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RNA-Seq analysis reveals that multiple phytohormone biosynthesis and signal transduction pathways are reprogrammed in curled-cotyledons mutant of soybean [Glycine max (L.) Merr.]
- Guixia Shi†1, 2,
- Fang Huang†2,
- Yu Gong2,
- Guangli Xu2,
- Jingjing Yu2,
- Zhenbin Hu2,
- Qingsheng Cai1Email author and
- Deyue Yu2Email author
© Shi et al.; licensee BioMed Central Ltd. 2014
Received: 18 January 2014
Accepted: 18 June 2014
Published: 21 June 2014
Soybean is one of the most economically important crops in the world. The cotyledon is the nutrient storage area in seeds, and it is critical for seed quality and yield. Cotyledon mutants are important for the genetic dissection of embryo patterning and seed development. However, the molecular mechanisms underlying soybean cotyledon development are largely unexplored.
In this study, we characterised a soybean c urled- co tyledon (cco) mutant. Compared with wild-type (WT), anatomical analysis revealed that the cco cotyledons at the torpedo stage became more slender and grew outward. The entire embryos of cco mutant resembled the "tail of swallow". In addition, cco seeds displayed reduced germination rate and gibberellic acid (GA3) level, whereas the abscisic acid (ABA) and auxin (IAA) levels were increased. RNA-seq identified 1,093 differentially expressed genes (DEGs) between WT and the cco mutant. The KEGG pathway analysis showed many DEGs were mapped to the hormone biosynthesis and signal transduction pathways. Consistent with assays of hormones in seeds, the results of RNA-seq indicated auxin and ABA biosynthesis and signal transduction in cco were more active than in WT, while an early step in GA biosynthesis was blocked, as well as conversion rate of inactive GAs to bioactive GAs in GA signaling. Furthermore, genes participated in other hormone biosynthesis and signalling pathways such as cytokinin (CK), ethylene (ET), brassinosteroid (BR), and jasmonate acid (JA) were also affected in the cco mutant.
Our data suggest that multiple phytohormone biosynthesis and signal transduction pathways are reprogrammed in cco, and changes in these pathways may partially contribute to the cco mutant phenotype, suggesting the involvement of multiple hormones in the coordination of soybean cotyledon development.
Cotyledon is the main nutrient storage area in soybean seed, which contains approximately 40% protein and 20% oil at maturity [1]. Flowering plants are divided into monocots and dicots based on the number of cotyledons. In Arabidopsis, as a paradigm for dicot embryonic development, cotyledons are specified from two lateral domains at the apical end of the embryo proper at the heart stage, showing bilateral symmetry [2]. Several studies have reported cotyledon mutants in Arabidopsis[3–7], Antirrhinum[8], tomato [9–11], and other plants [12]. These mutants have been used to identify a number of orthologous gene hierarchies that are involved in cotyledon development [13]. The Arabidopsis cup-shaped cotyledon (CUC) genes (CUC1, CUC2, and CUC3), belonging to the plant-specific NAC transcription factor, Picea glauca NAC01, and SHOOT MERISTEMLESS (STM) are required for shoot apical meristem (SAM) formation and cotyledon separation [4, 14–16]. STM induces the expression of CUC genes, and the induction is specific and independent of other meristem regulators [17]. STM negatively regulates ASYMMETRIC LEAVES1 (AS1), and the mutation of AS1 in Arabidopsis disrupts cotyledon development [18]. KNOTTED-like (KNAT6) is expressed at the boundary between the SAM and cotyledon later than STM and CUC, suggesting that KNAT6 plays a crucial role in SAM maintenance and boundary establishment in embryos via the STM/CUC pathway [19].
Phytohormones, primarily auxin, CK, BR, GA, ABA, and JA, orchestrate intrinsic developmental programs. Previous studies have greatly advanced the functions of individual hormone. During last two decades, extensive lists of genes involved in hormone synthesis, catabolism and signal transduction have been identified via the analysis of mutants [20–23]. Auxin plays a key regulatory role in the initiation of cotyledon in the apical margin of the globular embryo [24]. In many plant species, including Brassica napus[25], Arabidopsis[26], and Norway spruce[27], studies have shown that cotyledon and SAM development could be inhibited by various polar auxin inhibitors in embryogenesis. PIN auxin efflux regulators are the best characterised components of auxin transport. The polar location of PIN1 is associated with the position of auxin accumulation in incipient cotyledons, and auxin flows into the primordium interior during cotyledon outgrowth [28]. The pin1 pid double mutant completely lacks cotyledons and bilateral symmetry and exhibits increased CUC1, CUC2 and STM expression, indicating that directional auxin transport is important for the establishment of bilateral symmetry and the promotion of cotyledon outgrowth [26]. Similarly, the pid enp double mutant, which is similar to "laterne", also abolishes cotyledon development, reflecting failed auxin accumulation in the apex, and ENP is necessary for cotyledon development via the control of PIN1 polarity in the context of PID[29]. In addition to auxin, some studies have shown that CK affects cotyledon development. Exogenous application of benzyladenine (the synthetic cytokinin) onto hybrid larch somatic embryos reduced the cotyledon number [30]. The amp-arabidopsis mutant demonstrated polycotyly, indicating an elevated cytokinin level [31]. Arabidopsis ESR1 and ESR2, whose expression levels are induced by cytokinin, not only control shoot regeneration but also play a role in cotyledon development [32, 33].
In this study, we characterised a soybean mutant named cco, which displayed curled cotyledons. The cco embryos at the torpedo stage resembled the "tail of swallow". High performance liquid chromatography (HPLC) analysis showed that cco seeds contained higher IAA and ABA levels, but lower GA3 level. Transcriptome analysis revealed that multiple phytohormone biosynthesis and signal transduction pathways were reprogrammed in cco.
Results and discussion
Embryogenesis in the ccomutant
To determine the embryogenesis defects in cco mutant, we traced its phenotype back to the globular stage of embryogenesis. In higher plants, embryogenesis can be conceptually divided into three overlapping phases: morphogenesis, maturation and desiccation [35]. In general, for the wild type at the globular stage, an embryo comprises a spherical embryo proper and a suspensor (Figure 1D). The embryo proper is spherical during the pro-embryo and globular stage and eventually forms cotyledons, shoot meristem and hypocotyl region around the apical-basal axis, and this structure is connected to the embryo sac via the suspensor. During the globular-heart transition phase, a dramatic change occurs: two of the cotyledon primordia are symmetrically initiated from the lateral domains at the apical end of the heart-shaped embryo proper (Figure 1E). Subsequently, the cotyledons and hypocotyl elongate, and the embryo enters the torpedo stage in which the cotyledons continue to rotate. The morphogenesis phase of embryogenesis ends at the cotyledon stage (Figure 1F and 1G).
However, the development of embryos in cco was slower than that in WT (Figure 1). When WT embryos were at the heart stage, cco embryos remained morphologically at the globular stage (Figure 1E and 1H). The embryo proper in cco appeared smaller than that in WT (Figure 1D and 1H). At the heart stage, cotyledons were initiated from the cotyledon primordia. Figure 1E and 1I showed that the cotyledon initiation positions were identical in both the WT and cco. However, the WT cotyledons grew upwards, while that of the cco mutant demonstrated outward growth. Subsequently, the bifurcation of the cotyledons at the torpedo stage in the cco mutant was larger, and the tips of the cotyledons were sharper (Figure 1F to 1G and 1J to 1L). At the torpedo stage, cco mutant embryos resembled "tail of swallow", while the radicle and shoot apical meristem were also present and appeared normal. All in all, cco showed abnormal embryogenesis, especially at the torpedo stage, when the cco cotyledons became more slender and grew outward. However, SAM and the polar axis were normal. Cotyledons and leaves share homology in plants [13]. Cotyledon initiation and development in cco were defective, while leaf development was correct, suggesting partial homology between leaf and cotyledon.
