manufacturers.
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"Founded in 1991 by a group of Southern Californian cycling enthusiasts who envisioned bicycle lighting systems utilizing the latest technology with trend setting designs, Cygolite developed a line of systems that made it synonymous with innovation and quality. Over 20 years later, we still lead the way, creating rechargeable lighting systems inspired through test rides on the trails and roads then engineered and assembled in our California headquarters."
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These models are available for online purchasing and can be shipped directly to you.
In Store products are available to view in our showroom. In Store orders usually ship the same or next business day.
Buy this product and your entire order ships for free.
Some orders are inelligible for delivery discounts. See offer details for disqualifying products and destinations.


text:
PMI TerrAdaptor Lash Ring Kit
The TerrAdaptor QuickLash has been designed to work exclusively on the TerrAdaptor grey tube as an anchor point. With the QuickLash, TerrAdaptor users will now be able to install an auxiliary anchor point before, during or after set up. Simply connect the QuickLash by inserting the quick release pin and you have a high strength single or multi direction tieback. This new QuickLash is ideal for attaching pulleys or other gear to assist with tie backs, redirects, and/or tie downs. Quick, easy and high strength make this TerrAdaptor accessory a must have for all rescue teams who have come to love the TerrAdaptor.

domain: design


text:
In recent months we've felt so strongly that there had been a huge shift in reader habits that we commissioned You Gov to complete a research project examining in detail the new digital audience and its habits. What they've proved is that a completely new type of audience has developed. They are a distinct, articulate, aware group of smartphone readers, for whom news is. We call them Thrivers. Thrivers, quite simply, are flourishing in the UK. They are self-aware, inquisitive and enjoy new experiences. Thrivers are also tech savvy and media literate. They seek deeper brand relationships in exchange for experience and knowledge.
Today, we're leading with Aaron Eccles, head of social media of Cancer Research and how they turned around a meme that wasn't even theirs and have raised £8m so far off the back of it. We've then got comedy legend Ruby Wax with a fantastic post on how to use mindfulness as an internal weathervane to predict depression...
We are not only marking the tenth anniversary of the fall of Saddam but the 50th anniversary of the beginnings in 1963 of a campaign of demonisation of the Kurds that proceeded to full-blown genocide, most notably at Halabja where 5,000 people were killed and many more hideously injured by Saddam's Weapons of Mass Destruction.
Today I'm delighted to announce the launch of HuffPost UK Sport, the latest channel on our ever-expanding site. With London gearing up for the Olympics and Euro 2012 only a few months away, it couldn't be a better time for us to add Sport into the mix of topics we cover. Being British, we fully expect there to be plenty of footie written about in our blogs, however, we're also aiming to be a destination for niche sports, where everyone from synchronised swimmers to curling champions can debate the merits of their favourite pastime with other dedicated fans. In keeping with this wide range of sports, we have a real mix of voices from different walks of life blogging for us today, including Lord Coe on the Olympics, Gary Kemp from Spandau Ballet on cycling, and Kevin Cahill - founder of Sport Relief alongside Will Carling and Andy Murray.
After watching Occupy Wall Street from afar since mid-September (and being kept up-to-date on Twitter thanks to HuffPost US senior editor Craig Kannelly's constant stream of tweets), last weekend saw the protest move to London. We had editors there throughout the week (Business Editor Pete Guest even turning photographer for us while on the scene), but unsurprisingly many of our bloggers had opinions to share on the topic, too.
Mining accidents, such a regular part of life in industrial communities in decades gone past as to provide perfect plot twists in modern-day novels, became headline worthy again this week due to the tragic events in Swansea. I'm sure I wasn't the only one somehow naively expecting a repeat run of the jubilant scenes played out in Chile almost exactly a year ago. If only real life ran with such Hollywood-perfect endings. Calls for an enquiry have already been made, while Neath MP Peter Hain is appealing for donations to help the bereaved families of those brave men who lost their lives.
The US and the UK may be, as George Bernard Shaw once said, two nations "separated by a common language," but they're definitely joined by a set of common problems: economic crises that have left millions unemployed or underemployed, an economic debate that has been hijacked by deficit and austerity hawks, and politicians with a remarkable ability to propose solutions that are actually making the problems worse. Yet in both countries, there is a movement fueled by social media, community engagement, and the growing understanding that real solutions aren't going to be coming from our politicians any time soon. So, in the meantime, millions of people in thousands of communities are taking the initiative to connect, engage and solve problems themselves.
As I type this, the British government has just pledged to financially support the International Committee of the Red Cross as it steps up essential help for those injured during Libya's week of conflict, with aid for up to 5,000 wounded, along with food and household essentials for another 690,000. The announcement comes as a quiet reminder that in amongst the headline-grabbing images of rebels waltzing their way round Gaddafi's glitzy mansion, this uprising, like the others that preceded it across the Middle East and Africa this year, this has not been a conflict without horrific casualties.


