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A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus
Silke Schiebold1,
Henning Tschiersch1,
Ljudmilla Borisjuk1,
Nicolas Heinzel1,
Ruslana Radchuk1 &
Hardy Rolletschek1
Plant Methods volume 7, Article number: 19 (2011) Cite this article
The biology of the seed is complicated by the extensive non-homogeneity (spatial gradients) in gene expression, metabolic conversions and storage product accumulation. The detailed understanding of the mechanisms underlying seed growth and storage therefore requires the development of means to obtain tissue-specific analyses. This approach also represents an important priority in the context of seed biotechnology.
We provide a guideline and detailed procedures towards the quantitative analysis of laser micro-dissected (LM) tissues in oilseed rape (Brassica napus). This includes protocols for laser microdissection of the seed, and the subsequent extraction and quantitative analysis of lipids, starch and metabolites (sugars, sugar phosphates, nucleotides, amino acids, intermediates of glycolysis and citric acid cycle). We have also developed a protocol allowing the parallel analysis of the transcriptome using Brassica-specific microarrays. Some data are presented regarding the compartmentation of metabolites within the oilseed rape embryo.
The described methodology allows for the rapid, combined analysis of metabolic intermediates, major storage products and transcripts in a tissue-specific manner. The protocols are robust for oilseed rape, and should be readily adjustable for other crop species. The suite of methods applied to LM tissues represents an important step in the context of both the systems biology and the biotechnology of oilseeds.
Both the human and animal diet depends heavily, either directly or indirectly, on plant seeds, which are also used as a raw material for a number of industrial applications. A substantial research effort has therefore been focused on gaining a fuller understanding of seed development, and specifically of the mechanisms underlying the accumulation of the storage products oil, protein and starch. The seed is a complex structure, in which the various constituent organs (seed coat, endosperm and embryo) each have their specific function, determined by the integrated activity and specialization of groups of tissues/cells.
Treating the seed as a homogeneous entity inevitably ignores variation in the localized distribution of metabolites, gene expression, etc., and thus is not appropriate for investigating the spatial regulation of metabolism. Laser-assisted micro-dissection (LM), introduced by Emmert-Buck et al. [1], was developed as a means of sampling small groups or even single cells within plant tissues [2–4]. In this technique, the target is microscopically identified within a thin section, and then isolated via a computer-guided UV laser.
Currently, LM applications in plant research are predominantly concerned with the analysis of localized transcript abundance [4–9], including within the Arabidopsis thaliana embryo [10–12] or endosperm [13, 14], and the barley caryopsis [15, 16]. LM has also been successfully applied to analyse localized enzyme activity [17], identify proteins [18, 19] and metabolites [17, 20–23].
The combination of transcript profiling and biochemical analysis is particularly challenging, as these require contrasting fixation/extraction procedures [24]. However, such approaches would allow relating gene expression to the actual levels of metabolites and/or storage products in particular regions of the seed, making the link from genes to storage activities. Apart from the study of Thiel et al. [25] combining HPLC-based analysis of free amino acids with analysis of transcripts for the nucellar projection/endosperm transfer cell complex of barley caryopses, there are no studies published providing versatile analytical tools targeting seed tissues.
Oilseed rape (Brassica napus) is one of the leading temperate crops grown as a source of oil, and although much of the global physiology occurring over the course of its seed development is well understood [26–31], little attention has been paid to studying metabolism within specific tissues of the developing seed. The aim of the present research was to develop a set of procedures suitable for the parallel assessment and quantitation of metabolites, storage products and transcripts in LM tissues from developing seeds. We have established the necessary tools for achieving an integrated quantitative analysis of LM tissues and have demonstrated the application of LM for the study of tissue-specific metabolism in the oilseed rape seed.
Preparation and collection of tissue-specific samples via laser microdissection from oilseed rape seed
The first requirement was to optimize the preparation of tissue from the oilseed rape seed, appropriate for both transcriptome and biochemical analysis. Standard fixation and embedding procedures have been designed to preserve the morphological integrity of the sample, as well as to guarantee a sufficient quality of the RNA needed for gene expression analyses. However, these procedures entail the loss of many metabolites from the sample [20, 25]. A cryogenic approach was therefore adopted in which the whole seed was first snap-frozen in liquid nitrogen, before sectioning without any additional chemical treatment. Sections of thickness 15, 20 or 30 μm were mounted either on standard microscope glass slides, or on various types of membrane-covered slides. The sections were dried at -20°C and stored at -80°C until required. The resulting morphology was adequate to distinguish between the various seed organs and even, to some extent at least, to enable the recognition of cell types such as the vascular tissue inside the hypocotyl, or the various cell layers of the seed coat. The dried sections were then subjected to LM (Figure 1), in which the target was first identified microscopically and then excised from the section with a focused UV laser. Sections of thickness 15 or 20 μm proved to be the most appropriate for the LM procedure.
Experimental workflow. Schematic overview of the laser-microdissection based analysis of metabolites, storage products and transcripts in seeds of Brassica napus: (I) Seeds are removed out of the silique and immediately snap-frozen in liquid nitrogen. Two seeds mounted for cryosectioning are cut in 20 μm thick cross sections. (II) Sections are transferred to PET membrane slides and freeze-dried for one week. (III) Tissues of interest are laser microdissected from the cross sections. Light microscopy pictures show a cryosection of a 28 day old rape seed (bar = 1 mm) (IV) Isolated tissues are processed in parallel for subsequent biochemical and transcript analyses. Abbreviations: hy-hypocotyl; ic-inner cotyledon; oc-outer cotyledon.
Capture of the target was initially attempted by catapulting from a section mounted on a standard microscope glass slide into the cap of the collection vessel using laser-mediated tissue ablation (multiple laser pulses). However this method proved unsatisfactory as parts of the target remained attached to the surface of the slide, which compromised the estimation of the volume of the sample. Subsequently, laser pressure catapulting was used to effect the transfer from tissue mounted on membrane-covered slides. Here, a defocused laser pulse was used to catapult the sample into collection vessel cap. This method was only applicable for small target tissues, since the movement of bigger pieces was prevented by gravity. Finally, a micro-needle was employed to pick up and transfer the target, a method which also enabled the use of different collection vessels (plastic/glass tubes in different sizes) adjusted to the downstream biochemical analyses.
Various alternative materials were compared to act as the membrane covering the slide. The best results were obtained using PET (data not shown). Figure 1 illustrates the outcome of mounting 20 μm sections of seeds harvested 28 days after flowering on slides covered with a PET membrane. LM, in combination with the use of a micro-needle, allowed the isolation of the hypocotyl, and both the inner and the outer cotyledon tissue. Subsequent procedures for the extraction and the analysis of sugars, starch and lipid were adapted to suit a sample volume of ~0.008 mm3. An approx. five fold greater volume of tissue was required for the reproducible analysis of free amino acids, sugar phosphates, nucleotides as well as intermediates of glycolysis and citric acid cycle. For each measurement of metabolites and storage products, five technical replicates were taken from adjacent cross-sections of a single seed. Extractions and analyses of blank samples and samples containing only PET membrane were performed to provide an estimate of the background levels of analyte.
Analysis of lipid content and fatty acid composition in LM samples
Lipid extraction from LM samples was based on modifications of standard protocols (see Method section). Following extraction with organic solvents and transmethylation, the total lipid content in LM samples was assessed in the form of fatty acid methyl esters using gas chromatography (GC) (Figure 2). Where very small tissue samples are analysed, interference from contaminating impurities is a real issue [32]. As a precaution therefore, all plasticware (tips, tubes, etc.) was substituted with glass equivalents, pre-rinsed twice with solvent. This measure permitted the unequivocal detection of oleic and linoleic acids, and minimized the presence of impurities in the recovered fractions containing palmitic, stearic, vaccenic and linolenic acids (Figure 2a). A stable and low level of impurity was uniformly recorded from blank samples, and this background level was therefore subtracted from each sample reading, thereby avoiding the overestimation of individual lipid contents. The low standard deviations among chromatograph peak areas for each of the individual lipid species confirmed the experimental robustness of the analytical method (Figure 2b). A higher concentration of palmitic, vaccenic and linoleic acids was present in the hypocotyl compared with the cotyledons, while that of oleic acid was highest in the outer cotyledon and lowest in the hypocotyl. The least linolenic acid was present in the inner cotyledon, with similar concentrations present in the hypocotyl and outer cotyledon. When expressed in the form of total lipid, the three tissues differed markedly from one another (Figure 2c), with the hypocotyl being the most, and the inner cotyledon the least lipid rich. Application of NMR [for details see [33, 34]] on intact seed of Brassica napus further confirms our LM-based results. The NMR-based lipid map based on frequency-selective radiofrequency pulses (spin echo) in Figure 2d demonstrates max lipid levels in hypocotyl (red colored) and min levels in inner cotyledon.
Total lipids in laser-microdissected tissues of B. napus measured by gas chromatography. (a) Typical chromatograms of lipid standard, LM-sample and blank; IS = internal standard added to tissue samples during extraction. (b) Fatty acids in LM-samples of hypocotyl, inner cotyledon and outer cotyledon of one dissected element per tissue type (minimum tissue amount ~0.0084 mm3). (c) Comparison of total lipids in hypocotyl, inner- and outer cotyledon tissue. (d &e) Tissue localisation within a cryosection and the adequate lipid distribution map (based on spin-echo NMR). Lipid levels are color-coded. Mean values +/- standard deviation are shown (n = 5). Stars in b and c indicate statistically significant differences versus hypocotyl according to t-test (p < 0.01).
Combined extraction and measurement of starch and soluble metabolites
To enable the simultaneous quantification of particular metabolites and starch, the sample was first collected using Vivaclear mini clarifying filter (0.8 μm PES membrane). Soluble metabolites were extracted with methanol, and then starch was recovered from the pellet remaining on the membrane.
The rapid and effective extraction procedure developed was based on treatment of the pellet remaining on the membrane in 4.1 M HCl for 0.5 h at 80°C. The resulting glucose was quantified using ion chromatography (IC) coupled to amperometric detection. The treatment completely hydrolysed starch (Figure 3a) but left cellulose largely unaffected (Figure 3b). Acid solubilization and amyloglucosidase-based hydrolysis produced comparable estimates for the quantity of starch present in the sample (Figure 3c), but the former method proved the more reproducible, since the resulting standard deviation was lower. A small degree of contamination (~0.4 ng glucose per μl extraction solution) was associated with the use of the Vivaclear filter, but this can be simply corrected by subtraction when estimating the starch level of an LM sample. The treatment of a PET-membrane-blank sample with HCl produced no signals that interfered with the subsequent starch measurement. Statistically higher levels of starch were present in the outer cotyledon (75.6 ± 6.3 μg/mm3) than in either the inner cotyledon (39.1 ± 5.8 μg/mm3) or the hypocotyl (51.2 ± 6.3 μg/mm3) (Figure 3d). These levels agree with those derived from histological staining for starch in a tissue slice of oilseed rape seed sampled at the same physiological stage as used in the LM analysis (Figure 3e). A preferential accumulation of starch in the outer cotyledon has also been noted in the Vicia faba seed [35]. These inter-tissue differences in starch accumulation could be transitory, as the developmental stage analysed here (28 days after flowering) coincides closely with the period of peak transient starch storage in the oilseed rape seed [28]. Based on the starch concentrations measured here using the LM technique, the mean starch content of the embryo at this stage was ~60 mg/g fresh weight.
Total starch in laser-microdissected tissues of B. napus as measured by ion chromatography. (a &b) Effect of different HCl treatments on hydrolysis of starch and cellulose standards. (c) Level of starch detected in seed cross sections following either amyloglucosidase or HCl-mediated hydrolysis of starch. (d) Starch detected in dissected samples of 1 element per tissue type (minimum tissue amount ~ 0.0063 mm3). (e) Starch staining on a fixed seed cross section. Dark spots indicate accumulation of starch. (f) Laser dissected tissues showing hypocotyl (hy), inner cotyledon (ic) and outer cotyledon (oc). Mean values +/- standard deviation are shown (n = 5) in a-d. Stars in d indicate statistically significant differences versus hypocotyl according to t-test (p < 0.01).
Metabolites of core primary metabolism
Metabolite profiling typically is based on mass spectrometry (MS) coupled to either GC or liquid chromatography (LC) [36]. While a number of GC/MS-based profiling techniques have been developed for use with LM tissues [24, 36], they suffer from the problem that several compounds (e.g. most phosphorylated sugars) are either unstable or non-volatile, and thus are inaccessible. Thus we preferred to explore the potential of LC/MS-based metabolite profiling. The LC/MS protocol developed was shown to be sensitive enough to reliably detect the major compounds produced during primary metabolic pathways, specifically sugar breakdown, glycolysis, and the citric acid cycle, as well as nucleotides related to energy metabolism (Figure 4). The reduction of sugar phosphates and glycolytic intermediates occurred at a statistically different rate in the cotyledons compared to the hypocotyl. Although not pursued here, these metabolites could be readily quantified using external calibration procedures.
Metabolic intermediates in laser-microdissected tissues of B. napus measured by LC/MS. Levels of metabolites related to sugar breakdown (hexose-phosphates), glycolysis, citric acid (TCA) cycle and energy metabolism (nucleotides and pyrophosphate) were measured in LM-tissue of embryo (minimum tissue amount ~ 0.0063 mm3). 13C6-succinate was added prior extraction as internal standard. Data represent relative values and are given as mean +/- standard deviation (n = 3). Stars indicate statistically significant differences versus hypocotyl according to t-test (p < 0.05).
Soluble carbohydrates
Soluble carbohydrates were quantified by IC coupled to amperometric detection. Typical chromatograms derived from of an LM, a standard and a blank sample are presented in Figure 5a. Only traces of carbohydrates (60 pg/μl glucose and 0.65 ng/μl sucrose) were detected in the blank sample, and are assumed to have leached from the Vivaclear filter. The estimated carbohydrate concentrations in the LM samples were adjusted accordingly. At the 28 days after flowering stage, sucrose was the predominant soluble carbohydrate in the embryo, and was evenly distributed between the hypocotyl and cotyledons (Figure 5d). In contrast, there was a pronounced gradient of glucose concentration between the hypocotyl and the outer cotyledon (Figure 5b). Traces of fructose (Figure 5C) and some higher oligosaccharides (raffinose, stachyose and verbascose; data not shown) were also detected, as has also been noted in the A. thaliana embryo, where an increasing synthesis of higher oligosaccharides occurs as maturation proceeds [37].
Soluble sugars in laser-microdissected tissues of B. napus measured by ion chromatography. (a) Typical chromatograms of sugar standard, LM-sample and blank. Melezitose was added during extraction to all samples as an internal standard. (b-d) Levels of glucose (b), fructose (c) and sucrose (d) in LM-samples of 1 element per hypocotyl, inner cotyledon and outer cotyledon (minimum tissue amount ~0.0063 mm3). Mean values +/- standard deviation are shown (n = 5). Stars in b-d indicate statistically significant differences versus hypocotyl according to t-test (p < 0.01).
Free amino acids
HPLC was used to quantify the presence of 15 free amino acids in the LM samples. As most of these were present in very small concentrations, subtraction of blank sample levels was particularly important, and a somewhat larger size of LM sample was required. Relatively minor differences in amino acid composition were noted between the tissues (Figure 6a). Thus, glutamic acid was more abundant in the inner cotyledon, whereas arginine was least abundant in the hypocotyl. The most abundant amino acid across the whole embryo was glutamic acid. The free amino acid concentration in the hypocotyl was 5.3 ± 1.0 μg/mm3, compared to 6.6 ± 0.7 μg/mm3 in the inner cotyledon (Figure 6b).