Increased free IAA and ABA levels, while decreased GA3 level in cco
In addition, the endogenous GA3 and ABA content in WT and cco seeds at 7 DAF were quantified. The measurements demonstrated that the ABA content was significantly increased, while the GA3 content was decreased in cco (Figure 2). The increased ABA/GA ratio may lead to a low germination rate for the cco mutant.
Auxin sensitivity was depressed in cco
RNA-Seq of WT and the ccomutant
Summary of RNA-seq data from WT and cco pods at 7 DAF
Total reads
Total base pairs
Total mapped reads
Percent mapped
Unique match
Percent unique match
Fold changes of 15 genes selected from RNA-seq data
Gene ID
Gene description
Fold change
Transmembrane amino acid transporter protein
Transmembrane amino acid transporter protein
Membrane transport protein
WRKY transcription factor
HLH transcription factor
bZIP transcription factor
NPH3 family
YABBY protein
tryptophan synthase β-chain
ubiquitin-protein ligase activity
LBD transcription factor
DBB transcription factor
HLH transcription factor
In addition, we used semi-quantitative RT-PCR to analyse the expression of four selected genes (Glyma03g02620, Glyma14g39300, Glyma11g11580, Glyma17g14710) in WT and cco seeds at 7 and 15 DAF (Additional file 2: Figure S1C). Using qRT-PCR, we examined the expression of three genes (Glyma03g02620, Glyma14g39300, Glyma11g11580) in the roots, stems, leaves, flowers and seeds at the various developmental stages of WT and cco, and the results showed these genes were differentially expressed in these tissues (Figure 5B), indicating some of the DEGs that we obtained were differentially expressed in other tissues.
Functional classification of differentially expressed genes
Biological interpretation of the 650 DEGs was further completed using KEGG pathway analysis. Many of the DEGs were mapped to pathways essential for plant growth and development. Overall, 98 pathways were affected in the cco mutant, and some of these pathways were consistent with biological processes previously revealed in the GO analysis. The most frequently represented pathways primarily involved in secondary metabolism, including plant hormone biosynthesis and signal transduction (20.46%), such as auxin, CK, GA, ABA, ET, BR and JA (Additional file 3). Asakura et al. found that genes expressed in the early pod stage of soybean were the most numerous, compared with 2 and 5 mm seeds, and the number of expressed genes gradually decreased with seed development [39]. Commonly expressed genes in these stages accounted for 70–95% of the expressed genes in each stage, and only a small number of genes were differentially expressed, which mainly included β-conglycinin, lipid synthesis, lipoxygenase and seed-maturation proteins [39]. Therefore, most DEGs obtained in this study, including genes that were related to hormone biosynthesis and signalling, could still be identified in the early seed stage. However, some DEGs may have been missed because of tissue-dependent gene expression.
The role of GA in embryo morphogenesis has not yet been determined. In general, GA is required for seed development, which is determined through the analysis of GA-deficient mutants. In pea (Pisum sativum L.), the GA-biosynthesis mutant lh has provided the best evidence for the physiological role of GA in seed development. The LH protein, which encodes ent-kaurene oxidase (KO), is required for the three-step oxidation of ent-kaurene to ent-kaurenoic acid as an early step in the GA biosynthesis pathway altering seed development [45]. KS (ent-kaurene synthase) and KO are key enzymes in the GA biosynthetic pathway, and both are encoded by a single gene in soybean. GA2ox (GA2-oxidase) plays an important role in the last step of GA biosynthesis and catalyses the inactivation of GAs by conversion to bioactive GAs, thereby affecting the amount of bioactive GAs. Overexpression of GA 2 ox genes results in reduced GA level in Arabidopsis[46], rice [47], and poplar [48]. DELLA-domain proteins are transcriptional regulators that repress GA responses, and these proteins are rapidly degraded in response to GA. In this study, one KO gene and one KS gene were downregulated in the cco mutant, suggesting that an early step in GA biosynthesis was blocked (Additional file 5). In addition, four GA 2 ox genes and three DELLA genes were up-regulated, suggesting that the rate of conversion of inactive GAs to bioactive GAs was limited for the cco mutant (Figure 7 and Additional file 5).
ABA not only plays an important role in the stress responses and plant tolerance, but also controls seed development and germination. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) is involved in a rate-limiting step for ABA biosynthesis. Recently, Martínez-Andújar et al. reported that the NCED6 induction enhanced seed dormancy in Arabidopsis[49]. Here, we showed that NCED genes were up-regulated in the cco mutant, suggesting that ABA synthesis was promoted. However, another gene encoding abscisic acid 8′-hydroxylase, which catalyses a key step in ABA catabolism, was also up-regulated (Additional file 6). Snf1-related protein kinases (SnRK2s) are key positive regulators in ABA signal transduction pathway. In the absence of ABA, protein phosphatases type 2C (PP2Cs) physically interact with SnRK2s and negatively regulate ABA responses. However, interactions between PP2Cs and SnRK2s are disrupted when an ABA molecule binds to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) receptors, leading to structural changes in these receptors. When SnRK2s are released from PP2C inhibition, their downstream targets, including ABA-Responsive Element Binding Factors (ABFs), are activated [50]. Fujii et al. constructed an Arabidopsis line carrying mutations in all 10 members of the SnRK2 family. The decuple mutant snrk2.1/2/3/4/5/6/7/8/9/10 (srk2g/d/e/a/h/e/f/c/j/b) was defective in gene regulation and ABA accumulation [51]. In our data, the PP2Cs, SnRK2 and ABF genes were up-regulated in the cco mutant (Figure 7). In summary, ABA biosynthesis, catabolism and signalling were enhanced in cco.
In addition to auxin, GA and ABA, the pathways of other hormone biosynthesis and signal transduction (such as CK, ET, BR, and JA) were also influenced in the cco mutant. CK and ET, as major classes of plant hormone, are involved in various aspects of plant development, including organ development [52, 53]. In cco, we found two genes encoding adenosine-phosphate isopentenyltransferase (IPT), a rate-limiting enzyme in CK biosynthesis, expressed at significantly higher levels [54, 55] (Additional file 7). In CK signalling, type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) are transcriptional activators that regulate cytokinin targets, including type-A ARRs [56]. We observed the up-regulation of two type-B ARR genes (Figure 7). Aminocyclopropane-carboxylate oxidase (ACO), an "ethylene-forming enzyme" (EFE), catalyses the final step in ethylene biosynthesis, converting l-amino-cyclopropane-1-carboxylic acid (ACC) into ethylene, CO2, and cyanide. Four ACO genes were up-regulated in cco (Additional file 8). Antisense constructs of ACO genes can notably reduce ET production [57]. ET signalling was also influenced in the cco mutant, and the expression levels of ethylene-related genes encoding serine/threonine-protein kinase (CTR1), mitogen-activated protein kinase 6 (MPK6), and ethylene-responsive transcription factor (ERF1/2) were altered in cco (Figure 7 and Additional file 8). BRs regulate embryonic and post-embryonic development, adult homeostasis and expression of numerous genes [58]. Plant steroid hormones are recognised by brassinosteroid-insensitive 1 (BRI1), whose kinase activity is activated by exogenous BR application [59]. We also found that the expression levels of thirteen BR1 and four BRI1 Associated receptor Kinase 1 (BAK1) genes in cco were higher than those in WT (Figure 7). JA regulates plant growth and stress responses, biosynthetic pathway of which starts with α-linolenic acid [60]. The DEGs in JA biosynthesis and signalling pathway included those that encode phospholipase A1, 12-oxo-phytodienoic acid reductase, lipoxygenase, JASMONATE-ZIM (JAZ) and MYC2 proteins [61] ( Additional file 9 and Figure 7).