Price: PKR: 36299/-
Xiaomi is back with updated Mi 5s which is upgraded to the Plus version. This is a mammoth smart phone with almost up to date features and technology. Really a high end device without any rival in manner of its massive size. Xiaomi Mi 5s Plus is carrying 5.7 inch IPS capacitive display that brings you Quad HD resolution with the support of 2560 x 1440 pixels with a density of 1440p on screen. Xiaomi's Mi 5s Plus is stuffed with snapdragon 821 system on chip that produce such a massive amount of speed. To handle and utilize this speed in a proper way 6 GB of RAM is installed inside the Mi 5s Plus by Xiaomi. An android OS v6.0 marshmallow is running inside the phone to keep the everything in order. Just like its all other stunning specification the internal storage of Xiaomi 5s Plus is also impressive that brings you 256 GB of built-in storage for its users. Now they don't to put additional SD cards to expand their phone memory. Xiaomi Mi 5s's dual SIM setup meets all the smartphone standards and you can enjoy the packages of both networks at the same time. LTE technology and 3G support of Xiaomi Mi's 5s Plus with HSPA technology take the internet browsing experience to the whole now level. Live streaming is also improved and a video starts with just a blink of an eye. Other features of Mi 5s Plus includes GPS, A-GPS, GLONASS, Active noise cancellation with dedicated mic, photo/video editor, Document viewer, Google Maps, gmail, document viewer and speakerphone. 5s Plus is available in various colors and you can choose one according to your taste.
Xiaomi Mi 5s Plus Detailed Specifications:
|Battery||Capacity||3800 mAh||Type||- Non-removable|
|Build||Colors||Silver, Gray, Gold, Rose Gold||Dimensions||154.6 x 77.7 x 8 mm||OS||Android OS V6.0 (Marshmallow)||SIM||Dual-SIM, Dual Standby, Nano-SIM||UI||MIUI||Weight||168g|
|Camera||Features||Dual, F/2.0, phase detection, 1/2.3" sensor size, 1.55µm pixel size, geo-tagging, touch focus, face/smile detection, panorama, auto-HDR, Video (2160p@30fps, 1080p@30fps)||Front||4 MP, f/2.0, 1/3" sensor size, 2µm pixel size, 1080p||Main||12 MP, autofocus, dual-LED + dual tone flash|
|Connectivity||Bluetooth||v4.2 with A2DP, LE||Data||GPRS, EDGE, 3G HSPA, 4G LTE||GPS||Yes + A-GPS support & Glonass, BDS||Infrared||Yes||NFC||Yes||Radio||No||USB||Type-C 1.0 reversible connector||WLAN||Wi-Fi 802.11 a/b/g/n/ac, dual-band, Wi-Fi Direct, DLNA, hotspot|
|Display||Resolution||1080 x 1920 pixels (~386 ppi pixel density)||Size||5.7 inches||Technology||IPS LCD capacitive touchscreen, 16M colors, Multitouch|
|Features||Audio||3.5mm audio jack, Speakerphone||Browser||HTML5||Extra||Fast battery charging: 83% in 30 min (Quick Charge 3.0), Active noise cancellation with dedicated mic, Photo/video editor, Document viewer, Google Search, Maps, Gmail, Document viewer, MP4/DivX/XviD/WMV/H.265 player, MP3/WAV/eAAC+/FLAC player, YouTube, Google Talk||Games||built-in + downloadable||Messaging||SMS(threaded view), MMS, Email, Push Mail, IM||Sensors||Accelerometer, gyro, proximity, compass, barometer, fingerprint||Torch||Yes|
|Frequency||2G Band||SIM1: GSM 850 / 900 / 1800 / 1900 SIM2: GSM 850 / 900 / 1800 / 1900||3G Band||HSDPA 850 / 900 / 1900 / 2100||4G Band||LTE band 1(2100), 3(1800), 5(850), 7(2600), 38(2600), 39(1900), 40(2300), 41(2500)|
|Memory||Built-in||64GB built-in, 4GB RAM, 128GB built-in, 6GB RAM||Card||No|
|Processor||Chipset||Qualcomm MSM8996 Snapdragon 821||CPU||Quad-core (2 x 2.35 GHz Kryo & 2 x 1.6 GHz Kryo)||GPU||Adreno 530|
|Share With Friends:||Compare Xiaomi Mi 5s Plus|
DISCLAIMER: Mobile Prices are updated daily from local Pakistani Mobile shops & Mobile dealers in Pakistan but we can not guarantee that the information (Prices) on this page is 100% correct (Human error is possible). Always visit your local shop for exact mobile prices. *Above mobile prices and rates are only valid in Pakistan.
The exact price of Xiaomi Mi 5s Plus is PKR 36299/- from local trusted shops of Pakistan, however Human error is possible.More Xiaomi Mobiles | All Brands