Free amino acids in laser-microdissected tissues of B. napus measured by HPLC. (a) Comparison of single free amino acids per analysed sample of 5 elements per tissue (minimum tissue amount ~0.036 mm3). (b) Total amount of free amino acids calculated as the sum over all detected amino acids. Mean values +/- standard deviation are shown (n = 5). Stars indicate statistically significant differences versus hypocotyl according to t-test (p < 0.01). Abbreviations: hy-hypocotyl; in-inner cotyledon, oc-outer cotyledon
Procedure for RNA extraction and amplification from laser microdissected tissues
To maintain comparability of transcript and metabolite analyses, our goal was to find an efficient, robust and technically simple method to extract RNA from cryosectioned seed-material, prepared in the same way as for metabolite measurement.
We tested different RNA extraction methods from whole 20 μm thick cross-sections: a column based total RNA extraction in comparison to a bead based mRNA extraction method. Both extraction methods resulted in sufficient amounts (131.9 ng total RNA, 2.7 ng mRNA) of high quality RNA (Figure 7 a/c) for at least two technical replicates for subsequent linear RNA amplification using Eberwine Method [38].
Transcript analysis. Closeness of expression signals obtained after microarray hybridisation of conventionally extracted total RNA and amplified RNA samples of conventional, column-based and bead-based extraction methods. (a &c) Electropherograms reflecting quality of total RNA, isolated with RNeasy® (a) and mRNA, isolated with Dynabeads® (c) from a 20 μm seed cryosection (43. DAF). (b &d) Electropherograms of mRNA from the same samples (a/c) after 2 rounds of amplification. (e) Portions of signal intensities obtained from each hybridisation and divided into six groups. Y-axis represent % of observation. (f) Scatterplots comparing log2-transformed means of expression signals from the different sample treatments. Means are filtered against low signal intensities (≤ 10) and high coefficients of variation (≥ 20%).
After two rounds of amplification we were able to increase the RNA amount from approximately 44 ng input total RNA up to 5.1/10.4 μg and accordingly from 1.1 ng input mRNA to 2.8/10.4 μg high quality mRNA (Figure 7 b/d). Based on the Agilent Bioanalyzer electropherograms, the obtained transcript lengths were estimated between 50 and 4000 nt with most abundant transcripts being about 1000 nt long.
Different methods of RNA isolation and amplification will introduce some degree of bias into the population of amplified RNA. For evaluation, we compared: (1) total RNA isolated from fresh frozen rapeseed using a conventional phenol/chloroform method (previously described in Heim et al. [39]) (T-NA); (2) the same RNA but diluted and amplified (T-AM); (3) poly (A) mRNA isolated from one microdissected rapeseed tissue slice (20 μm) using Dynabeads®, amplified (mD-AM); (4) total RNA isolated from one microdissected rapeseed tissue slice (20 μm) using RNeasy®, amplified (TQ-AM).
For technical repeat the isolated RNA was divided into two parts, which were then separately amplified for two rounds. Hybridisations of the samples were performed on a 4 × 44 K array representing 43.803 Brassica probes. Normalised absolute signal intensities, which are corresponding to the expression level of the probes represented by the array, were arranged as shown in Figure 7e. About 50% of signals showed background intensity (less than 10) in all experiments what could be caused by the temporary and tissue specific expression of some genes. Portion of signal intensities above 1000, from the samples produced by amplified RNA after conventional phenol/chloroform RNA isolation method (T-NA and T-AM), was smaller than those of the kit based methods (mD-AM and TQ-AM). This indicates that kit based methods are in general more suitable for the sensitive amplification procedure and transcriptome analyses. The comparison of signal intensities between unamplified RNA (T-NA) and diluted and amplified (T-AM) demonstrates a loss of some transcripts during amplification. Consequently microarray data resulting from different pre-processing's regarding amplification should not be compared within one analysis. But nevertheless, the kit based RNA extraction derived from microdissected tissues, together with amplification of RNA, gives more high-signal-intensity spots than conventional methods of RNA isolation.
We evaluated the reproducibility of this method and the consistency of the propagation of the amplification bias by comparing the expression profiles of hybridizations of two independently amplified RNA samples (Additional File 1). The high degree of correlation between independent replicates (r ~0.98), suggests that the amplification is highly reproducible and that the amplification bias is introduced consistently when the protocol is repeated.
Log2(ratio) plots of fluorescence intensity in non-amplified total RNA (T-NA) sample was compared with amplified (T-AM) and amplified targets were compared to each other: T-AM vs. TQ-AM, T-AM vs. mD-AM, TQ-AM vs. mD-AM for correlation analyses (Figure 7f). The scatter plots showed that the degree of signal variation was a function of the signal intensity, with the variation increasing as the signal intensities decreased. Correlation of expression intensities between amplified and unamplified transcripts, originated from the same sample, was high (r = 0.909). The small bias is probably caused by the loss of transcripts during amplification as mentioned above. The intermediate correlation in comparisons T-AM vs. TQ-AM, r = 0.826 and T-AM vs. mD-AM, r = 0.820 show again that the kit based RNA is not well comparable to the RNA of conventional extraction method. However the direct comparison of the two kit based extraction methods (TQ-AM vs. mD-AM) show a strong correlation (r = 0.982) although RNA is originated from two distinct tissue slices. As a consequence of this, both methods are well suited for further application on B. napus seeds.
Taken together, the both methods of RNA isolation from cryosectioned seed material gain sufficient high-quality RNA. Amplification procedure generates long transcript and yields greater than 100-fold RNA amplification. Microarray assays performed with amplified RNA demonstrate that the method results in low amplification bias, is highly reproducible and can be useful in conjunction with a variety of experimental systems.
Standard extraction and analytical protocols have been modified to be applicable to LM tissues. As a result it has been possible to perform a parallel analysis of metabolic intermediates, major storage products and transcripts in LM tissues of seeds of Brassica napus. Protocols have been provided here for various analytical methods, differing in cost and analytical power. The approach was designed to enable a comprehensive analysis of LM tissues, and has been optimized for application to the important oilseed crop Brassica napus. We believe that the combination of metabolite profiling and the quantification of storage products, when combined with already established gene expression analyses from LM samples, should facilitate a comprehensive description of seed development under varying developmental/environmental conditions at a tissue-specific level, which would represent an important step in the context of both the systems biology and the biotechnology of oilseeds.
Plants of Brassica napus were grown on soil in a climate chamber at 16 h light/19°C and 8 h dark/16°C, 70% air humidity. Flowers were tagged at the time of opening for determination of the days after flowering (DAF).
Cryosectioning
Seeds were frozen in liquid nitrogen and transferred to a cryotome (Bright Instrument Co Ltd, Huntingdon, England) cooled down to -20°C. By the means of Tissue-Tek® O.C.T.™ Compount embedding medium (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) the frozen seeds were glued onto the sample plate and cut into 15, 20 or 30 μm thick cross sections. Immediately, cryosections were mounted on plain superfrost microscope slides (Carl Roth KG, Karlsruhe, Germany) or different types of membrane-slides like 1.0 mm PET-membrane frame-slides, 1.0 mm PET-membrane glass slides or 1.0 mm PEN-membrane glass slides (Carl Zeiss Microimaging GmbH, Bernried, Germany) and stored until complete dryness for 7-10 days in the cryostat chamber at -20°C.
Tissue preparation for metabolic analysis
Prior to laser microdissection, the freeze-dried cross sections were allowed to equilibrate to room temperature inside an airtight container to avoid condensation of moisture on the tissue. For microdissection, the target tissues were selected by the use of PALM® RoboSoftware and dissected by the PALM® MicroBeam System (Carl Zeiss Microimaging GmbH, Bernried, Germany). Following microdissection the tissue elements were picked by a microneedle and transferred into an 8 ml glass tube (Pyrex®, England) for fatty acid extractions. For the combined extraction of starch and soluble metabolites the dissected tissue elements were transferred directly on the 0.8 μm PES-membrane of a Vivaclear mini clarifying filter (Sartorius Stedim Biotech GmbH, Göttingen, Germany).
Analysis of total lipids
The overall fatty acid composition of microdissected tissues was extracted and transmethylated according to the method of Miquel and Browse [40] with some modifications. As internal standard we used glyceryl triheptadecanoate and accordingly for GC heptadecanoic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Samples were treated for 2 min with ultrasonic and then heated for 60 min at 80°C in 1 ml of 2.5% (v/v) H2SO4 and 2% (v/v) dimethoxipropane in methanol. After adding 200 μl saturated NaCL solution and 2 ml n-hexane, fatty acid methylesters (FAMEs) were transferred to the organic phase by intensive vortexing followed by centrifugation for 10 min at 1500 rpm. Subsequently the organic n-hexane-phase was transferred into a new 8 ml glass tube and extraction of the lower aqueous layer was repeated once. Afterwards 2 ml distilled water were added to the organic phase, vortexed, centrifuged (10 min/1500 rpm) and the organic layer was separated in a new glass tube where it was evaporated under nitrogen gassing and finally resuspended in 100 μl acetonitrile. For the whole extraction procedure it was necessary to avoid the use of plastics which were replaced by glass equivalents and additionally rinsed twice with distilled water and ethanol.
Analysis of FAMEs was carried out using a gas chromatograph GC-2014 equipped with auto injector AOC-20i and flame ionization detector (Shimadzu Corporation, Kyoto, Japan). The separation was performed on a 30 m × 250 × 0.25 μm DB-23 capillary column (Agilent Technologies, USA). The column temperature was set initially at 150°C (held for 1 min) then increased at 3°/min to 215°C, followed by 15°/min to 250°C which was held for 6 min. Identification and quantification of the detected fatty acids was done by comparison of retention times with FAME standards of different concentrations which were separated under same conditions. Signal intensities of impurities were subtracted by means of blank measurements. The amount of total lipids was calculated as the sum over all detected FAMEs.
Combined extraction of soluble metabolites and starch
For the combined isolation of soluble metabolites and starch 200 μl 10% methanol were added to the dissected tissue elements on the 0.8 μm PES-membrane. Additionally we added 2 nmol melezitose as internal standard. After 2 min ultrasonic treatment, samples were cooled for 5 min on ice and centrifuged at 13000 rpm for 1 min. This extraction was repeated twice followed by concentration of the flow through under vacuum. Dry samples were redissolved in 100 μl 10% methanol and stored at -80°C. For extraction of starch, 100 μl 15% HCl were added to the tissue residue on the filter membranes and incubated 30 min at 80°C. After cooling, samples were centrifuged for 1 min at 13000 rpm and diluted with 60 μl 10% methanol.
Analysis of soluble sugars and starch
Soluble sugars as well as glucose of hydrolysed starch were analysed using the Dionex ICS 3000 system coupled to an amperometric detector (Dionex, Idstein, Germany). Separation was performed on CarboPac™PA1 column (4 × 250 mm; Dionex, Idstein, Germany) under total flow of 1.3 ml/min distilled water (A) and 150 mM NaOH (B) in the following gradients: t = 0 min (91% A, 9% B); t = 15 min (60% A, 40% B); t = 18 min (100% B); t = 32 min (100% B); t = 32.5 min (91% A, 9% B); t = 38 min (91% A, 9% B) and column temperature of 30°C. Melezitose was used as internal standard and signal intensities of impurities were subtracted as means of blank measurements. Injection volume was set to 10 μl. Taken into consideration the tissue density (~1 g/ml) and volume-weighed contribution of the various tissues(hypocotyl, inner and outer cotyledons), the mean starch content of the embryo can be calculated and related to mean fresh weight.
Analysis of free amino acids
Derivatisation of the samples was performed using the AccQ-Fluor™ Reagent Kit (Waters, USA) according to the manufacturer's instruction. Separation was accomplished by AccQ-Tag™ column (3.9 × 150 mm, Waters, Ireland) in a Summit HPLC-system (Dionex, Idstein, Germany) equipped with a fluorescence detector. For separation the column temperature was set to 37°C with a total flow of 1 ml/min. As eluents we used buffer (A) with 7 mM triethanolamine hydrochloride and 140 mM sodium acetate, (B) acetonitrile and (C) distilled water, in the following gradients: t = 0 min (100% A); t = 0.5 min (99% A, 1% B); t = 27 min (95% A, 5% B); t = 28.5 min (91% A, 9% B); t = 44.5 min (82% A, 18% B); t = 47.5 min (60% B, 40% C); t = 50.5 min (100% A) and t = 60 min (100% A). Excitation wavelength was 250 nm and emission wavelength 395 nm. Identification and quantification of the detected free amino acids was done by external calibration with an amino acid standard mix (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) completed with asparagine and glutamine. Signal intensities of impurities were subtracted as means of blank measurements. The amount of total free amino acids was calculated as the sum of all detected amino acids.
LC/MS-based metabolite profiling
Metabolic intermediates were analysed using the ICS 3000 system (Dionex, Idstein, Germany) coupled to a API 4000 triple quadrupole mass spectrometer (ABSciex, Darmstadt, Germany). Separation was performed on a IonSwift MAX-100 column (1 × 250 mm, Dionex, Idstein, Germany) under constant column temperature of 40°C and total flow of 150 μl/min. With sodium hydroxide as the eluent we used the following gradients: t = 0 min (5 mM); t = 10 min (5 mM); t = 16 min (12 mM); t = 28 min (25 mM); t = 32 min (100 mM); t = 38 min (100 mM); t = 42 min (5 mM) and t = 56 min (5 mM). The identification of the detected metabolites was done by specific MS/MS transitions described in detail elsewhere [41].
Transcript analysis
Total RNA was extracted from whole seeds of 43 DAF by the conventional phenol/chloroform method as previously described [39]. Finally RNA was treated with RNase-free DNase I using TURBO DNA-free™ kit (Ambion, Austin, Texas). For mRNA extraction from a 20 μm thick cross section of 43 day old seeds Dynabeads® mRNA DIRECT™ Micro Kit (Invitrogen, Karlsruhe, Germany) was used according to the manufacturer's protocol. Total RNA from a cross section was isolated using RNeasy® Micro Kit (Qiagen, Hilden, Germany) with some modifications. 350 μl lysis buffer RLC was added to the cross section and incubated for 2 min at 56°C. Subsequently the lysate was transferred to a QIAshredder spin column (Qiagen, Hilden, Germany) and centrifuged for 2 min. The supernatant of the flow-through was mixed with 0.5 volume 100% ethanol and RNA extraction combined with on column DNA digestion was continued with step 6 according to the manufacturer's protocol pp39.
To get sufficient RNA for microarray analyses we performed a two-round linear amplification with Dynabeads® mRNA, RNeasy® total RNA and a diluted aliquot of conventional extracted total RNA, using C&E Version ExpressArt mRNA amplification Nano kit (Amptec GmbH, Hamburg, Germany) according to the manufacturer's protocol.
Quality and quantity of both the extracted and the amplified RNA was checked with Agilent 2100 Bioanalyzer, RNA 6000 Pico Kit and accordingly RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany) and in addition with NanoDrop™ 1000 (PeqLab GmbH, Erlangen, Germany).
Hybridisation of the 4 × 44 K microarray representing 43.803 Brassica probes (Agilent Technologies, Waldbronn, Germany) was performed by ATLAS Biolabs GmbH (Berlin, Germany). Normalised data were generated using the Agilents Feature Extraction Software (Version 10.5.1.1). For correlation analyses the normalised signal values were filtered against low signal intensities (≤ 10) and high coefficients of variation (≥ 20%) and subsequently transformed into Log2 values.
Quantitative imaging of storage oils in rapeseed was performed using magnetic resonance imaging (17.6-T wide-bore superconducting magnet; Bruker BioSpin, Rheinstetten, Germany) equipped with actively shielded imaging gradients. The experiments were performed as described in detail in [33, 34]. After non-invasive experiments seed were immediately frozen for further analysis. Starch staining was done using conventional staining with iodine solution as detailed in [42].
DAF:
Days after flowering
IC:
LC/MS:
Liquid chromatography-mass spectrometry
LM:
Laser microdissection
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We acknowledge BayerCropScience (Belgium) for funding.
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3 Gatersleben, 06466, Germany
Silke Schiebold, Henning Tschiersch, Ljudmilla Borisjuk, Nicolas Heinzel, Ruslana Radchuk & Hardy Rolletschek
Silke Schiebold
Henning Tschiersch
Ljudmilla Borisjuk
Nicolas Heinzel
Ruslana Radchuk
Hardy Rolletschek
Correspondence to Hardy Rolletschek.
LB and HR conceived the study. SS, HT, LB, RR, and NH carried out the analysis. All authors contributed to draft the manuscript, and approved the final manuscript.
13007_2011_140_MOESM1_ESM.PDF
Additional File 1:Reproducibility of transcript analysis. Scatter plot comparison of unfiltered signal intensities originated from technical replicates for the different sample treatments. Each axis of scatter plots represents independent amplification and hybridisation of one RNA sample. Correlation coefficient r reflects the high reproducibility of RNA amplification and array-analysis (PDF 267 KB)
Schiebold, S., Tschiersch, H., Borisjuk, L. et al. A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus. Plant Methods 7, 19 (2011). https://doi.org/10.1186/1746-4811-7-19
Free Amino Acid
Citric Acid Cycle
Storage Product