Auxin, BR, CK and GA are generally considered major developmental growth regulators, whereas ABA, ET and JA are involved in stress responses, all of these hormones converge on auxin [62, 63]. Auxin is not only a hormone but also a morphogen. Auxin accumulation is controlled by auxin biosynthesis and metabolism, the conjugation/deconjugation of active auxin to/from its inactive conjugated form and auxin transport [64–66]. Auxin maxima and activity have been implicated in embryo development and patterning [37]. In cco, the free IAA level and auxin polar transport were affected. Polar auxin transport promotes the formation of local auxin maxima and gradients within tissues that play important roles in auxin action, such as the patterns of cell division and differentiation in the root meristem [67] and the establishment of cotyledons [26, 29]. Plants show directional growth in response to environmental stimuli such as light or gravity (tropisms). It was found that auxin played an important role in tropisms [68] and unequal auxin distribution across organs may lead their bending [69]. The cotyledons of cco showed outward bending, which allowed us to hypothesise that PAT might cause an auxin imbalance between the abaxial and adaxial side in cotyledons of the cco mutant, resulting in the adaxial side developing faster than the abaxial side. However, in soybean embryogenesis, the endosperm surrounds the embryo and cotyledons, the curled cotyledons observed in the cco mutant might reflect the fetter of the endosperm. Many signals can regulate auxin polar transport including stress, protein phosphorylation, reactive oxygen, flavonols and other hormones [70–73], which among these signals affect(s) PAT in cco require further investigation. In cco, only the shape of cotyledons not including leaves was abnormal, indicating that signal(s) or mutated genes regulating PAT were specific for cotyledon initiation and growth.
GA and ABA have mainly been implicated in later stages of embryo development, acting antagonistically in the regulation of seed germination [74]. Compared with WT, cco showed higher IAA and ABA levels, whereas a lower GA3 level. Several reports indicate that auxin and GA overlap in the regulation of multiple aspects of plant development, and there is positive cross-talk interaction between them. Willige et al. have reported that a mutant deficient in GA biosynthesis and signalling showed a reduced PIN protein level, which could be recovered by exogenous GA application. Moreover, GA could interact with PIN1 and promote cotyledon differentiation [75]. Auxin can also induce GA biosynthesis. Root cells with lower auxin level have reduced GA biosynthesis [76, 77]. Interestingly, our results demonstrated a lower GA3 but higher IAA level in cco seeds. But beyond that, cco seeds had higher ABA content, which might correlate with lower GA3 level and may be the reason for lower germination rate in cco. Consistently, ABA negatively regulates bioactive GA level during seed germination.
Multiple phytohormone biosynthesis and signalling pathways are reprogrammed in the cco mutant, which is consistent with our HPLC analysis. Our results indicate that curled-cotyledons and reduced seed germination rate may result from the biosynthesis and signalling disorder of multiple phytohormones. Accumulating evidence supports the idea that hormones not only act in a linear pathway but also function in a complex network of feedback regulation and cross talk within or between different hormone pathways. At present, the studies of molecular mechanisms underlying hormonal cross talk mainly focus on postembryonic development, including the roots, shoots and leaves. However, adequate models of the hormonal network involved in cotyledon development are not well established in plants. Analysis of cco may provide a promising frame for further studies of hormone interactions and offer the potential discovery of new genes controlling cotyledon development in plants.
Plant materials
Soybean seeds from wild type cultivars Nannong 94–16 and its cotyledon mutant cco were provided by Soybean Research Institute, Nanjing Agricultural University, China. The seeds were cultivated in field under natural conditions in Nanjing, China. Total RNA was extracted from the WT and cco tissues, including the roots, stems, leaves, flowers and seeds, which were frozen in liquid nitrogen and stored at −80°C until further analysis.
Plant paraffin section
For paraffin sectioning, wild type and mutant seeds at various developmental stages were fixed in Carnoy's fluid (ethanol/glacial acetic acid, 3:1) at room temperature followed by dehydration in a graded ethanol series, staining with 1% haematoxylin solution, and clearance with xylene. The seeds were embedded in paraffin and sectioned at 10 to 15 μm (Leica, RM2135).
Semi-quantitative RT-PCR and quantitative RT-PCR (qRT-PCR)
To analyse the gene expression in soybean, total RNA was extracted with the Plant RNA Extract Kit (TianGen, Beijing, China) according to the manufacturer's instructions. PCR amplification was performed with PrimeScript 1st Strand cDNA Synthesis Kit (Takara). qRT-PCR was performed as described previously [78]. The Gmtubulin (GenBank no.AY907703) and Gmactin (GenBank no.XM_003531354) genes were used as reference genes for qRT-PCR and semi-quantitative RT-PCR, respectively. The primers used in this study were provided in Additional file 10.
Extraction of endogenous free IAA, GA3, ABA and HPLC analysis
A total of 0.5 g fresh seeds at 7DAF from at least 10 individual lines of WT and cco were ground in liquid nitrogen, phytohormones were extracted in 10 milliliter cold 80% (v/v) methanol and 1% (w/v) polyvinylpyrrolidone was added as an antioxidant. After 24 h of incubation at 4°C, the samples were centrifuged for 15 min at 12,000 g at 4°C. The supernatant was retained, and the pellet was re-extracted with 10 ml of cold 80% (v/v) methanol and recentrifuged as indicated above. The supernatants were pooled. After evaporation under a N2 gas flow, the pH of the remaining water phase was adjusted to 3.0. IAA, GA3 and ABA purification was performed using acetyl acetate. The acetyl acetate was also evaporated under an N2 stream. The resulting dried precipitate was collected in 0.5 ml of 100% methanol, filtered through 0.45 μm filter membrane and submitted to HPLC (Shimadzu, Kyoto, Japan) analysis. HPLC analysis was performed using an Alltima C18 column (4.6 mm × 250 mm). The IAA and ABA concentration was determined at 254 nm, and the GA3 concentration was determined at 210 nm. Hormone measurements were conducted in triplicate and subjected to statistical analysis.
IAA sensibility assay
WT and cco seeds were germinated in moistened vermiculite at 30°C. After 72 h, the seedlings were sprayed with 2.5 × 10−3 M 2,4-D solution (pH 6). The intact hypocotyls were then segregated from the seedlings and incubated in 10 ml of incubation medium in the absence or presence of 5 × 10−5 M and 2 × 10−4 M 2,4-D (pH 6) at 30°C with continuous shaking. At 0.5 and 1 h, the 1.2-cm sections obtained from the elongating region of the hypocotyls (0.5-1.7 cm below the cotyledons) were excised. The hypocotyl sections were rinsed in deionised distilled H20 and immediately frozen in liquid N2. The sections were stored at −70°C until further use for RNA extraction.
cDNA library construction for RNA-Seq and sequencing
Beads with oligo (dT) were used to isolate poly (A) mRNA after total RNA was collected. Fragmentation buffer was added to digest the mRNA to generate short fragments. These short fragments were used as templates, and random hexamer-primers were used to synthesise first-strand cDNA. Second-strand cDNA was synthesised in a reaction containing buffer, dNTPs, RNase H and DNA polymerase I. Short fragments were purified using the QIAquick PCR extraction kit (QIAGEN) and eluted in EB buffer for end preparation and poly (A) addition. Subsequently, the short fragments were ligated with sequencing adaptors. For PCR amplification, we selected suitable fragments to use as templates based on the results of agarose gel electrophoresis. The libraries were sequenced using the Illumina HiSeq™ 2000 system.
Raw read filtering and mapping to the reference genome and genes sequences
Dirty raw reads were discarded via the following four steps: 1. reads with adaptors were removed; 2. reads with unknown nucleotides larger than 5% were removed; 3. reads with low quality (more than half of the base quality less than 5) were removed; 4. clean reads were obtained. Clean reads were mapped to the reference genome and genes sequences respectively using SOAPaligner/soap2. Mismatches with no more than m (m default is 5) bases were used in the alignment. All subsequent analyses were based on clean reads.