text:
Glyceria maxima, commonly known as reed sweetgrass or tall mannagrass, is a species of grass in the Poaceae family. It is native to Europe, Asia, and North America, and can be found in wetland habitats such as marshes, swamps, and along the banks of streams and rivers. The plant can grow to be quite tall, up to 3m in height, and has long, narrow leaves and spikes of greenish-purple flowers. G. maxima is commonly used as a forage crop for livestock, as well as for erosion control and as a decorative plant in gardens.
Reed sweet-grass, scientifically known as Glyceria maxima, is a perennial grass species that is native to wetlands, marshes, and meadows throughout Eurasia and North America. The plant is also commonly referred to as tall mannagrass, reed grass, or reed meadow grass. Reed sweet-grass is an important plant species in wetland ecosystems, providing both habitat and food for a variety of animal species.
Appearance and Characteristics
Reed sweet-grass is a tall, robust plant, growing up to 10 feet (3 meters) tall, with a thick, sturdy stem that can reach up to 1 inch (2.5 cm) in diameter. The leaves are long, narrow, and flat, with a prominent midrib, and are usually about 1 inch (2.5 cm) wide. The flowers are arranged in large, dense panicles at the top of the stem, with each flower having two small, greenish-yellow florets. The plant blooms from June to August and produces a profusion of seeds, which are dispersed by the wind.
Reed sweet-grass is a keystone species in wetland ecosystems, providing habitat and food for a variety of animal species. Its tall, dense growth provides shelter and nesting sites for waterfowl, marsh birds, and other wetland animals. The plant also plays a crucial role in stabilizing wetland soils, preventing erosion, and filtering pollutants from the water.
Reed sweet-grass has been used by humans for a variety of purposes throughout history. In traditional medicine, the plant has been used to treat a variety of ailments, including fever, diarrhea, and dysentery. The fibrous stems of the plant have also been used for weaving baskets, mats, and other household items. In some cultures, reed sweet-grass has been used for thatching roofs and making wattle and daub walls.
Reed sweet-grass is a common and widespread species, but its populations are threatened by habitat loss and degradation. Wetland habitats are under increasing pressure from development, agriculture, and other human activities, which can lead to the destruction of the plant's habitat. Conservation efforts aimed at protecting and restoring wetlands are critical for the long-term survival of reed sweet-grass and the many other plant and animal species that depend on wetland ecosystems.
Reed sweet-grass is a fascinating plant with important ecological and cultural significance. Its tall growth, ability to stabilize wetland soils, and provide habitat and food for a variety of animal species make it a vital component of wetland ecosystems. Efforts to protect and restore wetlands are critical for the long-term survival of this important plant species and the many other species that depend on wetland habitats for their survival.
Reed sweet-grass is a highly adaptable species that can tolerate a wide range of environmental conditions, including waterlogged soils and areas with high salinity. In fact, it is often found growing in brackish marshes and along the banks of tidal rivers and estuaries. The plant is also resistant to grazing by livestock, making it an important food source for wetland wildlife.
One of the unique characteristics of reed sweet-grass is its ability to store large amounts of carbon in the soil. Wetland ecosystems are known for their high carbon sequestration potential, and reed sweet-grass plays a significant role in this process. As the plant grows, it stores carbon in its tissues, and when it dies, the carbon is released into the soil, where it can be stored for centuries or even millennia.
In addition to its ecological and cultural significance, reed sweet-grass has also been studied for its potential economic value. The plant contains high levels of cellulose and hemicellulose, making it a potential source of bioenergy. Research has shown that reed sweet-grass can be converted into ethanol, biogas, and other biofuels, making it a promising candidate for sustainable energy production.
Overall, reed sweet-grass is a fascinating plant with a rich history and important ecological and economic significance. Its ability to thrive in wetland habitats, provide habitat and food for a variety of animal species, and store large amounts of carbon in the soil make it a crucial component of wetland ecosystems. As wetland habitats continue to be threatened by human activities, conservation efforts aimed at protecting and restoring these important ecosystems are more important than ever.
Reed sweet-grass also has a role in phytoremediation, a process that uses plants to remove pollutants from the soil or water. The plant is known to accumulate heavy metals, such as lead and cadmium, from the soil and store them in its tissues. This ability to tolerate and accumulate pollutants makes reed sweet-grass a potential candidate for the remediation of contaminated sites.
Reed sweet-grass is not only an important species for the ecology and the economy, but it also has some cultural and spiritual significance. In some indigenous cultures, the plant is considered a sacred plant, and its use in ceremonies and rituals dates back centuries. For example, the Tlingit people of the Pacific Northwest traditionally use reed sweet-grass in basket weaving and other crafts, and it is often featured in their artwork.
Reed sweet-grass is a versatile and important plant species with many ecological, economic, and cultural values. As wetlands face increasing pressures from human activities and climate change, it is important to recognize and protect the vital roles that reed sweet-grass and other wetland species play in maintaining the health of these important ecosystems. By understanding and appreciating the many values of reed sweet-grass, we can work towards ensuring its conservation and sustainable use for future generations.
Reed sweet-grass is a highly productive plant species that can grow rapidly, producing up to 10 tons of dry matter per hectare per year. This high productivity makes it a potential source of feed and forage for livestock. In some areas, reed sweet-grass is cultivated as a forage crop, providing a valuable source of feed for dairy and beef cattle, sheep, and other livestock.
The plant's ability to tolerate and thrive in wetland habitats also makes it an important tool for wetland restoration and management. Restoring wetlands that have been degraded or destroyed can be a challenging and costly process, and using native plant species like reed sweet-grass can help to speed up the process and reduce costs. Planting reed sweet-grass and other native wetland species can help to stabilize soil, reduce erosion, filter pollutants, and provide habitat and food for wetland wildlife.
In addition to its ecological and economic benefits, reed sweet-grass is also valued for its aesthetic qualities. The tall, graceful stems and delicate flowers of the plant make it a popular choice for ornamental gardening and landscaping. It is often used in water gardens, ponds, and other aquatic environments, adding beauty and texture to these spaces.
Overall, reed sweet-grass is a versatile and valuable plant species that has many important ecological, economic, and cultural values. Its ability to thrive in wetland habitats, provide food and habitat for wildlife, store carbon in the soil, and be used for phytoremediation, bioenergy production, and wetland restoration make it a valuable resource for sustainable development. As we continue to face environmental challenges and work towards a more sustainable future, reed sweet-grass and other wetland species will play a crucial role in the conservation and management of these important ecosystems.