OTTAWA, March 28, 2019—Today's decision by the Court of Appeal of Newfoundland and Labrador is a victory for Justin Brake, and offers protection for journalists from broad injunctions.
Mr. Brake faces criminal charges of mischief and disobeying a court order after he followed a group of mostly Indigenous demonstrators onto the site of the Muskrat Falls hydro project site, following demonstrators for the purpose of reporting in October 2016. He also faced civil contempt proceedings in Newfoundland and Labrador Supreme Court, based on the same set of facts.
"Justin Brake should never have been named in the injunction," says Canadian Association of Journalists vice president Evan Balgord. "This was a naked attempt by a crown corporation to avoid accountability by preventing a journalist from doing their job. While we celebrate the verdict in the civil case, these charges have cost Brake time and money and are an attack on our shared Charter right to freedom of the press. The criminal charges he still faces based on the same set of facts should be dismissed immediately."
In today's decision regarding the civil proceedings, the Court stated that Mr. Brake's role as a journalist reporting on issues impacting Indigenous people was a factor of significance and as a result his status as a reporter should have been included on the court injunction and considered by the judge. The decision also recognized the key role media should play in fulfilling the goals of the Final Report of the Truth and Reconciliation Commission.
The Court agreed with APTN, who intervened in the case, that failing to recognize journalists as independent observers risks impeding the media function, and accepted that the chilling effects are real, significant and should be avoided.
In addition, the Court was concerned about broad injunctions and gave some guidance for the future regarding "John Doe" orders, while stating the real purpose of the injunction was to protect specific property rights by restraining protest activities that impeded or prevented access to the construction site. It concluded that the injunction prohibited "trespass", but the order shouldn't have been interpreted so broadly to prevent technical trespass unconnected to the protests. As a result they found Brake was not the object of or caught by the injunction.
Working for The Independent in the fall of 2016, Mr. Brake was reporting on concerns raised by local residents, many of whom were Indigenous, about the impact of the Muskrat Falls hydroelectric dam project on their land, health and rights.
On Oct. 22, demonstrators cut the lock on the gate to the construction site and entered. Mr. Brake followed the demonstrators and embedded with them until Oct. 25,after Nalcor named Mr. Brake on a court injunction with demonstrators but failed to identify him as a working journalist.
Mr. Brake now works for APTN.