Gene expression annotation
Gene coverage is the percentage of a gene covered by reads. This value equals the ratio of the number of bases in a gene covered by unique mapping reads to the number of total bases in that gene. The UniGene expression was calculated using the RPKM method (Reads Per kb per Million reads). The RPKM method eliminated the influence of different gene lengths and sequencing discrepancies on the gene expression calculations. Therefore, the calculated gene expression could be used to directly compare differences in gene expression between the samples. If there was more than one transcript for a gene, the longest transcript was used to calculate the expression level and coverage.
Differentially expressed gene analysis
Using "The significance of digital gene expression profiles" [79], we identified differentially expressed genes between WT and the cco mutant based on the following criteria: (FDR) < 0.001 and |log2Ratio| ≥ 1.
GO enrichment and KEGG pathway analysis
The differentially expressed genes were subjected to GO enrichment analysis. The analysis first mapped all DEGs to GO terms in the database (http://www.geneontology.org/), calculating gene numbers for every term, followed by the ultra-geometric test to identify significantly enriched GO terms in DEGs compared with the genome background. KEGG Pathway (http://www.genome.jp/kegg/pathway.html) enrichment analysis was used to identify significantly enriched metabolic or signal transduction pathways in DEGs compared with the whole genome background.
Availability of supporting data
The RNA-seq data of this article have been deposited in the Gene Expression Omnibus (GEO) Database, with the following access number: GSE58354 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58354). Other supporting data are included as additional files.
This study was supported in part by funding from the National Basic Research Program of China (973 Program) (2010CB125906), the Key Transgenic Breeding Program of China (2013ZX08004-003), and the National Natural Science Foundation of China (31171573, 31371644).
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


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Happy Thanksgiving!
You have to try this delicious recipe for Pumpkin Pancakes that can be eaten for breakfast, as a side for dinner or even as a snack! Thank you Diane- I love your Practical Paleo book!
Pumpkin Pancakes
Prep Time: 10 minutes
Cooking Time: 20 minutes
Yield: Approximately 8 small
Pancakes or 2 servings
|4 eggs
|1 teaspoon pumpkin pie spice
|½ cup canned organic pumpkin
|1 teaspoon cinnamon
|1 teaspoon pure vanilla extract
|¼ teaspoon baking soda
|1 tablespoon of organic molasses
|2 tablespoons butter, ghee or coconut oil (add extra for pan frying)
Whisk the eggs, canned pumpkin, pure vanilla extract, and molasses together. Sift the pumpkin pic spice, cinnamon, and baking soda into the wet ingredients.
Melt 2 tablespoons of butter in a large skillet over medium heat. Then, mix the butter into the batter.
Grease the skillet and spoon batter into the skillet to make pancakes of your desired size. When a few bubbles appear, flip the pancakes once to finish cooking.
Serve with grass-fed butter or ghee and cinnamon or sliced bananas.
Recipe is by my friend, Diane Sanfilippo, author of Practical Paleo. Some ingredients have been revised for Dr. Susanne's community!

subset: commoncrawl


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By Robert Barnes and Sandhya Somashekhar
Washington Post Staff Writer
Thursday, August 5, 2010; A01
A federal judge in California ruled Wednesday that the state's ban on same-sex marriage violates the constitutional right to equal protection. The decision is the first step in a legal struggle that is widely expected to end at the Supreme Court.
In the nation's first federal trial on same-sex marriage, Judge Vaughn R. Walker said that California's Proposition 8 "fails to advance any rational basis in singling out gay men and lesbians for denial of a marriage license."
"Indeed, the evidence shows Proposition 8 does nothing more than enshrine in the California Constitution the notion that opposite-sex couples are superior to same-sex couples," wrote Walker, chief judge of the U.S. District Court for the Northern District of California in San Francisco. In 2008, 52 percent of the state's voters approved Proposition 8, an amendment to the state Constitution.
(Read Judge Vaughn's full ruling)
The decision set off joyous celebrations in California and elsewhere, outrage among conservative activists and a solemn determination among those opposed to same-sex marriage to appeal the decision to the nation's highest court. Walker said opponents have until Friday to convince him that the decision should be stayed during the appeals process, or the marriages will resume. About 18,000 same-sex couples in California married in the five-month period between the time the state Supreme Court allowed such unions and the voter initiative curtailed them.
(Post poll: Do you agree with the decision?)
The U.S. Supreme Court has never directly addressed the question of same-sex marriage, and most think that the case is headed there after it is appealed to the U.S. Court of Appeals for the 9th Circuit.
Walker, a veteran judge who was first nominated by President Ronald Reagan and confirmed under President George H.W. Bush, accepted virtually every argument advanced by opponents of Proposition 8. They said the prohibition on same-sex marriage violated the U.S. Constitution's 14th Amendment guarantees of due process and equal protection.
(Appeal of ruling could delay weddings)
"To characterize plaintiffs' objective as 'the right to same-sex marriage' would suggest that plaintiffs seek something different from what opposite-sex couples across the state enjoy -- namely, marriage," Walker wrote. "Rather, plaintiffs ask California to recognize their relationships for what they are: marriages."
(Obama: I'm against gay marriage and Prop 8)
Walker said the decisions of voters must be respected. But because the right to marriage is fundamental, he wrote, "voters' determinations must find at least some support in evidence. This is especially so when those determinations enact into law classifications of persons."
The ban's supporters accused Walker of interfering with a decision that ought to be left to the people, potentially inflaming the culture wars much as the Roe v. Wade decision of 1973 did in legalizing abortion.
"Under our Constitution, the definition and meaning of marriage is a decision left in the hands of the people, not given to that small fraction of the population who happen to be judges," Robert George, chairman of the anti-gay-marriage advocacy group National Organization for Marriage, said in a statement.
(Seea roundup of religious responses to Proposition 8)
One of the attorneys who represented Proposition 8's backers said they would immediately appeal the ruling.
"What's at stake here is bigger than California," Andrew Pugno said in a statement released by the Alliance Defense Fund, an advocacy group participating in the case. "Americans in numerous states have affirmed -- and should be allowed to continue to affirm -- a natural and historic public policy position like this. We are prepared to fight all the way to the U.S. Supreme Court if necessary."
(See a roundup of religious responses to Proposition 8 ruling)
Five states and the District of Columbia allow same-sex marriages, and 30 states have constitutional prohibitions such as California's.State officials respond
Pugno's group defended the voter initiative because the state declined to. Even though he is the named defendant in the suit, Gov. Arnold Schwarzenegger (R) said Wednesday, "For the hundreds of thousands of Californians in gay and lesbian households who are managing their day-to-day lives, this decision affirms the full legal protections and safeguards I believe everyone deserves."
State Attorney General Jerry Brown (D), who had declined to defend Proposition 8, said that Walker "came to the same conclusion I did."
It was the second major court decision in recent weeks to go the way of gay rights activists.
Last month, a federal judge in Massachusetts struck down a 1996 law barring the federal government from recognizing gay marriages. He said it was unconstitutional to disqualify couples in states where such marriages are allowed from receiving marriage-based federal benefits under the Defense of Marriage Act.
(Read more about the Massachusetts case.)
Challengers of Proposition 8 were represented by a political odd couple: conservative legal stalwart Theodore B. Olson and liberal trial lawyer David Boies. The two men were on opposite sides in arguing Bush v. Gore in 2000 before the Supreme Court, and Olson went on to become President George W. Bush's solicitor general.
(Read about how Olson's role in the case surprised conservatives.)
Washington lawyer Charles J. Cooper, who usually is on the same side as Olson, represented the group behind Proposition 8.
Walker had presided over a 2 1/2-week trial in January, but procedural matters delayed closing arguments until June.
Olson and Boies provided an elaborate record for Walker and future courts, with their own clients, social scientists, psychologists and experts on marriage providing testimony. Cooper called only two witnesses, and, in his questioning at closing arguments, Walker seemed peeved that Proposition 8's backers did not do more.
