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Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process
© The Author(s). 2017
- Received: 7 September 2016
- Accepted: 26 December 2016
- Published: 13 January 2017
Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively.
Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer.
Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.
- Archival tissue
- CpG island methylator phenotype
- DNA methylation
- Formalin
- Heat treatment
LINE-1 is repeated half a million times in the human genome, interspersed throughout, and comprises approximately 17% of the human genome [1]. It has a high density of CpG dinucleotides in its 5′ untranslated region, and these CpG sites are usually heavily methylated in normal cells. Because of both the extremely high frequency of LINE-1 and the heavy methylation in its 5′ CpG sites, the level of LINE-1 methylation has been thought to be closely associated with genomic DNA methylation levels. In 2005, Weisenberger et al. demonstrated a strong relationship between the levels of LINE-1 methylation and genomic DNA methylation [2]. In that study, the LINE-1 methylation level was assessed using the MethyLight assay, a probe-based real-time PCR assay for the detection of methylation. More recently, however, instead of using the MethyLight-based LINE-1 methylation assay, a pyrosequencing-based LINE-1 methylation assay has become widely used because the pyrosequencing-based assay has been validated for its precision and reliability in formalin-fixed, paraffin-embedded (FFPE) tissue samples by Irahara et al. [3].
LINE-1 hypomethylation in tumors has been demonstrated in virtually all tissue types of human cancer except for thyroid cancer and renal cell carcinoma [4–11]. However, prior to the report of Tournier et al. in 2012, there had been no study which compared the results of the PCR-based LINE-1 methylation assay between paired fresh-frozen (FF) and FFPE tissue samples [12]. Tournier and colleagues analyzed paired FF and FFPE tissue samples for their LINE-1 methylation levels using a pyrosequencing assay to identify whether formalin fixation induced deviations in the measured value of methylation levels in individual genes or in LINE-1. Tournier et al. found that a significant discrepancy existed in the measured LINE-1 methylation levels between paired FF and FFPE tissue samples. This discrepancy raised doubts regarding the utility of the pyrosequencing LINE-1 methylation assay in FFPE tissue samples.
In formalin-fixed tissue samples, formaldehyde induces several types of DNA damage on either double strand or single strand, including formaldehyde-induced crosslinks, DNA fragmentation, abasic sites, and deamination of cytosine bases [13]. Of various formaldehyde-induced crosslinks, inter-strand DNA crosslinks and protein-DNA crosslinks are thought to affect the efficacy of bisulfite modification which is an essential step for genomic DNA methylation analysis. Because the reaction of bisulfite with cytosine residues is highly single strand-specific and cannot occur on double-stranded DNA [14], inter-strand DNA crosslinks are thought to cause some resistance against heat and alkaline denaturation. Incomplete denaturation of DNA leads to incomplete bisulfite conversion, which might cause the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. Because formaldehyde-induced crosslinks are known to be reversible by heat treatment [15, 16], the application of prolonged heat treatment during DNA preparation process might increase the performance of bisulfite modification in DNA samples obtained from FFPE tissues [12]. The present study aimed to identify whether formalin fixation is related to the increased values of LINE-1 methylation detected in FFPE tissues and whether the addition of a heating step during the DNA extraction process helps to decrease the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. From five cancer cell lines, we generated 10 xenograft samples that were analyzed for the effects of formalin fixation and for heating in the pyrosequencing-based assay of LINE-1 methylation. Finally, we attempted to identify the effect of heating in the methylation analysis of LINE-1 or of single genes in human FFPE tissue samples.
Effect of formalin fixation in the assessment of LINE-1 methylation
Heating decreases the formalin-associated elevation of LINE-1 methylation levels
The discrepancy of LINE-1 methylation values between paired FF and FFPE tissue samples was found to decrease with the application of a heating step (95 °C, 30 min) during the FFPE DNA extraction process. With the addition of a heating step during DNA preparation, the differences in the measured levels of LINE-1 methylation between paired FF and FFPE tissue samples became insignificant (Fig. 2). However, FFPE tissue samples with delayed fixation showed significantly increased measured values of the LINE-1 methylation level despite the application of a heating step. The correlation of the measured values became stronger between paired FF and FFPE tissue samples with the application of a heating step. Although we tested another condition of heating, heating at 95 °C for 1 h, we did not find any difference in LINE-1 methylation levels between samples treated with heating at 95 °C for 30 min and those with heating at 95 °C for 1 h (Additional file 1: Figure S1).