Broomfield, Colo., May 15, 2018 - Lineup Systems, the leading media sales technology provider, has launched the Adpoint App Market to provide media customers with an online app directory of publishing solutions that have been integrated into Adpoint, its media sales solution. The new marketplace enables Lineup's customers to easily browse more than 50 of Adpoint's pre-built integrations to publishing applications being used by its customers worldwide.
Broomfield, Colo., Jan. 10, 2018 — Lineup Systems, a leading provider of advertising sales technology, has appointed Markus Feldenkirchen as its executive vice president of North America. As a member of the senior leadership team, Feldenkirchen will help guide the company's North American expansion as it prepares for continued success in 2018.


In order to enhance communication with parents, our text messaging service (Remind) will "push out" information specific to Elementary North. Click Here for more information.
Elementary North will be hosting it's information night for parents of incoming kindergarteners Tuesday, January 30th at 7PM.
Arushi P. won Elementary North's Spelling Bee last week. Ryan D. was the runner up. Arushi will go on to the sectional competition on February 27 in Deerfield. Congratulations to all the students for a job well done.


B2B means "business-to-business", and is also known as e-biz. It is e-commerce, or the exchange of products, services or information, between businesses. Since B2C (business-to-customer) has grown so rapidly that many customers around the world with busy schedules now do a large chunk of their shopping online, and even order their groceries online, it is not a surprise that the traditional B2B sector is ripe for disruption.
Evolving buyers' expectations are pushing the need to develop B2C-like functionalities for B2B, and when push comes to shove, companies should wise up and move with the times. Of course, as with all changes and upgrades to the way things have been done for the last few decades, there will be teething problems, but this is a necessary part of progress.
Furthermore, the growth of the digital-savvy millennial workforce, mobile ubiquity and continual improvements in eCommerce technology is accelerating the process. The B2B sector is set for change.
To remain current, B2B companies must prioritize the development of easy-to-use buying experiences with real-time response. In addition, they must also incorporate intuitive options for repeat orders, and online accounts that allow customers to view their order history and status. Last but not least, all these functionalities must be integrated into a seamless system capable of responding to demands in real time. All these functionalities must be available across the spectrum of devices and buying platforms.
An integrated approach to systems development will yield a seamless omnichannel – a multichannel approach to sales that will be able to provide the customer with a seamless shopping experience. This seamless experience will be uniform regardless of whether the customer is shopping online from a desktop, laptop or mobile device, through the phone or in a physical store.
The goal is to create a system that enables a business to connect its many data points to deliver an omnichannel and personalized experience in its B2B dealings.
The first step is to implement a software platform that allows data to flow seamlessly across the supply chain, including commerce, customer relationship management, and manufacturing. This seamless connectivity will allow businesses to cross-sell and upsell, augmented by an improved understanding of their buyers' requirements and needs.


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At $200 Patreon Goal – Audio Feed Cometh
June 9, 2017 June 9, 2017 by Matt Hartley
I will add an audio feed as a Patreon reachable goal of $200 per month. Once we hit this, we will be introducing an audio feed via RSS for those who like to listen while commuting. This will be made available to all $3 and above patreons.
Once this kicks in, it will include both For The Record and the Unleaded (Hangout community) shows – each episode, in your ear holes.
Thanks everyone.
Matt Hartley
Freedom Penguin's founder & talking head – Matt has over a decade working with Linux desktops, his operating system experience consists of both Windows and Linux operating platforms. In addition to writing articles on Linux and open source technology for Datamation.com and OpenLogic.com/wazi, Matt also once served as a co-host for a popular Linux-centric podcast.
Matt has written about various software titles, such as Moodle, Joomla, WordPress, openCRX, Alfresco, Liferay and more. He also has additional Linux experience working with Debian based distributions, openSUSE, CentOS, and Arch Linux.
WannaCry and OS Security | Feedback Hangout
Linux Is Everywhere (The Shirt)

redpajama_set_name: RedPajamaCommonCrawl


Here is complete rework of the map for Janardûn. For those familiar with the CC2 version of this map, there are immediate differences. The river systems have been completely reworked and the island-continent's terrain has been diversified.
It took me a long time to finalize the names of icons for this map. Note that there aren't any names for Janardûn's terrain, yet. At most, I've added new names for the most important islands and island groups. The islands names Welinmar and Zaladmaraul are new additions to the world, as are a few of the Utaran Isles.