domain: biology


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The idea for green number plates from Grant Shapps is straight out of nudge theory:
How can we best encourage zero-emission vehicles? Green number plates could help…
✅ highlight electric vehicles
✅ raise awareness
✅ promote clean energy
— Dept for Transport (@transportgovuk) October 22, 2019
This is at the sensible end of green policymaking: it doesn’t cost much, encourages rather than coerces, and will allow people to virtue signal over paying top money for a car that can’t get them to the shops and back… yet.
The Guardian’s political correspondent Peter Walker, however, reckons “giving electric vehicle drivers free parking just perpetuates yet another problem, inequality.” It does seem paradoxically iniquitous to give the richer owners of the most expensive cars lower taxes and driving costs…
UPDATE: It turns out this is a re-announcement of the scheme Chris Grayling unveiled in September of last year. Unlike
Michael Green Grant Shapps to simply rename and relaunch something like this…
Reminds Guido of Shapps’ suspiciously similar speech to Chris Grayling over airline collapse. Is this a DfT that is a little too keen on recycling?..


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--:-- • --:--Пример экскурсии
You can't have missed the high clock tower on the odd-numbered side of the street. It's the Tower of the City Duma. Before the 1917 Revolution the City Duma met in the adjacent building – an elected body who determined matters of local city governance in St.Petersburg.
Considered tall by St.Petersburg standards, the City Duma Tower did double duty as a fire-watch tower, and there used to be a team of ten firefighters based here. The openwork construction on the roof was used to raise signal-balls, which reported on the location and strength of blazes. The tower also gave warning of floods.
In 1838 the City Duma Tower was one of the links in the world's longest optical telegraph chain. The telegraph ran for 1200 kilometers, linking the Winter Palace with TsArskoye SelO, KronstAdt and Warsaw. The system worked by a system of specially-mounted mirrors. A message sent from the roof of the Winter Palace would pass through 149 relay stations, and in good weather could be delivered to Warsaw in fifteen minutes. Messages could even be delivered by night – special lights were fitted to the mirror-frames. However, the optical telegraph didn't last long – within fifteen years it was scrapped in favour of the more conventional electric telegraph.
St. Petersburg is known as Russian cultural capital. It is also called “Northern Palmira” or “Northern Venice” as it was founded on islands in the delta of the Neva River and has a lot of architectural masterpieces.
Nevsky Prospect is the main avenue and one of the best-known streets in Russia. It is a real treasury of Russian culture. The street was planned by Peter the Great as beginning of the road to Novgorod and Moscow. It goes through the historical center of the city, firstly - from the Admiralty to the Moscow Railway Station and then, after making a turn at Vosstaniya Square, to the Alexander Nevsky Lavra.
Life on Nevsky Prospect has always been the object of attention of writers. For example, it was described by Nikolai Gogol in his story "Nevsky Prospect". Fyodor Dostoevsky often employed the Prospect as a setting within his works, such as Crime and Punishment and The Double: A Petersburg Poem.
It is best to start your walk from famous Palace Square where you can visit one of the most prominent world museums – the Hermitage. In the center of the square you will see the Alexander Column, a very popular among tourists place. With Your Audio Guide you will listen to the history of the square and all its attractions (The New Hermitage, The Winter Palace, the Alexander Column and The General Staff Building).
During the tour you will walk through the Alexander Gardens, past the Admiralty, see a lot of historical houses – real architectural “pearls” - Wawelberg's Mansion Apartments, The Sivers-Treiberg House, The Chaplin Brothers House, Chicherin's House, etc. The chief sights of the Prospect include palaces, churches and cathedrals, shopping centers and department stores. Among palaces are the Rastrelliesque Stroganov Palace, the Anichkov Palace, The Beloselsky-Belozersky Palace, Grand-Princess Yusopova's Residence and the Mikhailovsky, or Michael Palace.
Nevsky Prospect deserves the name "the street of religious tolerance". Religious buildings are presented by the huge neoclassical Kazan Cathedral, the picturesque Russian-style Cathedral of Our Savior on Spilt Blood – a place where terrorists exploded the Emperor Alexander II, the Alexander Nevsky Lavra, St.Catherine's Cathedral - the oldest Catholic Church in Russia, and The Armenian Church.
Nevsky Prospect is also the city’s central shopping street and the hub of the city’s entertainment and nightlife. For that You can visit the Art Nouveau Bookhouse,Elisseeff Emporium, half a dozen 18th-century churches, , an enormous 18th-century shopping mall “Gostinny Dvor”, a mid-19th-century department store “The Passage” and The Alexandrinsky Theatre, in the yard of which a monument to Catherine the Great is standing.
Your audio guide is glad to present you a wide audio tour of the Nevsky Prospeсt with map and descriptions. With this map, you will never get lost and you will always know what surprise awaits you ahead.

domain: history


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On her goals for the future: “I want to make furniture. I want to complete the album, go on tour. I have a documentary that I’ve been filming secretly for three and a half years. Maybe write another book. Tons of things. I’ve really gotten into entomology—I bought a lot of butterflies and I’m learning how to pin them and make lampshades, like, Victorian era. If I were to write out a to-do list, it would fill the entire wall.”
EXCLUSIVE OUTTAKES: Camila Cabello's March/April 2017 Cover Shoot!
March 17, 2017


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Day 13 – Tioga Pass and Yosemite – Photos
Thanfully the pass was open and we could drive directly to Yosemite National Park. It had been closed a couple of days before due to early season snow, and that would have meant a very long drive round the mountains.
Despite the near freezing temperatures, the drive up was done with the roof down, all the better to admire the mountauin views.
Part way up, we found our first snow of the trip, a real change from the heat of the dessert we had been in just the day before!
There were also some very pretty lakes.
We went over the Tioga Pass and entered Yosemite. I climbed an extra few metres just to make sure I had gone over 10,000 feet.
As we descended, the scenery changed rapidly, and we soon arrived in Tolumne Grove for a walk and to see some Sequoias and a few deer.
We headed out of the park to our hotel, the Yosemite View Lodge. Our advice is to stay in the park, not here. See our Tripadvisor Review.
We then drove back into the park (which is a long way) and up to Glacier Point, a very twisty road, where we (and plenty of other people) caught the end of sunset.
We returned to our hotel for an annoying pizza dinner and to encounter the racoons (see Tripadvisor review…)
Day 14 – Yosemite – Photos
The valley was pretty as the drove up on this misty morning.
The walk up past both falls was great, and we came back down the John Muir trail side. It is hard work though, and going all the way on to Half Done would be tough.
We then went to the main visitors centre and had some big rolls for lunch from the deli, which were very tasty after our long walk.
A quick wander down the valley floor and a paddle in the river was pleasant, followed by dinner in the Yosemite Lodge Cafeteria (in the park), which looks like a more sensible place to stay.
Day 15 – Yosemite to home – Photos
Sadly our holiday was coming to an end, and it was time to head to San Francisco for our flight home.
The drive was fairly interesting, through varied scenery, and we arrived at the airport carhire drop-off in good time.
The flight back was as much fun as can be expected from an overnight flight from the West Coast, but all went well, we drove home feeling rather tired, and were in work the next day!
This was a great trip. There were some truly lovely hotels (Desert Pearl Inn, Signature at MGM Grand), some hotels than were clean and were good value, and then some poor hotels…
The variation in scenery was great. Cities, deserts, mountains, lakes, rivers, canyons, forests. These really are must see places.
It would have been good to have spent more time there. We ended up driving 2414 miles from hire car collection to drop-off. We like driving, but many people might find that a bit much, and a few extra days in some of the locations would be good.
If you are thinking of doing a trip like this, you should.