In his 136-page opinion, Walker returned to that, saying, "Proponents' evidentiary presentation was dwarfed by that of plaintiffs."Backers' arguments
Cooper had argued that it is "crucial to the public interest" to limit marriage to opposite-sex couples. It is "fundamental to the very existence and survival of the human race," he said, that society promote marriage to ensure that procreative relations are in "enduring, stable unions," with a goal that children be raised by both parents.
But Walker said California had no requirement that couples who wanted to marry be capable of procreating or willing to procreate. He echoed the arguments of Olson and Boies that laws limiting marriage to opposite-sex couples were comparable to previous restrictions that disallowed interracial marriage or subjugated a woman to her husband.
The exclusion of homosexuals from marriage "exists as an artifact of a time when the genders were seen as having distinct roles in society and in marriage," Walker wrote. "That time has passed."
Curt Levey, executive director of the conservative Committee for Justice, denounced the ruling as "a particularly shameless example of judicial activism." He also raised the question of Walker's sexuality. The San Francisco Chronicle and the Los Angeles Times have reported that Walker is gay. The judge has neither confirmed nor denied those reports.
Boies called the ruling a "great result" saying that "discrimination against gays and lesbians is the last area where you have state-enforced discrimination, and this ruling goes a long way to eliminating that."
Boies and Olson were representing two couples -- lesbians from Berkeley and gay men from Burbank -- who had tried to marry but were denied licenses.
When asked at a news conference when the couples planned to marry, plaintiff Paul Katami responded, "I need time to order a cake."
The case is Perry v. Schwarzenegger.
Read more on PostPolitics.com.


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On March 3, we told you about H.R. 1424, the the Paul Wellstone Mental Health and Addiction Equity Act of 2007. This measure passed the House on March 5 by a vote of 268-148.
A Motion to Recommit the bill to committee was offered by Republicans in an effort to amend the bill "forthwith" by replacing it with the bipartisan Senate-passed Mental Health Parity bill (S.558) and clarifying language on abortion-related coverage.
The Senate mental health parity bill does not include any of the references to the DSM-IV that were included in the House bill and removes the employer mandate regarding coverage. Therefore, the Motion to Recommit would resolve concerns regarding conscience protections for employers and health insurance groups.
A conference will be held on both the Senate and House versions in order to resolve differences between the bills. Members must be aware that the language in the House version is dangerous. Not only does it cover such mental disorders as gender identity disorder and pedophilia, but it does not include a conscience clause. Further, abortion proponents have asserted there can be "therapeutic abortions" done to "preserve women's mental health."


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Changes expected at MLB Well-being of college athletes secondary Cartoon: Hoops scrutiny Brooks NBA's chief innovation officer MAC at home in non-Saturday slots Rich Eisen's Top 5 Labor and Agents: Player's NFL lawsuit Lefton: Kaepernick's tough task Legends selling PSLs for Chargers Nomadic's Super Bowl party
SBJ/Aug. 25-31, 2014/CollegesPrint All
ASU draws on NFL experience
From the time Ray Anderson considered leaving the NFL to go to work at Arizona State University, he's heard the Sun Devils referred to as a sleeping giant.
The school is the largest public university in the country, yet ranks in the bottom half of the Pac-12 in annual revenue. It attracts a Who's Who of athletic directors, only to see them leave and become collegiate power brokers elsewhere.
Among athletic departments that could be considered an enigma, ASU is the most curious of cases. More than 200,000 alumni live within a half-hour of the campus, but the Sun Devils routinely play before less-than-capacity crowds.
Ray Anderson (right) introduces ASU baseball coach Tracy Smith in June.
Photo by:ARIZONA STATE UNIVERSITY
"The sleeping giant stuff has got to go away," said Anderson, who studied political science at Stanford and earned his law degree from Harvard. "We're waking up."
The alarm began to go off three months ago when Anderson, former executive vice president of operations at the NFL, reached into his past to hire Greg McElroy from the Dallas Cowboys.
In subsequent weeks in June, Anderson again dipped into his past and brought in two more veteran administrators with NFL ties — Scottie Graham from the NFL Players Association and David Cohen, who spent time with the Falcons and Vikings before moving to the Valley of the Sun.
Each hire filled a newly created position.
McElroy joined ASU in May as chief business development officer and associate vice president. The former head of the Cowboys' sales and marketing will bring his trademark enthusiasm in spearheading all aspects of revenue generation and marketing.
Cohen, a senior associate AD, was selling premium products at the Vikings' new stadium for Van Wagner Sports and Entertainment, and will work on stadium sales, ticketing and Olympic sports for the Sun Devils.
At the start of this year's football season, ASU's athletic department has begun to look like an NFL front office. And that was by design.
"I wanted to bring in guys with experience and the mindset of urgency that comes from the pro level," Anderson said. "These hires elevate us, in terms of how we run this place like a business that creates revenue. I don't want to have to keep running back to the university for subsidies."
ASU's $64 million annual budget falls far behind the financial pacesetters in the Pac-12, especially considering that close to $10 million of the Sun Devils' revenue comes from campus subsidies. Annual revenue at Southern California, California and Stanford surpasses $90 million, while Oregon, UCLA and Washington top $80 million (see chart below).
Anderson doesn't see any reason why the Sun Devils can't be in that range, and that was a primary motivation in hiring McElroy and Cohen, two proven revenue generators from the NFL.
"They're charged with improving existing revenue streams and finding new streams," Anderson said. "This is part of the process of doing things differently. We're not going to be about the status quo."
McElroy and Cohen are joining the Sun Devils at a good time. The school initiated a $225 million renovation of Sun Devil Stadium earlier this year, with architect HNTB and local firm Gould Evans working on the design. Capacity will shrink from 70,000 to 60,000, but the premium products will increase significantly.
"It's not just a renovation," McElroy said. "It's a reinvention of the stadium. We'll have what amounts to a brand-new stadium by the kickoff of the 2017 season."
They are in the process of developing a plan and pricing for suites, club seats and possibly loge boxes that will give the stadium far more revenue-generating power.
It's not unlike the role McElroy played in the development of the Cowboys' AT&T Stadium.
"What I took away from having done this before is to push the envelope, as far as developing revenue," McElroy said. "Let's not take the safe way out. Let's be aggressive when we price it. As Jerry [Jones] used to say, 'Let's get as close to breaking the glass as we can.' And what we wound up with [at AT&T Stadium] was one of the most successful projects in all of sports."
One of the core challenges for Arizona State has been selling a college product in a pro sports town. The Phoenix area also is home to an eclectic mix of retirees who don't necessarily have a connection to the school.
Some of the brightest and most influential ADs in the country — Duke's Kevin White, Ohio State's Gene Smith and, most recently, Texas' Steve Patterson — tackled those challenges at ASU with varying degrees of success before leaving for other high-profile jobs. The turnover in the Sun Devils' AD office, however, never dissuaded Anderson, 60, who calls ASU a "destination job."
Countering those endemic challenges in the desert starts with creating a pro-like game-day experience with the premium products, connectivity and customer service fans have come to expect across town.
"We're looking at everything that touches the fan," Cohen said. "We just had a meeting about traffic patterns. We'll make sure, however we price it, that it matches the expectations and the amenities we can provide."
With a total student enrollment approaching 80,000 this year, ASU is cultivating an alumni base that will serve as a massive customer base in the future.
But Anderson shows little interest in looking too far down the road. It's with a "mindset of urgency" that he's rousting the sleeping giant.
Anderson has charted a course "to try new things, be innovative," he said. At a time when many athletic departments are contemplating fewer sports, Anderson wants to add to ASU's 22 varsity sports.
He's also working with McElroy and Cohen to evaluate the school's multimedia rights deal with IMG College and its ticketing deal with Aspire Group. McElroy said he'll be working with IMG College sales executives on a daily basis, while Cohen will take on more of a day-to-day role in the ticketing operation.