Effect of heating on LINE-1 methylation analysis of archival tissue samples and survival analysis
Effect of heating on the methylation analysis of single genes in archival tissue samples
FFPE tissue is an invaluable resource for oncologic epigenetic studies because large collections of FFPE tissue samples with clinical annotations are managed in hospitals. Histologic examination and delineation of areas with the highest tumor cell content is important for the quantitative methylation analysis of cancers to prevent dilution of the neoplastic cell signal by contributions from non-neoplastic cells in the cancer tissue samples. FFPE tissue samples are more suitable for manual microdissection than FF tissue samples. Minimal amounts of tissue, even a 10-μm-thick section from an endoscopic biopsy specimen, are required for the PCR-based quantitative methylation analysis of single genes or repetitive DNA elements. Tissue samples dissected from the mounted tissue slices are subjected to incubation in lysis buffer containing proteinase K at 55 °C for 2 days. Then, the lysed tissue solutions are subjected to bisulfite conversion using commercially available kits, including the EpiTect or EZ DNA methylation kit, which generate bisulfite-modified DNA samples. In our study, the addition of a heating step, 95 °C for 30 min, between the 2-day incubation step and the bisulfite conversion procedure was found to decrease the discrepancy between the measured values of LINE-1 methylation of paired fresh and fixed tissue samples.
In the present study, formalin fixation was found to increase the measured value of LINE-1 methylation levels compared to the paired FF xenograft tissue samples. But, the measured value of the LINE-1 methylation levels was not different among FFPE xenograft tissue samples of varying fixation lengths from 1 to 5 days. These findings suggest that formation fixation, rather than duration of fixation, plays an important role in the causation of deviation in the measured value of LINE-1 methylation level. However, because we did not examine the effect of fixation duration >7 days or <1 day on the performance of pyrosequencing-based LINE-1 methylation assay, we could not insist that the duration of fixation does not affect the pyrosequencing assay of LINE-1 methylation. However, to the best of our knowledge, there has been no study which investigated how duration of formalin fixation affects bisulfite conversion of genomic DNA and its performance in downstream methylation analysis. Formaldehyde reacts with amino groups in nucleobases, leading to the formation of methylene bridges between complementary strands of DNA. Because the generation of methylene crosslinks is a time-dependent process [17], longer fixation time is expected to form more inter-strand crosslinks. However, we did not find any difference in the measured value of LINE-1 methylation among fixation time from 1 to 5 days.
As a surrogate marker for genomic DNA methylation content, methylation of LINE-1 has been measured by various assays, including the combined bisulfite restriction assay [18], the MethyLight assay [2], pyrosequencing, and absolute quantitative analysis of methylated alleles [19], which target CpG sites located in the 5′ untranslated region of LINE-1. However, 85% of LINE-1 elements are truncated in their 5′ sequences [20], and thus, PCR-based assays only assess 15% of the LINE-1 elements in the human genome. Weisenberger et al. demonstrated that the MethyLight assay-based measurement of LINE-1 methylation level correlates with genomic DNA methylation content as measured by high-performance liquid chromatography (HPLC). However, before the study of Lisanti et al. [21], there had been no study that directly analyzed the correlation between the pyrosequencing-based measurement of LINE-1 methylation and genomic DNA methylation content. Lisanti et al. showed a strong correlation between the methylation levels in LINE-1 measured via a pyrosequencing assay versus a high-performance liquid chromatography.
Studies have shown that hypomethylation of LINE-1 in tumors is closely associated with poor patient prognosis for many types of human cancers, including gastric carcinoma [6, 22], colorectal carcinoma [9], esophageal squamous cell carcinoma [23], and lung adenocarcinomas [24, 25]. In particular, the association between LINE-1 hypomethylation in gastric carcinoma and poor prognosis has been reported by three studies [6, 11, 22]. However, all three of these studies, including Shigaki et al. [6], used FFPE tissue samples. In the Shigaki study, DNA was extracted from FFPE tissue samples using the QIAamp DNA FFPE tissue kit, which includes a heating step for 60 min at 90 °C in its protocol. However, our previous studies did not use commercial DNA purification kits, and the extraction protocol did not include any prolonged heating step. In the present study, we analyzed the effect of heat during DNA extraction from FFPE tissue samples by comparing the survival curves of four groups for both heat-treated DNAs and untreated DNA. With heat-treated DNA samples, PFS and OS decreased as tumoral LINE-1 methylation decreased. This trend was not observed with unheated DNA samples. Regardless of whether the heating step was included during DNA extraction, tumoral LINE-1 hypomethylation was found to be an independent parameter for identifying gastric cancer patients with a poor prognosis.