domain: geography


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Top bosses at Network Rail are in line for bonuses worth thousands of pounds a year just days after the company was fined £53 million for poor punctuality.
NR’s members, the equivalent of shareholders for a company that is a not-for-dividend firm, have overwhelmingly voted in favour of a new bonus scheme for the top executives.
The scheme replaces, and is less generous than, the old bonus scheme, but chief executive Mark Carne and his fellow top bosses could still get up to 20% of their annual salary in annual bonuses should performance targets be reached.
Mr Carne is currently on a salary of £675,000 a year, so could get as much as £135,000 in bonuses for 2014/15, while group finance director Patrick Butcher, on £412,000, could get £82,400.
The recent fine was imposed by the Office of Rail Regulation and took the form of an ordered payment by NR to the Treasury following failure to meet some punctuality targets.
After MPs investigated deaths at level crossings, House of Commons Transport Committee chairman Louise Ellman called for NR chiefs’ bonuses to be suspended.
Following today’s vote at NR’s annual meeting in Reading, Berkshire, Manuel Cortes, leader of the TSSA transport union said: “This is a slap in the face to all those bereaved (level cross death) families which the Transport Committee found had been treated with “callous disregard” down the years by NR.
“Mark Carne should have re-set the company’s moral compass by cancelling these bonuses or, better still, donating the money to a rail safety charity.
“We welcome his move to cut back on bonuses in the future but he has missed a golden opportunity to demonstrate in practical terms his genuine commitment to improve level crossing safety on his watch.”