Across campus, athletics is working with university Chief Marketing Officer Dan Dillon on a plan to create campuswide sponsorships for corporate partners. Such a program would include combined sponsorship assets from athletics and the university side.
"The enticing part of this job, in addition to working with Ray, is that ASU has a great brand, but there's also tremendous opportunity for growth," McElroy said. "I can't wait to see where we are three years from now."
Retailers buy into CLC College Colors platform
Gary Dubiel walked the floor at the massive, two-story Barnes & Noble in Baton Rouge, La., last week, impressed by the square footage the bookstore had devoted to LSU gear.
Underneath a purple and gold tent were logo chairs, wine glasses, Tervis tumblers, car flags, T-shirts and just about anything else a licensee could make with the Tigers' marks.
"The back-to-school period is our biggest promotional time," said Dubiel, who joined
This college football season marks the 10th year of CLC's College Colors Day, which designates the Friday of the first college football weekend as the day to wear college colors. "Back to school" had always provided a sales boost, but College Colors Day gave CLC an annual marketing platform with TV and online advertising, special point-of-
Dubiel said extending distribution for the licensees of CLC's 200-plus college clients is the chief objective.
Bringing the Stage Stores group and its 900 stores, which include Peebles and Goody's outlets, into the mix this year stands as one of CLC's big wins for 2014. Kohl's activation across 490 stores is another new chain in the College Colors Day promotion. CLC said more than 12,000 store locations are participating with in-store signage and online awareness, such as College Colors Day banner advertising. Online advertising on ESPN.com and print ads in ESPN The Magazine are promoting the day as well.
Sales through the first three weeks in August show that the supercenter stores — Wal-Mart, Sam's Club, Target, Meijer and others — are seeing the biggest gains, with sales up by nearly 20 percent over last year, Dubiel said. The big sporting goods channels — Dick's, Academy, Sports Authority — are pushing double-digit growth as well.
The bigger stores that can devote ample floor space to displays — like the Barnes & Noble in Baton Rouge — tend to move more product, Dubiel said.
For some of the larger retailers, CLC has created unique marketing platforms for its point-of-purchase displays. Wal-Mart, for example, encourages shoppers to wear their "Saturday Best," while Dick's uses a "Saturday Ritual" tag line.
"Sometimes we will separate out a slogan because not every retailer wants what every other retailer has or wants," Dubiel said.
New to the program this year is a sweepstakes that will send two fans to the College Football Playoff championship game in North Texas. Entries will be accepted through Thursday night.
"This is the first time we've had a consumer contest," Dubiel said. "It gives us one more way to reach our consumers and fans."


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The Earthquakes have signed Honduran defender VictorBernardez, the team announced Wednesday. The 29-year-old center back comes tothe Bay Area from top Belgian club Anderlecht, though he spent 2011 on loanwith another Belgian outfit, Lierse, and most recently with Indios de CiudadJuarez in Mexico. The move comes pending receipt of Bernardezs P-1 Visa, aninternational transfer certificate and a team-administered physical. Weve scouted Victor for several years, said EarthquakesGeneral Manager John Doyle. We saw him play at the CONCACAF Gold Cup and werevery impressed with his game. Hes a great addition to the offseason movesweve made so far and we look forward to having him in San Jose.
Bernardez has featured 52 times for Hondurassenior national team, and has scored two goals for his country. While with Anderlecht, the 62 defender made 22 appearances,scoring one goal, and was part of the team that won the Belgian championship in2010. Prior to his stint in Europe, Bernardez played for Motagua,one of the most popular teams in Honduras, where he was part of a team that wonthe 2006 Apertura championship and the 2007 Central American club championship.In addition to his height and defensive ability, Bernardezhas established himself as a capable free kick taker throughout his career. Victor gives us another quality player in the center of ourdefense, said Earthquakes head coach Frank Yallop. Hes a big, tough andathletic defender who can also play well with his feet and distribute the ball.Were excited to have him on the squad next season. Bernardez is the Earthquakes fourth addition of theoffseason and joins fellow Honduran Marvin Chavez as a new addition to headcoach Frank Yallops team. The acquisition also fills the hole left by thedeparture of Bobby Burling, who was selected by Montreal in Novembers ExpansionDraft.For more Earthquakes, MLS and world soccer chatter,follow Nick Rosano on Twitter: @nicholasrosano.


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Check out the chapter 'The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans' from WormBook in GENETICS, by
May 2016: 8 new labs joining the community
8 new labs have registered with WormBase and the CGC. Please join us in welcoming these labs to the community!
|Alex Mendenhall
|University of Washington, Seattle, WA
|Hiroaki Miki
|Osaka University, Osaka, Japan
|Megan Hwa Brewer
|Sydney Medical School, Concord, NSW, Australia
|Suhong Xu
|Zhejiang University, Hangzhou, Zhejiang, China
|Tamara Mikeladze-Dvali
|Biozentrum der LMU Mnchen, Planegg-Martinsried, Germany
|Matt Crook
|Whitman College, Walla Walla, WA
|Anna K Allen
|Howard University, Washington, DC
|Wolfgang Fischle
|Max Planck Institute for Biophysical Chemistry, Goettingen, Germany
Passing of Robert Stuart (Bob) Edgar, (1930 – 2016)
One of the pioneer spirits of our field, Bob Edgar, has died. Bob started the Worm Breeder's Gazette in 1975 and helped organize early worm meetings. He played an essential role in fostering the collegiality that has distinguished the C. elegans field. Read about him here.
Focus on Parasite Papers
For the upcoming WS246 release, a set of new papers have been added to the WormBase database. Some papers of interest to the parasite community are shown below.
Zang X, Atmadja AK, Gray P, Allen JE, Gray CA, Lawrence RA, Yazdanbakhsh M,
Maizels RM. The serpin secreted by Brugia malayi microfilariae, Bm-SPN-2, elicits strong, but short-lived, immune responses in mice and humans. J Immunol. 2000 Nov 1;165(9):5161-9.
linked genes: Bma-srp-2
ParaSite: Bma-srp-2
Zang X, Yazdanbakhsh M, Jiang H, Kanost MR, Maizels RM. A novel serpin expressed by blood-borne microfilariae of the parasitic nematode Brugia malayi inhibits human neutrophil serine proteinases. Blood. 1999 Aug 15;94(4):1418-28.
linked genes: Bma-srp-2
ParaSite: Bma-srp-2
Manoury B, Gregory WF, Maizels RM, Watts C. Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing. Curr Biol. 2001 Mar 20;11(6):447-51.
linked genes: cpi-2 , Bm5160 , Bm10669
ParaSite: Bm5160 , Bm10669
Focus on Parasite Papers
For the upcoming WS246 release, a set of new papers have been added to the WormBase database. Some papers of interest to the parasite community are shown below.
Yadav S, Gupta S, Selvaraj C, Doharey PK, Verma A, Singh SK, Saxena JK. In silico and in vitro studies on the protein-protein interactions between Brugia malayi immunomodulatory protein calreticulin and human C1q. PLoS One. 2014 Sep 3;9(9):e106413.
linked genes: Bm6412
ParaSite: Bma6412
Galvin BD, Li Z, Villemaine E, Poole CB, Chapman MS, Pollastri MP, Wyatt PG, Carlow CK. A target repurposing approach identifies N-myristoyltransferase as a new candidate drug target in filarial nematodes. PLoS Negl Trop Dis. 2014 Sep 4;8(9):e3145.
linked genes: Bma-nmt-1 , nmt-1
ParaSite: Bma-nmt-1
Terry FE, Moise L, Martin RF, Torres M, Pilotte N, Williams SA, De Groot AS. Time for T? Immunoinformatics addresses vaccine design for neglected tropical and emerging infectious diseases. Expert Rev Vaccines. 2014 Sep 5:1-15.
linked genes: Bma-prdx-2
ParaSite: Bma-prdx-2


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Fila Heritage Sleeveless Polo
A classic look for tennis with a fit every woman will want. This polo style shirt offers a five button placet with contrast color buttons, horizontal self fabric taping on sides and Fila Heritage patch logo.