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All Eyes On Alumni
Alumni & Community Awards
Raymond Leopold
Dr. Raymond J. Leopold was the chief technologist and co-inventor of The Iridium System, the first realization of worldwide, wireless, personal communications. As the Vice President and Chief Technical Officer of Motorola's Satellite Communications Group he guided the creation, development, deployment, and initial operation of Iridium. He is an IEEE Life Fellow, an Aviation Week and Space Technology Laureate for Space and an inductee in their Hall of Fame at the National Air and Space Museum in Washington, D.C. He is a recipient of The IEEE Third Millennium Medal, The AIAA Biennial Communications Award, and The Mobile Satellite Users' Association's Pioneer Award. He is also a Motorola Master Innovator and holds Motorola's highest technical honor, The Title of Dan Noble Fellow. He has 27 US Patents and 75 foreign patents.
A retired Air Force Lieutenant Colonel, he directed the development of military communications systems, twice served in The Pentagon, taught at the USAF Academy, performed R&D at the Air Force Weapons Laboratory, and earned The Defense Meritorious Service Medal, The Air Force Meritorious Service Medal with two oak leaf clusters, The Air Force Commendation Medal, and The National Defense Medal.
He was the James C. Hunsaker Visiting Professor of Aerospace Systems at MIT's Department of Aeronautics and Astronautics and delivered their Golden Anniversary Minta Martin Lecture in 2004. He previously chaired MIT's Industry Advisory Board for their Conceive-Design-Implement-Operate Initiative. He earned his BS in Electrical Engineering from the USAF Academy in 1967, a Master's of Electrical Engineering from NC State in 1968, and his PhD from the University of New Mexico in 1973, and was honored with a Doctorate in Telecommunications Management from South Dakota School of Mines and Technology in 1997.


Maoists on Sunday gunned down a ruling TDP MLA in Andhra Pradesh's Visakhapatnam district, police said.
The incident took place during the ultras' weekly celebrations of CPI Maoists Formation Day. Maoists gave a call to celebrate the event for a week till September 27.
VISAKHAPATNAM: Telugu Desam Party (TDP) MLA Kidari Sarvewara Rao of Araku constituency and former MLA Siveri Soma were gunned by the Maoists at Liviriputtu in Dumbriguda mandal on Sunday afternoon. Sarveswara Rao is survived by wife and two sons.
According to sources, Kidari Sarveswara Rao, who was elected as Araku MLA on the ticket of YSRCP party, had later joined the TDP. He along with another former MLA Soma of same constituency and other henchmen were going to Dumbriguda mandal when the incident took place.
Around 60 Maoists, including huge number of women cadres, were involved in the attack. The Visakha range DIG Srikanth told TOI that they have some inputs of the incident but are verifying the facts. Rural SP Rahul Dev Sharma said that they have already issued notices to all the politicians on the Naxals hit-list to inform the administration prior to their visits to villages. But MLA Kidari and former MLA Soma's visit to Dumbriguda mandal was not informed to the police, he said.
Kidari Sarveswara Rao, the former MLC of Congress, has been facing severe allegations of illegal mining activity in the agency area. He was allegedly warned by the Maoists earlier for supporting the Bauxite mining.
According to sources, Maoists had conducted reccee and observing the movements of the MLA for the past few days.


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Business News›Magazines›India's biggest film star Shah Rukh Khan on why numbers limit success
India's biggest film star Shah Rukh Khan on why numbers limit success
Shah Rukh Khan, who has been retained by TAG Heuer as brand ambassador for the 9th year, discusses his unique philosophy of success.
ET Bureau|
Updated: Aug 17, 2012, 02.21 AM IST
SRK, who has been retained by TAG Heuer as brand ambassador for the 9th year, discusses his unique philosophy of success.
As we are ushered into the 24th floor suite at a posh Bandra hotel, the Badshah of Bollywood greets us warmly, the ever-present cigarette smouldering in one hand. As his aides buzz around to get him a sandwich, Shah Rukh 'King' Khan strolls to the sea-facing window and muses aloud, "Too bad Sea Rock hotel is gone. I'd always dreamt of buying it during my struggler days."
Staring far into the sea, watching the waves splash around, it looks as if he is trying to relive those frustrating days of sitting on Bandra's bandstand with nothing more than just a dream. But with success, SRK's dreams have only gotten bigger.
Circa 2012, he is not only the biggest star in Bollywood, but also owner of the IPL-winning Kolkata Knightrider team, and promoter of Red Chillies Entertainment and Red Chillies VFX. SRK, who has been retained by TAG Heuer as brand ambassador for the 9th year, discusses his unique philosophy of success. Edited excerpts:
So what drives you — a higher goal, insecurity or success itself?
You know, I delivered a speech on success for Young President's Organisation a few years ago. Success is an extremely strange thing. It has two elements. First, it cannot be passed on, however much you try, whether you are a CEO of a company, or a parent. And the second thing about it is that the successful person actually actually does not know why he or she is successful. Of course, you can always say things like: the more I practice, the easier it becomes, etc. People get cagey trying to explain the reason for success and they hide behind big words. Someone asked Warren Buffet, how he was so successful in his investments. Buffet said: 'I take the right decisions'. The person asked: 'And how do you take right decisions?' He replies: 'Experience'. He was further asked: 'How do you get experience?'
Buffet said: 'By making the wrong decisions'. So even Warren Buffet cannot explain the reason for success — no one can. So when you ask me what drives me, it's really none of the five top things: profit, fame, the quest for excellence and perfection, wanting to do better than I did last time, or just because this is what I do. I think it is something as basic as, feeling good about what you do. And then everything stems from it. Whenever I've felt good doing something, it has always been successful. I felt good about getting into cricket. Fine, it wasn't successfully immediately.
People criticise you because their analysis of your success from exterior forces is based on the five peripherals. And I explain that yes, I lost money on RaOne but it feels good, and I will make it again. I could not have done RaOne 20 years back, but now I can afford to do it, and lose a bit of money if I need to, as long as it felt right. Of course I am in a lot of businesses now. Some of those things I do not feel good about, so I don't like to do them, but once I feel good about my core business, I go for it.
You have created an ecosystem of success around you, right producers, right directors, right co-star's etc. Do you owe your success to this ecosystem, or was it because of your success that you were able to make those choices?
Actually, most of the work I have done is with first or second-time directors — Farah Khan, Shimit Amin, Rajiv Mehra, Aziz Mirza, Kundan Shah — and it felt good to work with them. The new actors I work with, like Anushka, Katrina, Priyanka, Deepika, etc. get very surprised, because I don't interfere in the script or change this song and that scene. My philosophy is, don't fix it if it ain't broke. Any amount of fixing won't work if it was going to go bad anyway. So there is a huge amount of instinct.
I believe your first instinct will never go wrong. Like my new film with Rohit Shetty. He came to narrate a film to me two years ago through some mutual business friends. The film is called Chennai Express and I said yes, I will do this film. Even with Munnabhai, which I didn't do eventually because of my surgery…I'd said yes to it. When Raju (Hirani) came to me, I was shooting the climax scene in Devdas. As we were chatting, he said, 'I have got a film, it is called Munnabhai MBBS'. And after hearing the title, I said I am doing it. Raju asked me if I was joking. I said no, I am doing this, finished. I do not want to hear anything. Sometimes instinct works and sometimes it goes wholesomely wrong.
Did being an outsider in the industry help you break rules more often?
Actually it doesn't and it does. I do not get the feedback that industry people get, because I am still an outsider. But I am very proud of it. I did not know how to be a hero and have not been groomed by my senior colleagues. I find it a little wrong when people assume my son will become an actor. Why? He will get a good education and then decide what he wants. I would love it if he became one, because I would be able to at least have a conversation. If they become economists, I may not.
But my logic is that because I was not groomed by people as to how a hero should be, I did atypical films. People turn around after 20 years and say, oh he is a romantic hero, he only works in these kind of films. But if I look back at my 72 films, there's a lot of different commercially viable films I've done. I can do a hockey film with 13-15 girls (Chak De India). If I was an insider, I would think, there is no hero-heroine or song — now, all those considerations do not come in my head.
In the entertainment industry, as well as business, you have to network extensively. Are there days when you wake up and just do not feel like doing it anymore?
As a matter of fact, I detest networking, which is a wonderfully American concept. Of course, when I am doing a business, I sit with the business head and listen to him. But I never get emails from them. I never meet advertisers. I don't spend even two minutes a month to sit down and say, 'Should we pitch for this?' If it happens, it happens. You just need to give the impression to people that you mean business, and will give it your best shot. Nothing else matters, I have never, ever gone for a networking meeting though I have been offered that time and again. If I meet someone and they assume that I am meeting them for work, they assume completely wrong. I do not have a PR drive, except when a film is due to release. Even now a lot of people say I should do a little image-building. My answer to them is this is what I am and this is what I do. It won't feel good if I have to ask for work. And there is no ego, but I am not going to ask for work, ask around what is happening or which business to get into.
How do you keep growing as a person?
I have got this huge greed for learning new things. Like yesterday at 5:30 am, I saw India lose to Korea in a hockey game. Though I know a lot about hockey, I realised the rules have changed. So I sat up for one and a half hour to learn the new rules, because it is important to know it. I am not going to play but I need to know, I have this huge yearning for wanting to know. That helps me a lot.
So how do you manage all the diverse businesses under Shah Rukh Inc?
I cannot give a dishonest answer — actually, I do not manage anything. I think I am just extremely fortunate. My friends tell me: You know, Shah Rukh, you should give one hour for this business, two hours for this one, one hour for this one and then meet them again next week. I have never done that.
Because time is the most valuable commodity that you have, and it is money
I do not think so, I think people who talk about time management and getting organised are talking crap. If Usain Bolt wants to practice at 3 am in the morning, that's great for him. My meetings can happen at 3 in the morning. If it is important, I know it will find its way to me. It is highly important that I remain completely uncontrolled by time.
In any business, how much is dictated by the head and how much by the heart?
For me personally it is 95% heart, I have never done a business in my life and I will never do it with my head. I just do not understand number-crunching.
Even the IPL?
I could not even afford it actually. I remember that Jay and Lalit came to me and said you should bid for it, it will be very interesting. The price they quoted five years ago was $40 million, which was Rs 240 crores. In my life, I had not earned that money. And nobody knew the model of the business. But I just love the English Premier League and I felt, these leagues should be there in India, they will help kids. Luckily it worked out fine and the brand was eventually worth $70-75 million. But I remember I stayed up all night thinking that even if my heart is into it, I cannot sell my family off. But the business model was very well designed by the IPL guys, and finally it was very good. But it could have gone the other way. Losing Rs 35-40 crore rupees in one year is something I can ill afford. I am not one of those corporate guys who have lot more money than me. Actors do not take such risks for a bloody film, let alone for a cricket team. Most of it was love for sports.
So you aren't driven by numbers, even in business?
For me, if you have a desire to win a business in terms of numbers, you are limiting yourself. Guys like Steve Jobs, Bill Gates and Mukesh Ambani have amazing minds. When I sit with them, I come to this conclusion that what they want to do is transform the lives of people through their products and services, because it makes them feel good. People like to somehow analyse everything in tangible terms, but if your business is going to be intangible, the sky is not the limit. I think the numbers create a finish line. I've made Rs 100 crore; should I aim for, Rs 150 crore or 200 crores next? If you ask me, 'Your film has done 100 crores are you happy?' I'll say I am not, I did not make everyone feel good. Numbers are like pet dogs, they will follow. But if you follow numbers, I do not think you'll get far.
Shah Rukh Khan to produce horror series for Netflix; likely to be titled 'Betaal'
Kent ropes in Shah Rukh Khan for new product
Shah Rukh Khan to be honoured with an honorary doctorate degree from La Trobe University next month
Shah Rukh Khan-starrer 'Zero' to close Beijing International Film Festival
Shah Rukh Khan will be the chief guest at 10th Indian Film Festival of Melbourne
Shah Rukh Khan congratulates PM Modi, asks Indians to work with election winners
Reliance Foundation to adopt 4 flood-hit villages in Gujarat
Reliance Jio, demonetisation led to revenue drop: Vodafone
Mutually discuss Interconnection issue and sort it out: Manoj Sinha to telcos
Tatas approach sovereign wealth funds to buy Cyrus Mistry family's stake
Kalyan Jewellers to spend Rs 20 crore to build 2000 homes for poor
Global mergers and acquisitions hit all-time high in 2015 at $4.86 trillion: Dealogic report
Work done in the first five months gave Paytm a strong foundation, says founder Vijay Shekhar Sharma
In a startup environment, there is no clear structure or process, says Faasos's Revant Bhate
Built a fantastic team of over 3000 people across 22 countries, says Zomato's Deepinder Goyal
Professionals struggle with letting people go, but entrepreneurs don't as much, says Shaadi.com's Anupam Mittal