When we examined the methylation frequencies and levels of eight CIMP panel markers between DNA samples with and without heat treatment, all of the genes except for MLH1 showed increased methylation frequencies and levels in the heat-treated DNA samples relative to the unheated DNA samples. Consequently, heat treatment enabled identification of more cases of CIMP-high CRCs. At present, it is unclear why assessment of methylation at the MLH1 CpG island locus was not affected by heat treatment. Interestingly, the increased methylation level of individual genes in association with heat treatment is in contrast to the decreased methylation levels of LINE-1 under the same conditions. The question arises why heat treatment led to a decrease in methylation levels in repetitive DNA elements but an increase in individual genes. The reason of this apparent discrepancy is unclear, but it might be attributable to the difference in the methylation assays employed: LINE-1 methylation was analyzed using a pyrosequencing assay, which measures the mean methylation level of all the DNA alleles at individual CpG sites [2], whereas the methylation levels of individual genes were assessed by the MethyLight assay, which evaluates the relative amount of specific DNA alleles with concurrent methylation of serial CpG sites [26]. When the region of interest is incompletely converted and thus contains non-converted CpG and non-CpG cytosines, the MethyLight probe cannot anneal to the incompletely converted region of interest and does not generate a fluorescent signal. Thus, on the condition that all CpG sites located in the region of interest are methylated, complete conversion leads to an increase in the measured value of methylation levels compared to an incomplete conversion. In contrast with the MethyLight assay, enzymatic cascade of the pyrosequencing reaction cannot discriminate incompletely the converted template sequence (region of interest) and generates luciferase light signals not only from methylated CpG cytosines but also from non-converted unmethylated CpG cytosines, which causes an increase in the measured value of methylation levels compared to complete conversion.
We have demonstrated that formalin fixation can result in elevated values of the LINE-1 methylation level, irrespective of the duration of fixation, but prolonged heating of the DNA solution prior to bisulfite conversion helps decrease the discrepancy between paired FF and FFPE tissue samples. However, heating did not offset the discrepancy in FFPE tissue samples prepared using delayed fixation. Our results indicate that prolonged heating of DNA samples obtained from FFPE tissues is necessary for proper evaluation of DNA methylation levels, regardless whether single genes or repetitive DNA elements are assayed.
Cell culture
The human gastric cancer cell lines, MKN-45 and SNU-638, and colorectal cancer cell lines, SW620, SNU-C5, and LoVo, were obtained from the Korean Cell Line Bank (Seoul, Korea) and were cultured in a 37 °C incubator with 5% CO2. Cells were grown in RPMI 1640 with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Culture medium was replaced approximately every 48 h.
In vivo xenograft experiment
A total of 10 6-week-old normal BALB/c-nu mice were used for the tumor xenograft experiment. To generate two tumor masses per mouse, 2 × 106 cells in phosphate-buffered saline were bilaterally injected subcutaneously into the flanks of the mice. The mice were maintained in a pathogen-free barrier facility and fed a standard diet. All mice were euthanized 8 to 10 weeks after subcutaneous injection. The xenograft experimental plan and protocol were approved by the Biomedical Research Institute of Seoul National University Hospital (15-0111-C1A0).
DNA extraction from xenograft tissue samples
FFPE tissue mouse xenograft tissue slides were made according to one of five formalin-fixation conditions: no fixation (fresh frozen), fixation for 1, 3, or 5 days; or delayed fixation (room temperature for 2 days prior to fixation for 1 day). Ten-micrometer-thick sections were cut from the FFPE tissue blocks or from the FF tissue blocks. Deparaffinization was accomplished by first heating the glass slides mounted with the paraffin section to no more than 60 °C. The paraffin was dissolved in xylene. Deparaffined and rehydrated sections of the FFPE tissue blocks or sections of the FF tissue blocks were stained with hematoxylin and eosin. The FFPE or FF tissue slides were examined under a microscope, and the areas with the highest tumor cell density were selectively dissected using a knife blade. The scraped tissues were collected in microcentrifuge tubes containing 50 μL of tissue lysis buffer (0.5% Tween 20 (Sigma, St Louis, MO, USA), 100 mM Tris HCl buffer (pH 7.6), 1 mM EDTA, and 20 μg of proteinase K (Sigma)). After incubation at 55 °C for 2 days to ensure complete lysis, the microcentrifuge tubes were centrifuged at 10,000×g for 1 min to remove insoluble debris. The supernatant was transferred to a newly labeled microcentrifuge tube. DNA samples were prepared from the human FFPE tissue blocks using the same protocol for the xenograft FFPE tissue blocks.