- Color: Methyl Blue
- 88% polyester/12% spandex
- Classic Fit: Looser more relaxed fit
- Overall Rating: 5.0 / 5.0
- Write a Review
Love this top!
I am very happy I decided on this tennis top, it's so soft to the touch and fits perfectly, it even made me looked slimmer. I received a lot of compliment from my teammates the first time I put it on. The color looks better than on the picture.


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West Hempstead, New York (PressExposure) June 07, 2011 -- DAY Elevator and Lift offers quality accessibility equipment for customers in New Jersey. Our services are also available in New York and Connecticut. At DAY Elevator and Lift, customer care and satisfaction is given top priority. Keeping this in mind, the company delivers quality accessibility equipment from leading manufacturers in the industry such as Savaria, ThyssenKrupp Access, Adaptive Engineering Inc, Summit Lifts, Federal Elevator, AlumiRamp, Bruno, Matot, Inclinator Company of America, Aquatic Access, Pride Mobility, and Giant Lift Equipment Mfg Co Inc.
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Results 1 to 2 of 2
03-13-13, 14:11 #1Registered User
- Join Date
- Mar 2013
- Posts
- 2
Unanswered: Macro to Find Data in Another Sheet
I have Sheet1 with Column A being a list of all US ZipCodes.
Sheet2 has Column A listing out only the zipcodes where we have sales and Column B has the amount of sales in that zip code.
Ideally, here's what I want it to do:
I want it to select A2 on Sheet1
Search for that term on Sheet2
If it doesn't exist on Sheet2, place a "0" in B2, then automatically move on to A3 and continue the process
If it does exist in Column A on Sheet 2 - select the cell to the right of the result (Column B)
Then paste that cell's contents in B2 on Sheet 1
Then continue until ALL of column A on Sheet1 has been searched for on Sheet2.
I have tried tons of macros, but they don't seem to work because not all of the zip codes in Column A Sheet1 exist in Sheet2...
Here's one the one I've been using... it only works for about 5 lines before it comes up with an error... ANY HELP would be greatly appreciated. It's frustrating not being able to figure it out :/ I really don't want the MsgBox to pop up at all...
Sub abc()
' abc Macro
'Keyboard Shortcut: Ctrl+b
Do Until IsEmpty(ActiveCell)
Dim MyString As String
MyString = ActiveCell
Set RangeObj = Cells.Find(What:=MyString, After:=ActiveCell, _
LookIn:=xlValues, LookAt:=xlPart, SearchOrder:=xlByRows, _
SearchDirection:=xlNext, MatchCase:=False)
If RangeObj Is Nothing Then MsgBox "Not Found" Else: RangeObj.Select
ActiveCell.Offset(0, 1).Range("A1").Select
Application.CutCopyMode = False
ActiveCell.Offset(0, 1).Range("A1").Select
Selection.PasteSpecial Paste:=xlPasteValues, Operation:=xlNone, SkipBlanks _
:=False, Transpose:=False
ActiveCell.Offset(-1, -1).Select
End Sub
Please let me know what I'm doing wrong
03-18-13, 13:36 #2Grumpy old man (training)
Provided Answers: 17
- Join Date
- Sep 2006
- Location
- Surrey, UK
- Posts
- 1,101
Why not just use VLookup? You can wrap it in IF(ISERROR(...)...) [if you're working with Excel pre-2007] or IFERROR(...) [if you're working with Excel post-2007] to return 0 for zip codes with no sales.10% of magic is knowing something that no-one else does. The rest is misdirection.
Beers earned: 2


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Listing Courtesy of Berkshire Hathaway Alliance
Pictures shown are of former displays. Picture shown shows optional 3 car garage. Some features shown are optional. What a great floor plan for the money. This ranch home has approximately 1478 +/- Sq ft, 3 bedrooms. 2 full baths, 2 car garage, colonial grids on front windows, 20 ft wide concrete driveway, front yard sodded to front corners, six panel white colonist doors and trim package, smooth wall finish, hardwood entry foyer, electric range, dishwasher, garbage disposal, Moen faucets, 1 year builders warranty and a 2-10 Home Warranty.


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Gilat Satcom today announced that it is providing a satellite solution with connectivity over the Iridium network to Cattle-Watch, a new location, behavior monitoring and tracking service for the cattle industry that is being deployed around the world. Reliable broadband is vital to the success of Cattle-Watch which provides herd owners with real-time reports about the location, walking, grazing and resting time, water consumption, reproductive condition and other health issues of individual cows, sheep and goats, as well warnings about possible thefts. Every animal is fitted with an Ear Tag device that feeds information wirelessly to the 4-5 percent of the herd which have solar-powered collars containing the Gilat Satcom M2M satellite solution. Gilat Satcom transmits information from the collar to a web-based and mobile platform developed by Cattle-Watch. From here, information is sent—again using Gilat Satcom's satellite service—immediately to the rancher's computer or smartphone...more
Issues of concern to people who live in the west: property rights, water rights, endangered species, livestock grazing, energy production, wilderness and western agriculture. Plus a few items on western history, western literature and the sport of rodeo... Frank DuBois served as the NM Secretary of Agriculture from 1988 to 2003. DuBois is a former legislative assistant to a U.S. Senator, a Deputy Assistant Secretary of Interior, and is the founder of the DuBois Rodeo Scholarship.
Wednesday, December 16, 2015
Counting Collared Cows...
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North Texas Food Banks Need Holdiay Help!
The need at the Tarrant Area Food Bank is up 49% from last year.
And North Texas Area Food Bank needs to raise almost $4 million and receive nearly 10 million pounds of food to keep up with need over the holidays.
To find out how you can help go to:
Published at 3:15 PM CDT on Jul 20, 2009 | Updated at 10:51 PM CDT on Jul 28, 2009


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Separate names with a comma.
Discussion in 'General Firearms Forum' started by SC_Dave, Nov 9, 2012.
In my neck of the woods, they'd get heavy resistance.
Doesn't the government know what we have from non-private sales and purchase because of the federal form we fill out and background checks?
From the background checks? No, they don't know anything (make, model, serial number, etc) from that.
From the form? The form stays with your dealer. (unless dealer goes out of business within a certain number of years of your purchase)
Here as well
I can think of three states right off the bat that would be a real challenge for them to disarm....FL, TX and AZ.
Here in FL it would be a bloodbath!
4473's are not kept on file by the ATF, thus allowing them to be able to say "there is NO federal registry of gun owners". However, FFLs ARE required to retain these forms, and surrender them on demand to the ATF.
Thus, they have denial, and have shifted the responsibility off to the FFL.
Don't panic. They have rattled this saber before to get people to react with fear, panic buying, ect. ( besides the fact that the source is questionable)
But also, be ready, have a plan that is not just" shoot them at my door'. Think.
We have TIME to plan. use it.
Best thing to do is NOT be there when they knock...be on vacation with your guns.
Will they maybe send you a postcard advising when they will come and knock? If you are not there do they forget about you completely or maybe come back in a few weeks? What if you are in the shower when they come?
If the deadline for voluntary turn in is July 1, 201x at noon then the battle starts July 1, 201x at noon.
Sitting around waiting for them to come is a fools move.
But one person attempting to fend off an entire tactical team is not a fool's move?
No, it is a patrot's move, and his duty to defend the Constitution.
If enough of us do it then they will retreat back to Washington with their tails between their legs!
DO NOT be afraid to stand up for your rights! THAT would be a fool's move.
After the Katrina nightmare, Kentucky's General Assembly had the sense to pass laws protecting citizens from LE confiscation of firearms during times of disaster (as long as the person isn't using the weapon illegally, of course).