Clifford the Big Red Dog, the star of the WCNY-TV animated children's series, will greet children from 11 a.m. to 3 p.m. Saturday, March 10, at Sangertown Square in New Hartford.
A similar appearance on March 3 at The Shops at Ithaca Mall attracted more than 100 groups of parents and children who wanted to meet their favorite TV character.
Children can meet Clifford March 10 and receive free Clifford stickers and activity sheets. Parents can also receive information about WCNY's move to a new facility in downtown Syracuse and learn how they can help Clifford "build a new doghouse.'' For a small donation, children can have their picture taken with Clifford and WCNY will provide the photo.
Clifford will also appear at the Sangertown Mall on Saturday, May 19. Clifford's visits to the Mohawk Valley are part of his mall tour that also includes stops at the Salmon Run Mall in Watertown (March 17 and May 12), The Shops at Ithaca Mall (May 5), Shoppingtown Mall in DeWitt (April 7), Carousel Center in Syracuse (May 26), and Great Northern Mall in Clay (June 2).
Visit www.wcny.org/events for more information about WCNY's events and programs. You can watch Clifford the Big Red Dog at 2 p.m. Monday through Thursday and 9 a.m. Sundays on WCNY channel 24.1.


A Chapter 7 bankruptcy (liquidation) does not disqualify a borrower from obtaining an FHA-insured mortgage if at least two years have elapsed since the date of the discharge of the bankruptcy.
"I filled for a Chapter 13 bankruptcy in 2009, then converted it to a Chapter 7 which was discharged in 2012. I would like to buy a house, but was told I had to wait 2 years from the 2012 date. What process should I take to try and qualify for an FHA loan under the 2 year wait period after bankruptcy?"
chosen not to incur new credit obligations.
The rules state that two years must elapse from the time the bankruptcy is discharged, not when it is filed--that is a very important distinction to make.
has since exhibited a documented ability to manage his/her financial affairs in a responsible manner.Note: The lender must document that the borrower's current situation indicates that the events which led to the bankruptcy are not likely to recur."
It might be easy to overlook that these FHA loan rules do not require any bank or lender to offer a shorter wait time; this shortened "seasoning period" is at the lender's discretion.
Any borrower who wants an FHA loan in the wake of a bankruptcy should be prepared to provide the required proof mentioned here. If you have at least one year of on-time payments and has documented the same, you might stand a better chance at getting an FHA loan approved based on your circumstances--but there is definitely a waiting period of some kind after the bankruptcy, regardless. Much depends on your circumstances--discuss your needs with a loan officer for more information.


All network services at the mentioned locations will experience 5 short interruptions, each in less than 1 minute.
The voicemail service for all phone numbers prefixed by 28315 will be suspended.
The RCGAS will be suspended.
There will be two 1-minute interruptions to the Internet service in the Cyberport Campus.
There will be two 5-minute interruptions in accessing the DLP servers.
Emails sent to all mailing lists supported by listserv.hku.hk will experience delay. Besides, the web-based system for use by mailing list administrators will be suspended.


Group Swim Lessons are a great way for children to learn to swim while enjoying the camaraderie of making new friends.
Our Swim Team Coaches will teach classes for Newbies, Beginners and Advanced Beginners. The program will teach students to be comfortable and safe in the water, and instill an enjoyment of swimming. Basic swimming skills are taught through demonstration, practice and games.
To register for classes, please complete the Registration Form found here or at the Guard Desk.
*Friday is reserved for make-up class if a class is cancelled due to weather.
There will be at least one instructor for every 4 children, and classes will be no larger than 8 students.
The cost is $65 per student per session ($32.50 for one week).
Child has never had lessons before. Class teaches child to be comfortable and safe in the water. Will learn to put face in the water, open eyes underwater, submerge, blow bubbles, control breathing, float and glide. Will learn kicking and arm movements for freestyle. Must be at least 3 years.
Child is confident in the water and knows freestyle and backstroke basics but technique needs improvement. Will learn proper technique in freestyle, backstroke and breaststroke in preparation for joining the swim team. Butterfly will be introduced. Will develop deep water skills including treading water, swimming underwater and diving into the pool.
Private lessons will be offered by our Lifeguards and Swim Team Coaches. The program will provide focused one-on-one instruction and parent choice of instructor. A list of instructors and telephone numbers is posted on the bulletin board above the drinking fountain to help parents identify and select the instructor of their choice. Parents will make arrangements and payments directly with the instructor. Costs are expected to range from $15 to $35 per 30-minute private lesson –depending on the credentials/experience of the instructor. Private lessons for beginners between 11:30 AM and 12:00 noon on weekdays between June 22 and July 30. This timeslot is reserved strictly for learn-to-swim instruction, not for fine-tuning of those who already know how to swim.
For swimmers of any/all abilities, parents can schedule private lessons for any other time during normal pool operating hours –but to avoid crowded conditions and distractions, it is best not to schedule lessons when the pool is very busy (3:30-6:30 daily). All evenings (6:30-8:00/9:00) are typically very quiet, as are weekend mornings when there is not a home swim meet. Private Lessons can continue through the September closing of the pool during normal pool operating hours. Please do not discuss lessons with lifeguards while they are life guarding the pool –wait until they rotate to the front desk. As always, lessons conducted for fee may only be conducted by someone from our Lifeguard and Coaching staff. Any other lessons for fee are prohibited at our pool.


Cascade Engineering announced the promotion of Kenyatta Brame to executive vice president. Most recently, Kenyatta served as CE's group services senior vice president and chief administration officer.
In his new position, he will remain chief administrative officer and continue overseeing the services group, which includes legal/risk management, human resources, marketing, IT and several other key departments. His additional responsibilities include managing the following business units: IWS, Decade, Xtreme RFID, Cascade Consulting Group and Triple Quest.
Prior to joining Cascade Engineering in 2006, Kenyatta was senior vice president for operations and merchandizing at Sysco Food Services of Grand Rapids, LLC and he served as a practicing attorney for Miller, Johnson, Snell & Cumminsky, P.L.C. He has also served as an adjunct professor at Thomas M. Cooley Law School.
Following his 1992 graduation from Wabash, Kenyatta earning a J.D. from the University of Michigan Law School and a master's degree from Michigan State University.