WASHINGTON — The Air Force wants to retire 310 aircraft, including 42 A-10 Warthogs, as part of its fiscal 2024 budget to free up money for its modernization programs.
The proposed FY24 budget calls for buying 72 fighters — 48 Lockheed Martin-made F-35As and 24 Boeing F-15EX Eagle IIs — and spending more on the Northrop Grumman B-21 Raider stealth bomber, the Boeing E-7A battle management and command-and-control aircraft, the Northrop Grumman-made next-generation nuclear missile known as the Sentinel, and the network of drone wingmen the Air Force calls collaborative combat aircraft, meant to eventually fly alongside crewed fighters.
The Department of the Air Force is requesting $215.1 billion in 2024, $9.3 billion, or 4.5%, over the amount enacted for fiscal 2023. That includes a proposed budget of $185.1 billion for the Air Force itself, up $5.4 billion from 2023, and $30 billion for the Space Force, up $3.9 billion increase from 2023.
Nearly $5 billion of that total budget growth would go to research, development test and evaluation spending, bringing that section’s funding up to $55.4 billion in 2024.
Massive retirement wave
The Air Force’s plan to retire 310 aircraft in 2024 would be far greater than the 150 retirements it recommended in the 2023 budget, though Congress ultimately approved just 115 of those. If lawmakers approve all, or even most, of these retirements, it would represent a significant step forward in the service’s effort to weed out older, outdated airframes from its fleet.
If approved, the proposed retirement of 42 A-10s in 2024 would follow this year’s retirement of 21 Warthogs, and would leave the service with 218 of the attack aircraft. The Air Force has tried for years to retire the Warthog, arguing it would be too vulnerable against an enemy with advanced air defenses, and instead use the money to pay for newer fighters.
Air Force Chief of Staff Gen. CQ Brown told reporters at the Air and Space Forces Association’s symposium in Colorado March 7 the service wants to have all A-10s retired by 2029. Until recently, Congress has blocked the Air Force’s attempts to retire the A-10.
The Air Force also wants to retire 57 F-15 C and D fighters. Some those Eagle jets are around four decades old, and are reaching the end of their lives. The Air Force plans to bring on new F-35s, as well as newly-made F-15EX Eagle II fighters from Boeing, to replace those retiring fighters.
And the service is already withdrawing dozens of older F-15s from Kadena Air Base in Japan for retirement, replacing them with rotational fighters such as F-22s.
The Air Force is taking another shot at retiring 32 Block 20 F-22A Raptor fighters, which are not combat-capable and are now primarily being used for training purposes. Congress last year blocked the Air Force’s first attempt at retiring 33 of those F-22s.
The Air Force also wants to take more steps toward retiring the E-3 Sentry airborne warning and control system, or AWACS, aircraft in 2024.
The Air Force suggested retiring 15 of its 31 AWACS in its 2023 budget proposal, but lawmakers granted only 13 of those retirements. And Congress placed conditions on those retirements, requiring the service to produce a report on the E-3 retirement as well as its acquisition strategy for replacing it with the E-7.
Maj. Gen. Michael Greiner, the Air Force’s deputy assistant secretary for budget, told reporters in a March 10 briefing at the Pentagon that the Air Force believes it has met the requirements for the 13 AWACS retirements this year, which would bring its total fleet down to 18.
And in the 2024 budget proposal, the service said it wants to retire two more E-3s, leaving it with 16.
The Air Force has flown AWACS, with the unmistakable massive rotating radome mounted on its fuselage, since the 1970s. But the Air Force says the E-3′s air frame is increasingly difficult to keep flying and its sensor technology is outdated.
The Air Force plans to replace the AWACS with a new fleet of 26 E-7s by 2032. The service awarded Boeing a contract Feb. 28 to begin working on the U.S.’s first rapid prototype E-7, which is expected to begin production in fiscal 2025 and be fielded two years later.
In the briefing last week, Air Force Secretary Frank Kendall downplayed the likelihood of a capability gap emerging by retiring some AWACS before E-7s arrive to replace them.
“The E-3 is essentially not effective in the environments we’re most worried about,” he said. “The sensor is pretty ancient at this point, and the aircraft are very expensive to maintain.”
Retiring about half the current E-3 fleet will make available spare parts and other resources the Air Force can use to keep the remaining AWACS fleet in the air, Kendall added, so it can meet critical requirements such as NORAD’s air defense mission.
“It’s a question of making the transition as smooth as possible, freeing up the resources, starting to retrain the people, and then move them over [to E-7s] with a minimal operational impact from taking [AWACS] down,” he said.
The budget also proposes adding $254 million to continue rapid prototyping work on the Boeing E-7A, set to replace the aging E-3 Sentry airborne warning and control system aircraft, which would bring its FY24 funding to $681 million.
Greiner said the increased spending on E-7 would allow the Air Force to procure a second rapid prototype aircraft.
The budget calls for retiring the final 24 KC-10 Extender refueling tankers and the remaining 48 Block 1 MQ-9 Reapers. It would also retire the last three E-8 Joint Surveillance Target Attack Radar System, or JSTARS.
The Air Force also wants to retire 37 HH-60G Pave Hawk combat rescue helicopters, which are being replaced by HH-60W Jolly Green II helicopters made by Sikorsky and Lockheed Martin; 52 T-1 Jayhawk twin-engine jet trainers.
The Air Force has also decided to retire one B-1B Lancer bomber that suffered an engine fire in April 2022, rather than try to repair it. That would leave the service with 44 B-1s. The force aims to have the entire fleet retired by the early 2030s.