Of course, that doesn't mean they cannot take them, it just means that they cannot take them legally.
They sure like to elect wackos and commies out there.
Leftist housewives seem to be their favorite at election time.
We had a Pelosi clone running in Iowa but sent her packing.
We should all understand one very important thing. A treaty with the UN trumps states rights. Doesn't matter what each state has passed reference law regarding confiscation. The treaty will dictate the final verdict in the minds of the feds. With the treaty in place, our innocent, compassionate federal government can then divert blame to the UN, all the while gently coaxing (read FORCING) your state into abiding the mandates of the treaty.
It will be the individual that guarantees your own right, knock-by-knock and arrest-by-arrest. BTW, they USUALLY knock after o'dark thirty.
International treaty does not trump the constitution.
It doesn't in my eyes either. But this isn't the same world we used to live in. You wouldn't think municipal or state law would trump the Constitution either but I can think of places in the US you can't have a handgun, or it has to be "approved" first.
20 states have already filed petitions to secede from the Union. If the UN treaty passes you will see things that you never thought you would see happen in America.
When the knocking starts the shooting begins.
Sorry liberals, it has to be this way, you should have been smarter!
Or SAFELY!!!!
Again, when the patriots war starts, do not be sitting around the house waiting to be scooped up by a tactical team.
The president can't just do whatever he wants be executive order. Their has to be some legislation in place to authorize him (well the executive branch, but it goes up to him) to issue such orders under the legislation. AFAIK, none of the gun control laws in give him anywhere near the level of power we're talking about.
It's a pretty big if. If it did happen it's would be less unlikely. But that still doesn't get it out of the unlikely category.
Even if the two recent pro 2A rulings got overturned, the Congress would still have to pass legislation. That would then be challenged, and win. Besides the 2A, there's the whole 4A (search and seizure) issue.
I would take my AR and remove the upper, stock, grip, trigger group and take the stripped upper out to the driveway with a BFH and beat the hell out of it. Then turn it in
I have a lot more respect for this guy, than somebody who would allow the SS to subdue them and take away their rights and dignity by force. Anybody who would be enforcing the dismantling of the Constitution by the socialists would be SS.
I don't think it is going to happen. The SCOTUS has spoken. We are a nation of laws even though we have a lawless administration.


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i hate cardiff central station half of the time. not because it is a bad station, nope! it's really quite attractive and it's in two languages.
it's just half of the time that i'm there it means that i have to leave cardiff and that rips me to shreds. because cardiff feels like home? ugh, sidebar with me here a minute.
sidebar: i adore both cardiff and london. like, major city crushes. i could live in either place. but while i love london, it is hard. like, london could beat the shit out of you if you lived there too long. i mean, i'd consider it a fair trade for all of the benefits, but it would wear me out.
cardiff has a more laid back feel? i mean, it's ABSOLUTELY a city but it doesn't steal nearly as much of your soul as london. this is why i love it. i feel more at home there than anywhere i've ever lived in the states. heart you, 'diffy. for realsies.
but back to our story.
shed met us at the station to see us off. this, of course, led to much texting on the platform. well, i mean, he typed and held up the phone to the train window and i just laughed because i text like a slow loris. (srsly, i type 180 + wpm on a full sized keyboard. there is no excuse.)
ugh, it was still sad though. i hate leaving cardiff. i really, really do.
STILL! i'm not going to icepo all over this post (even though i could.). let's move on to bath and rudyard pipling? yeah, let's do that.
pippi met us at the station. yay!! i'm glad because 1) it's always nice to see him 2) he knew the way to bath backpackers (leff and i were too tired to navigate on our own.)
um, PRO TIP time. if you are taking luggage to bath but are not staying overnight, drop your bags off at bath backpackers. (bath spa train station does not have storage lockers). it's only three pounds per bag per day and they are locked in a secure room. that way you won't be lugging 11 kg around the town.
if you take more than 11 kg worth of luggage with you for a two week stay, what is your problem?! seriously WHAT ARE YOU TAKING WITH YOU?!? (11 kg is actually too much. i will downsize even more next time.)
right, right. sooooo, leff and i made pips go with us to sally lunn's. HAHA! IT IS SO TOURSITY! but, you know, they have cream tea. AND I WOULD CUT A BITCH FOR SOME CLOTTED CREAM.
this is not clotted cream. it is milky tea.
this is also not clotted cream. it is pip's lunch.
ah! now THERE'S the stuff in a little ramekin. i had a cinnamony bath bun with my clotted cream. leff's (on the left) was more lemon curdy? i think? idk, i was too intent on trying to use utensils to really notice.
i am not kidding. i am all the thumbs in the uk when it comes to fork 'n' knives. *shoulder shrug*
so how touristy is sally lunn's? SO touristy. this is the view out the window. HELLO, ARCHITECTURE STUDENTS! I CAN SEE YOU!
and here are more people taking photographs outside.
still, you should do this at least once, right? then you never have to do it again!
near sally lunn bunn is, of course, the avon river. (or the "river river" as gareth has pointed out. heh.) we somehow missed this on our first trip to bath?!? don't look at me like that. i was tired.
it was here that we informed philip pirrip of the existence of the piss dungeon. (ed note: sorry again, dad and chris. forget that you read that!) you can hear me encouraging leff to talk about it in this video. (also, you can hear the way that my accent is starting to skew slightly british. DEFENSE MECHANISM!!)
at the avon, we encountered an eavesdropper. embiggen for the full effect.
LESSON LEARNED, EAVESDROPPING LADY! DO NOT LISTEN TO OTHER PEOPLE'S CONVERSATIONS UNLESS YOU WANT TO LEARN ABOUT PISS DUNGEONS!! (ed note: sorry, dad and chris!)
the last time we saw pip he told me that there was no such thing as sidewalk chalk in the uk. clearly, this was a lie. heh.
but, you know, i can't hold it against him. for starters, it's called something other than sidewalk chalk. also, he got us to gay street before he had to run to his meeting.
THANK YOU, PIPLETTE!! some time we will visit you when you are not slammed with work responsibilities!
you know what's at the top of gay street? the CIRCUS!!! here are the trees that stopped bombs during wwii.
and here is a shot of the circus that you would not see if i wrote nice travel posts like a normal person. yes! it is a large construction tip! THE PEOPLE IN BATH HAVE TO DO CONSTRUCTION WORK TOO! OMG!!! IT IS NOT EURO DISNEYLAND AFTER ALL!! *eyeroll*
for serious though, it is such a beautiful place.
it even has all three kinds of columns
AND a sense of humor. this dog waste bin has "i'm full of shit!" written on top of it! ha!
aren't you glad you aren't reading one of those normal travel type posts now? yeah, thought so.
very near the circus is, of course, the royal crescent. *happy, happy sigh at the sweeping beauty of it all*
there is a ha ha located part of the way down the lawn to make it appear flat. IT IS AN OPTICAL ILLUSION! here it is from the side.
leff and i were completely wiped out at this point so we walked to the city center where we stopped at a convenience store/off license for sandwiches and drinks
before we rested at bath abbey. god, the light was tasty that day!
and the abbey was just as cool and relaxing as last time.
right, like YOU don't use places with impressive architecture as a place to cool your boots.
train to london. love the spacing!
love how london this announcer guy is.
DID NOT love having to wait around at paddington station for an additional hour eating yo sushi, drinking diet cokes and glaring at pigeons due to our next host's "work emergency". OMG!! THE HORRID FOURTH FLOOR WALK UP STUDENT FLAT!! i do not have the energy to tell you about it today. let's just look at the high point of the night, 'k?
tomorrow on rakka and leff unintentionally recreate scenes from "withnail and i" even though they had not gone on this holiday by mistake: i will tell you about the horrid student flat. maybe. we revisit the hardy tree! camden locks! THE CANALS!! regent's park again! WE FINALLY CRUISE THE THAMES! and then there was another nando's...