Baking soda, or bicarbonate of soda, can be used as a natural, non-toxic alternative for many cleaning and bath products. Drastically reduce your consumption, eliminate your use of toxic products, and save a lot of money with simple baking soda solutions.
Using baking soda for bath and beauty needs, cleaning, deodorizing and other eco-friendly uses is easy. For solutions, stir together about 4 tablespoons (1/4 cup) of baking soda with 1 quart of water until dissolved. For pastes, stir together three parts of baking soda with one part water. For sprinkling, simply store baking soda in a jar or bottle with a shaker-type cap.
Shampoo: Use baking soda as a shampoo, rinse, then use apple cider vinegar as a conditioner.
Spa Bath: Add baking soda or bath salts to your bath.
Toothpaste: Dip your wet toothbrush into baking soda to brush your teeth, whiten your teeth and freshen your breath.
Teeth Whitener: Create a paste with a teaspoon of baking soda and water. Rub paste on your teeth once a week, let sit for five minutes, then rinse.
Deodorant: Lightly pat baking soda under your arms.
Mouthwash: Add one teaspoon of baking soda to a small glass of water.
Exfoliant: Mix three parts baking soda with one part water to use as an exfoliant to gently remove dead skin cells. Rub in a circular motion, then rinse.
Insect Bites: Make a paste out of baking soda and water and apply to skin.
Clean Combs And Brushes: Remove oil build-up by soaking combs and brushes in a glass of warm water mixed with one teaspoon of baking soda. Rinse and let dry.
Oral Appliances: Clean retainers and dentures with two teaspoons of baking soda dissolved in a cup of warm water.
Hand Softener: Mix baking soda with warm water and rub on your hands to clean and soften.
Rash: Use two tablespoons of baking soda in bathwater to relieve rash.
Antacid: Use baking soda to relieve heartburn, stomach upset and acid indigestion by drinking half a teaspoon of baking soda mixed with half a cup of water.
Canker Sores: Used as a mouthwash to relieve canker sore pain.
Windburns: Moisten baking soda with water and apply.
Feet: Soak your feet in a warm bowl of water with three tablespoons of baking soda.
Sunburn: Apply a paste of baking soda mixed with water.
Bee Stings: Create a poultice of baking soda mixed with water.
Measles And Chicken Pox: Relieve general skin irritations such as measles and chicken pox by adding baking soda to your bath.
Itchy Skin Relief: Mix baking soda with water to create a paste – then rub it on your skin.
Splinters: Splinters come out naturally after a few days of soaking in baking soda twice a day.
Ulcers: Baking soda neutralizes stomach acid and is beneficial for ulcers. Add a pinch of baking soda to your drinking water.
Cancer Prevention: Eating baking soda can offer nutritional and immune support for people with cancer. Add a little baking soda to your drinking water. Baking soda increases the pH of acidic tumors without affecting the pH balance of healthy blood and tissues. A pH imbalance causes unhealthy organisms to flourish, damaging tissues and organs and compromising immune systems.
Exercise Enhancer: Mix a pinch of baking soda in your drinking water before workouts. Baking soda absorbs lactic acid in muscles during vigorous workouts, prolonging fatigue and enhancing athletic performance.
Kidney Function: Low-functioning kidneys have difficulty removing acid from the body. Baking soda buffers acids and maintains balanced pH levels in your body.
Soft Scrub: Sprinkle baking soda on a damp sponge to scrub bathtubs, showers, tiles and sinks – then rinse and wipe dry.
Vinyl Shower Curtains: Sprinkle baking soda on a damp brush to scrub shower curtains, rinse and allow to dry.
Toilet Cleaning: Add one cup of baking soda to the toilet and scrub.
Clogged Drains: Unclog your drain with one cup of baking soda and one cup of vinegar.
Laundry Detergent: Use half to 1 cup of baking soda in the wash cycle to get clothes clean and smelling fresh naturally.
Laundry Detergent Boost: Add half a cup of baking soda to detergent to get clothes brighter.
Pre-Soak: For heavy odor and dirt issues, use baking soda as a pre-soak. Dissolve 1 cup of baking soda in warm water. Fill the washer or sink with water and add the dissolved baking soda and clothes to soak overnight before washing.
Fabric Softner: Add half a cup of baking soda to the rinse cycle to balance pH levels and suspend detergent or mineral deposits in the water that make clothing feel stiff.
Iron Cleaner: Remove built-up starch and scorch deposits from irons with a mix of baking soda and water, then wipe the plate with white vinegar.
Cloth Diapers: Add half a cup of baking soda to 8 cups of water to soak cloth diapers.
Floors: Mix half a cup of baking soda in a bucket of warm water. Mop and rinse clean.
Microwave: Sprinkle baking soda on a damp sponge or cloth to clean inside of microwaves and remove odors.
Cookware: Shake baking soda onto pots and pans, add hot water and soak for 15 minutes before washing.
Oven: Sprinkle baking soda on the bottom surface of your oven and spray with water. Allow to sit overnight, then scrub and rinse.
Cookware Oil And Grease: Add a heaping scoop of baking soda to your regular dish soap to help cut oil and grease.
Dishwashers: Deodorize and cleanse your dishwasher by adding baking soda to the wash cycle.
Dishcloths: Sweeten sour dishcloths with baking soda.
Cutting Boards: Sprinkle baking soda on cutting boards, scrub and rinse.
Drains: Unclog your sink with one cup of baking soda and one cup of vinegar.
Polish Silver: Mix baking soda and water to create a paste and rub onto silver with a clean cloth, then rinse and dry.
Stainless Steel And Chrome: Rub with a moist cloth and dry baking soda. Rinse and dry.
Fridge And Freezer: Clean with baking soda sprinkled on a damp cloth, then rinse.
Food And Beverage Containers: Wash food and beverage containers with baking soda and water.
Melted Plastic Bread Bags: Use baking soda to remove melted plastic from bread bags by dampening a cloth and creating a mild abrasive with baking soda.
Counters: Clean with baking soda sprinkled on a damp sponge.
Thermos Bottles: Wash out with baking soda and water.
Coffee Pots: Clean glass or stainless steel coffee pots (but not aluminum) with 3 tablespoons of baking soda mixed with one quart of water.
Coffee Makers: Run coffee maker through its cycle with a baking soda solution, then rinse.
Garbage Disposals: Eliminate odors by slowly pour baking soda down the drain while running warm water.
Barbecue Grills: Sprinkle baking soda on barbecue grills, let soak, then rinse off.
Garage Floors: Sprinkle baking soda on greasy garage floors. Allow to stand, then scrub and rinse.
Repel Rain From Windshields: Apply gobs of baking soda to a dampened cloth and wipe windows.
Patio Furniture: Sprinkle baking soda under chair cushions to freshen patio furniture.
Weeds: Sprinkle baking soda between the cracks of your walkway to keep weeds away.
Cars: Mix baking soda with warm water on a soft cloth, brush or sponge to clean off dirt and bugs.
Garbage Cans: Wash garbage cans with baking soda and water.
Hands: Remove odors from hands by wetting hands and rubbing them hard with baking soda, then rinse.
Furniture: Sprinkle baking soda on a damp sponge and rub furniture lightly. Wipe off with a dry cloth.
Surfaces: Clean and remove stains from marble, formica and plastic surfaces by scouring with a paste of baking soda and water.
Batteries: Create a baking soda paste and apply with a damp cloth to scrub corrosion off batteries. Use caution as batteries contain acids. Disconnect battery terminal before cleaning, and to prevent corrosion wipe on petroleum jelly.
Oil And Grease Stains: Sprinkle baking soda on oil and grease and scrub with a wet brush.
Crayon Marks On Walls: Add baking soda to a wet cloth to remove crayon marks on walls.
Air Freshener: Add one tablespoon of baking soda to water and a little essential oil.
Refrigerator: Place an open box of baking soda in the refrigerator.
Rugs And Carpeting. Sprinkle baking soda on rugs and carpet, wait 15 minutes or overnight, and vacuum.
Garbage Cans: Sprinkle baking soda on the bottom of garbage cans.
Sports Gear: Sprinkle baking soda into gym, sport and golf bags.
Closets: Place an open box of baking soda in closets. To ward off moths, add a few drops of lavender oil.
Toilets Odors: Add one cup of baking soda to the toilet and allow to sit an hour before flushing.
Stuffed Animals: Clean stuffed toys by sprinkling them with baking soda; brush off after 15 minutes.
Fireplaces: Reduce soot odor by cleaning the ashes out of your fireplace and placing a bowl of baking soda inside.
Vacuum Cleaners: By vacuuming baking soda into the vacuum cleaner, you deodorize the vacuum.
Shoes: Shake baking soda into shoes.
Dry Bath: Sprinkle baking soda on dry fur, brush it in then brush it out. Keep away from eyes.
Wet Bath: Bathe your dog with a solution of 1 tablespoon of baking soda for every 1 1/2 cups of warm water. Let it soak into fur for a few minutes. Thoroughly rinse, then apply apple cider vinegar to condition fur – 1 tablespoon of apple cider vinegar per cup of warm water – allowing to sit for a few minutes. Rinse thoroughly, then dry. Keep both solutions away from eyes.
Accidents: Clean up companion animal "accidents" by scrubbing the area with club soda, then allow the area to dry. Sprinkle baking soda on the area and let stand for a while, then vacuum up.
Teeth And Gums: Brush your companion animal's teeth by dipping a damp, soft brush in baking soda and brushing gently.
Animal Bedding: Sprinkle baking soda liberally onto pet bedding, allow to sit for 15 minutes before vacuuming.
Animal Carpet Odors: Sprinkle baking soda on the carpet, let dry, then vacuum.
Litter Box Odor: Layer the bottom of the box with baking soda, add litter on top.
Litter Box Cleaning: Empty old litter and pour in a mixture of baking soda and vinegar. Let stand for 15 minutes, then scrub, pour out and dry.
Cage And Crate: Scrub with a solution of baking soda dissolved in warm water. Rinse and dry.
Dishes: Scrub dog and cat bowls with baking soda and water.
Toys: Dissolve baking soda in warm water to wash pet toys. Rinse well and dry.
Blankets And Towels. Add half a cup of baking soda to the wash.
Skunk Odors: Combine 1 quart of hydrogen peroxide with 1/4 cup of baking soda and 1 teaspoon of grease cutting dish detergent. Wash your animal with the solution.
Bee Stings: Remove stinger from animal if needed, then apply a baking soda paste.
Nail Bleeding: If you cut your animal's nails too close and draw blood, dip the nail in baking soda and apply pressure.
Bad Breath: Mix half a teaspoon of salt and half a teaspoon of baking soda with one cup of water in a spray bottle. Spray your animal's mouth regularly.
Produce: Scrub produce with baking soda under water to remove pesticides and residue.
Baking: Baking soda, as its name implies, can be used as a leavening agent in baked goods. It causes dough to rise.
Beans And Bloating. Sprinkle a teaspoon of baking soda in water while soaking dry beans to reduce bloating.
Tea: Add a pinch of baking soda to a gallon of freshly brewed tea to remove bitterness and cloudiness.
Lunch Boxes: Place a spill-proof box of baking soda in a lunch box between uses to absorb odors.
Ants: Mix equal parts baking soda and salt and sprinkle in areas where ants are entering your home.
Baby Bottles: Clean baby bottles with baking soda and hot water.
Cloth Diapers: Dissolve half a cup of baking soda in two quarts of warm water and soak diapers thoroughly before washing.
Diaper Rash: Add two tablespoons of baking soda to your baby's bath water to help relieve diaper rash.
Play Clay: Combine 1 1/4 cups of water, two cups of baking soda and one cup of cornstarch.
Baby Spit Ups: Moisten a cloth, dip it in baking soda and dab at the dribbled clothing.
Baby Pools: Add baking soda to the bottom of a mildewed baby pool, then hose it down.
Cut Flowers: Add a teaspoon of baking soda to a vase of flowers to expand their life.
Fill Wall Holes. Mix baking soda with white toothpaste to fill holes in a plastered wall.
Small Fires: Toss baking soda at the base of the fire to help put a fire out.
Ashtrays: Remove odors from ashtrays with baking soda and water. Sprinkle dry baking soda in ashtrays to prevent smoldering and reduce odor.
Canvas Bags: Use dry baking soda with a brush to rub canvas handbags clean.