The proposal includes $4.8 billion in budget growth for 20 new or significantly boosted programs to transform the Air Force, including CCAs.
Funding for the CCA program would dramatically increase by $470 million to $522 million, to speed up development and testing of the autonomous drones. CCAs would fly as part of the Next Generation Air Dominance family of systems that will make up the Air Force’s sixth-generation fighter, as well as the F-35 and possibly other crewed aircraft.
That funding boost would cover the cost of developing the platform itself, developing the autonomous programming that will fly the drones, and standing up an experimental operations unit to plan how the service will use CCAs, Greiner said.
Greiner said the budget proposal is where the “acceleration” of work on CCAs can be seen. “We start to work on CONOPS, concept of operations, and how we would actually employ these,” he said.
Kendall said at AFA March 7 the service is planning for 1,000 CCAs as it starts to flesh out the basing needs, organizational structures, sustainment, and other requirements for such a fleet.
Kendall said in the March 10 briefing the Air Force wants to start production on CCAs by the end of the decade. He also said that, as the Air Force has increasingly discussed its plans to team crewed fighters with autonomous CCAs, the defense industry has ramped up its own research and development on this technology.
“I’m encouraged by what industry’s been doing on their own,” Kendall said. “This is a serious program. It’s a multibillion-dollar program.”
Even so, Kendall stressed each CCA must cost “a fraction” of an F-35, and said he has seen enough work to indicate that’s a “very reasonable goal.”
Kendall in September said the Air Force might hold a competition for CCAs in 2024, but the service has not settled on when that will happen.
“We’re going to start with competition and we’ll downselect at some point,” Kendall said. “We haven’t figured out exactly when that will be.”
The Air Force also wants to boost the budget for NGAD by $276 million in 2024 for technology maturation and risk reduction. That growth would bring its total research and development funding to more than $1.9 billion.
Kristyn Jones, the Air Force’s assistant secretary for financial management and comptroller, who is performing the duties of undersecretary of the Air Force, said the service needs to change to maintain its air dominance, which is being challenged by China. The service needs to look for cost-effective ways to carry out its missions, including teaming crewed fighters with the autonomous drones it refers to as CCAs.
“We cannot afford complacency, nor can we afford an Air Force composed largely of fighters that cost as much as our fifth-gen aircraft,” such as the F-35, Jones said.
More fighters, tankers and B-21 Raiders
The Air Force hopes to buy 48 F-35As in 2024, more than the 43 Congress approved in 2023.
The proposed budget would also pay for 24 F-15EX fighters and 15 Boeing KC-46A Pegasus refueling tankers in 2024, which would be the same number of each bought in 2023.
The Air Force originally planned to buy 144 F-15EXs, but in documents accompanying last year’s budget request, the service dropped that expected procurement to 80. In the March 10 briefing, Kendall told reporters the Air Force has partially reversed that cut, and now plans to buy 104 F-15EXs, though he did not explain why the Air Force’s stance had changed.
The Air Force also wants a $673 million spending boost for the B-21 in 2024, which would bring its procurement funding to more than $2.3 billion to continue low-rate initial production. An Air Force official told reporters the Raider’s procurement funding would buy more than one aircraft, but would not say how many, and also includes money for spares, training and advance procurement for long lead parts for future production lots.
The Air Force said it is still anticipating the B-21′s first flight sometime in 2023.
Northrop Grumman executives said in a January earnings call the company expected the Air Force to award the first B-21 production contract later this year. In a March 7 roundtable with reporters at the AFA conference, Kendall and Air Force acquisition chief Andrew Hunter declined to say if they were planning on a B-21 contract this year, and said they were focused on the first flight milestone.
Kendall told reporters in Colorado he wants the B-21 to avoid the problems associated with concurrency that plagued the F-35 in its development period. Concurrency refers to when an aircraft moves through development and into procurement at the same time.
“The excessive concurrency [of] the F-35 program, I was famous at one time for calling that acquisition malpractice,” Kendall said March 7. “We’re not going to do acquisition malpractice [with the B-21]. We want to have some confidence.”
The Air Force also plans to buy seven MH-139 Grey Wolf helicopters, two more than in 2023, and one E-11 aircraft that can carry out the Battlefield Airborne Communications Node, or BACN, mission. It also bought one E-11 in 2023.
The proposed budget includes about $8 million for the Air Force’s Next Generation Air Refueling System, or NGAS, so the service can finish its analysis of alternatives research. The Air Force announced at AFA it is revamping its plans for future aerial refueling tankers and will cut in half the number of tankers it planned to buy as an interim step.
The budget also would for the first time add procurement money for the LGM-35A Sentinel program, meant to succeed the Cold War-era Minuteman III intercontinental ballistic missile later this decade. Greiner said the proposed $539 million would pay for long lead item parts to help prepare for the first lot of the Sentinel, expected in fiscal 2026.
Stephen Losey is the air warfare reporter for Defense News. He previously covered leadership and personnel issues at Air Force Times, and the Pentagon, special operations and air warfare at Military.com. He has traveled to the Middle East to cover U.S. Air Force operations.