Patient specimens
We retrospectively analyzed the clinicopathologic data of 476 patients who underwent surgery and extended lymph node dissection (D2) for advanced gastric cancer in the Seoul National University Hospital, Seoul, Korea, from January 2007 to December 2008. Patients who had a history of other primary malignancies within 5 years or were treated with neoadjuvant chemotherapy were excluded. The following pathological parameters were evaluated by gross and microscopic examination: tumor location, tumor differentiation, histological type, lymphatic invasion, perineural invasion, venous invasion, and TNM stage (American Joint Committee on Cancer, 7th edition). A total of 497 colorectal cancer patients who received curative surgery and adjuvant chemotherapy in the Seoul National University Hospital between June 2005 and November 2011 were included. Because each FFPE tissue block was made soon after the surgery, the tissue blocks of gastric cancer and colorectal cancer ranged in age from 6 to 7 and 4 to 10 years, respectively, at the time of DNA extraction. Microscopically, tumor areas with high tumor density and representative histology were marked for each case, were manually dissected, and were collected into microcentrifuge tubes containing tissue lysis buffer and proteinase K. The tissue solution was kept at 55 °C for 2 days.
The study protocol was reviewed and approved by the institutional review board of Seoul National University Hospital (1312-051-542) and was performed in accordance with the recommendations of the Declaration of Helsinki (2013) for biomedical research involving human subjects. Patient records/information were anonymized and de-identified prior to analysis.
Bisulfite conversion and Alu-based MethyLight control reaction
We used 20 μL of the supernatant for the bisulfite modification which was performed using the EZ DNA methylation kit according to the manufacturer's protocol (Zymo Research, Irvine, CA, USA). In order to measure input DNA (bisulfite-modified DNA), we performed Alu-based MethyLight control reaction which is a CpG-independent, bisulfite-specific control reaction [2]. We determined the threshold cycle (C(t) value) of this reaction in which the Alu reaction fluorescence was detected. To keep the C(t) value of bisulfite-modified DNA samples in the range from 18 to 20, we added distilled water to dilute bisulfite-modified DNA samples with C(t) values lower than 18. MethyLight PCR was performed in a 25-μL reaction volume with 200 μM dNTPs, 0.3 μM forward and reverse PCR primers, 0.1 μM probe, 3.5 mM MgCl2, 0.01% Tween 20, 0.05% gelatin, and 0.2 units of Taq polymerase on a 96-well plate (BioRad) using the following PCR program: 95 °C for 10 min, then 50 cycles of 95 °C for 15 s followed by 60 °C for 1 min.
Pyrosequencing methylation assay
The converted DNA samples were PCR-amplified with oligonucleotide primers that were designed against a consensus LINE-1 sequence by the Issa group for pyrosequencing [13]; the forward primer was 5′-TTTTGAGTTAGGTGTGGGATATA, and the reverse biotinylated primer was 5′-biotin-AAAATCAAAAAATTCCCTTTC. The PCR reaction was carried out in a 25-μL final volume comprised of 2 μL of bisulfite-treated DNA (input DNA was approximately 33 ng), 2.5 μL of CoralLoad PCR Buffer, 1.5 μL of 25 mM MgCl2, 1 μL of the forward and biotinylated reverse primers (0.4 μM final concentration), and 0.75 U of HotStarTaq Plus DNA polymerase (Qiagen, Valencia, CA, USA). The PCR cycling conditions were as follows: initial denaturing at 95 °C for 10 min, 50 cycles of 94 °C for 30 s, 57 °C for 40 s, and 72 °C for 40 s followed by a final extension at 72 °C for 5 min. The PCR products were added to the binding buffer (Qiagen) and the Streptavidin Sepharose High Performance beads (GE Healthcare Bio-Sciences Corp., Uppsala, Sweden). The biotinylated DNA-bound beads were collected and retained using the PyroMark Vacuum Prep WorkStation (Qiagen). The purified single-stranded PCR product was added to the annealing buffer (Qiagen) with 0.3 μM of sequencing primer (5′-AGTTAGGTGTGGGATATAGT), and the pyrosequencing reaction was performed using the PyroMark Q24 platform (Qiagen). The level of methylation at each of the four analyzed CpG sites (GenBank accession number X58075 sites 1–4: nucleotide positions 328, 321, 318, and 306) was determined by the percentage of methylated cytosines. The pyrosequencing assay was repeated in triplicate, and the median value of the three replicates was reported as the representative value of LINE-1 methylation.