According to new research conducted by Edinburgh Napier University for Scotland's Digital Technology Skills Group and partners, women account for only 18 per cent of the digital technologies workforce in Scotland.
The Tackling the Gender Technology Gap Together study found that few girls are pursuing digital technologies-based university study and employment.
Lower levels of female participation in the area are echoed at a school level. Girls make up only 20 per cent of Computing Studies National 5s and women hold only 16 per cent of degree places on university computing courses.
Over 65 per cent of students, employers and employees in the digital technologies sector cited a lack of women studying the required subjects as a reason for this unequal representation.
However, the industry also attracts women from different educational backgrounds, with around half of women in digital roles receiving their degrees in the creative arts, business studies and natural sciences sectors.
Speaking to The Student, Chris Belous, Women's Liberation Group Convener at the University of Edinburgh, said: "Unfortunately it's not as normalised in our society for women to be successful in tech – because science and tech is still seen so much as a 'boy's world'.
In response to the gender imbalance, a group of experts, chaired by Evelyn Walker of Hewlett Packard Enterprise, has been set up by Scotland's Digital Technology Skills Group to develop an action plan to attract more women into technology.
Walker told The Student: "Addressing the gender imbalance will not be a short term issue and significant work will be required by a range of stakeholders including education, industry and public agencies.
"Initiatives such as extracurricular coding groups, mentoring schemes, and the Digital World campaign which is raising awareness and changing perceptions of digital technology careers, are making progress but we need to do more and refine our strategies to reach more females, more effectively", she added.
In order to increase the number of women in the tech industry, Belous suggests that "normalising the idea that women should be in the tech workforce, not just among other women, but among men as well, would go a long way to helping.
"There are multiple schemes getting women into STEM subjects throughout the school system which are also really positive, but they are clearly not doing enough because there is still so much of a culture change which needs to happen in the workplace as well as in education", Belous continued.
The Digital Technology Skills Group are currently developing actions to combat the gender gap. They hope to tackle the issue by including and improving the use of role models in schools, promoting the benefits of gender parity and workplace flexiblity, extending the reach of technology into other subjects and supporting employers to attract, retain and promote female participation.
The group will also continue to ensure that it engages girls and women across all of its initiatives including Scotland's Digital Xtra Fund for extracurricular computing projects.
Walker told The Student: "the imbalance starts while girls are in school so it is important that solutions are developed to tackle this issue from early years onwards.
"However, attracting more females to enter digital technology careers is only part of the solution. Women should be encouraged to remain in the sector and to progress into interesting, rewarding and senior roles," she concluded.


We all know that traveling to different time zones or working the night shift shakes up our body clocks. A new URMC study also shows that tobacco smoke can harm circadian rhythms, by changing gene expression patterns in lung tissue.
The study, led by Irfan Rahman, Ph.D., professor of Environmental Medicine, was published online today by The FASEB Journal, and reports for the first time that chronic exposure to cigarette smoke caused mice to act sluggish – as if they just took a red-eye flight -- an effect that persisted for up to 30 days even after the exposure stopped.
Circadian rhythms are centralized in a specific region of the brain called the suprachiasmatic nucleus (SCN), but circadian clocks also exist in peripheral tissues including the liver, heart, and lungs. The latest research confirms that in addition to daylight, other environmental cues can put stress on the peripheral clocks. Rahman's lab found the mechanism by which tobacco smoke changes the core clock gene in the lung – BMAL1 – boosting lung inflammation.
Patients with chronic obstructive pulmonary disease (COPD/asthma) and other lung ailments usually have daily respiratory symptoms that run in cycles, such as nighttime breathlessness followed by severe early-morning coughing. In collaboration with circadian biologist Michael T. Sellix, Ph.D., assistant professor in the Division of Endocrinology and Metabolism, Paige B. Lawrence, Ph.D., professor of Environmental Medicine, and Patricia J. Sime, M.D., professor of Medicine, Rahman's group is also studying whether the flu virus similarly alters daily sleep cycles and lung function.


I'm a hand piecer and hand quilter. I read somewhere that it's difficult to hand quilt with batiks. Has anyone ever used batik for the quilt top and then used 100% cotton for the backing. Wonder if this would work better for hand quilting.
I've never hand pieced a batik quilt. I've machine pieced then handquilted a batik quilt. I used a cotton backing. It was still a little more difficult hand quilting thru the batik but it's doable. I think using batik for the backing may make it really difficult (just my humble opinion).
I've hand quilted a batik top with a muslin backing without difficulty. I wouldn't use my hand-quilting time on batik fabric where the quilting design wouldn't show. I've done machine and hand quilting go the same quilt. Don't tell the Quilt Police!
I am hand quilting a Pacific Rim Hawaiian quilt. I am having one heck of a time. After talking with Nancy Chong @ a quilt show I learned never put a batik backing on a batik quilt!!!!!!!
I have not hand pieced with batiks but I have hand quilted them with regular fabric for backing and have also used flannel backing. One I had a pieced backing that had a very small square of batik on the back as well. No problems but I do feel regular quilting cottons are easier to needle. Think the difference of room temperature butter vs butter straight out of the fridge. This is a bit extreme but it is much easier to spread room temp butter on to warm toast than it is cold. Both are quite doable, one just takes a wee bit more effort. Kind of the same with hand quilting batiks I do use batiks for hand applique and I love using them for applique, mostly for the color selections but they tend to not ravel very easily either which is very nice for needle turn and prepared hand applique. I use a Roxanne applique needle and silk thread for this and have no problems.
Have used batiks in quilt tops plenty. Don't recall using any for backing. Hand quilter as well and have never had issues with the batiks. Just make sure you're using a new, sharp needle and you should be fine.


text:
Band: KOMOR ROMOK
Title: Cryptoworm
Label: Fekete Terror Productions
Usually when I get to listen a new Hungarian Black Metal band it is a melodic, atmospheric or ambient one, but in this case things are different since Komor Romok (Hungarian for gloomy ruins Google translate tells me) are swimming in way more brutal waters. This is the band's debut EP and so far only release, a 4 tracks effort clocking 15 minutes of brutal, uncompromising, fast paced, evil Black Metal that reminds me of the South American scene to be exact. The echoes added to the screams here are the only trace of atmosphere if we don't consider the guitar riffs, but apart from that there are no keys or other melodic instruments to enrich the soundscapes, this is pure, raw, hateful and primitive Black Metal, but unfortunately this is not enough for the band to make a name for themselves outside of Hungarian borders since there are countless other bands playing like this all over. The third tracks's guitar riff reminded me a lot of Ancient's Svartalvheim, a more brutal version of it nonetheless, but you get the point. By now it's about time the band follows up with a new release and brings something of their own to the table, but in the meantime this is a good occasion to meet a non typical Hungarian Black Metal band. Released on professional tape with matte factory printed cover in limited edition by Fekete Terror.
Reviewed by Adrian
facebook.com/romok.komor
facebook.com/feketeterrorprod