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Batman Kids Bat and Blue Pajama Set
If you see the Caped Crusader as he appears on the 100% polyester Batman Kids Bat and Blue Pajama Set then you know that justice is soon to follow! Or perhaps it already happened and this is the result. Either way, this fine shirt and pair of pants inspired by the Dark Knight is what you need for the little one. That way they'll appreciate justice and the Batman Kids Bat and Blue Pajama Set!
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domain: entertainment


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Blue Decorative Sand
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- 1.25 lbs
- AP Nontoxic
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Not suitable for children under 3 years of age.
Our Price:$3.19 Price was: $4.59 You Save: 31%You're only $50.00 away from FREE SHIPPING *(USA only)This Blue Decorative Sand adds a hint of sparkle to any project!
Both children and adults will enjoy using it in their sand art and craft and decorating projects. Add to everything from terrariums, to dioramas, and miniature beach scenes. It can add color and fill for vases. But can also be used to embellish cards, jewelry or sand painting. Teachers will enjoy using it for sensory tables.
Try using it in a wedding sand ceremony for a pretty alternative to candle lighting, too. Pick your favorite colors, one for each of you. Then instead of lighting unity candles, pour the sand to show how your two lives have now become one blended like the sand. Afterwards you have a beautiful accent piece for your home decor!
Do not use with water. Contains no free silica or quartz. AP Nontoxic: Approved by the Art & Creative Materials Institute (ACMI)Blue Decorative Sand - Average Rating 5 out of 5 ( 1 reviews).
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