Measurement of single gene methylation level
For evaluation of the DNA methylation status in individual genes, the MethyLight assay was performed as previously described [27]. The converted DNA samples were analyzed for methylation status in eight individual genes (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1). A complete list of MethyLight reaction probes and primers has been previously reported [28]. The MethyLight assay was repeated in triplicate, and the median methylation level (determined by the percentage of methylated reference (PMR)) was obtained.
Statistical analysis
All statistical analyses were performed using SPSS for Windows (version 21.0) (International Business Machines Corp., Armonk, NY, USA). Two-sided P values <0.05 were considered significant. The clinical database for gastric cancer patients was updated in January 2014. Progression-free survival (PFS) was calculated from the date of resection of advanced gastric cancer to the first date of documented recurrence or the date of death from any cause. Overall survival (OS) was measured from the date of resection to the date of death or the date of the last clinical follow-up before January 2014. Kaplan-Meier survival analysis was performed to compare OS and PFS using the log-rank test. Because the data on the level of LINE-1 methylation in CpG sites 1 to 4 did not follow the normal distribution, mean values across two or more groups were compared using both parametric and non-parametric tests. The Mann-Whitney U test and ANOVA test were used for the comparison of mean values across two groups, while the Kruskal-Wallis test and Student's t test were used for the comparison across three or more groups. Pearson's correlation test was used to assess the correlation between the LINE-1 methylation levels in paired FF and FFPE xenograft tissue samples. The Wilcoxon signed-rank test and paired Student's t test were used to analyze the paired differences.
Not applicable.
This work was supported by grants from the National Research Foundation (NRF) funded by the Korean Ministry of Science, ICT and Future Planning (2011-0030049 and 2016M3A9B6026921), a grant from the Priority Research Centers Program through the NRF (2009-0093820), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute funded by the Korean Ministry of Health and Welfare (HI14C1277).
Availability of data and materials
The protocols are detailed in the manuscript for scientists wishing to use them for their research work. Also, the supporting data will be made available to editors and peer reviewers, if required for the purposes of evaluating the manuscript.
Authors' contributions
JHK and GHK conceived the idea; XW and GHK designed the experiments in-depth; XW, SJ, and NYC performed the experiments; YK and JMB provided tissue samples and related clinical data; YK and GHK analyzed the data; XW and GHK contributed to the figure and analysis tools; and XW and GHK wrote the paper. The paper was critically read by all the authors and approved for publication.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
The animal study plan and protocol were approved by the Biomedical Research Institute of Seoul National University Hospital (15-0111-C1A0). The human study protocol was reviewed and approved by the institutional review board of Seoul National University Hospital (1312-051-542). Informed consent was exempted because of the retrospective nature of the study and minimal risk of harm to the study subjects. This study was performed in accordance with the recommendations of the Declaration of Helsinki (2013) for biomedical research involving human subjects.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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.
Authors' Affiliations
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- Axis2 Modules
- Avoiding Mistakes Made Using Axis2 (Developer)
- Invoking Web Services using Apache Axis2 (Java.net)
- services.xml for Axis2 Web Services (WSO2)
- Axis2 Session Management (Developer)
- Embed an Axis2 Web Service in your Webapp (WSO2)
- Apache Axiom – The XML Object Model
- Introducing Axiom (TheServerSide)
- XML processing with AXIOM (developerWorks)
- Digging into Axis2 AXIOM (developerWorks)
- Apache Rampart – security module of Axis2
- Web Services Security with Apache Rampart (WSO2)
Xerces + – XML parser
- XMLBeans – XML data binding
Cloud computing seems to be all the rage these days, with magazine articles and news stories discussing the potential benefits and pitfalls.
As with any new computing concept, cloud computing is not easily defined.
Even the experts give varying definitions.
However, cloud computing basically boils down to the following:
It consists of computing activities that take place on a remote server.
- Navigate the cloud computing labyrinth – developerWorks
- Storage made easy with S3 – developerWorks
- Picking from a profusion of platforms – developerWorks
Eclipse is an open development platform comprised of extensible frameworks, tools and runtimes for building, deploying and managing software across the lifecycle.
The Eclipse IDE is widely used by developers across many different programming languages and technologies.
Grails is an open-source web application framework that leverages the Groovy language and complements Java web development.
- Grails Home
- Build your first Grails application
- Getting Started with Grails
- GORM – Understanding databases and Grails (developerWorks)
- Many-to-many relationships in Grails
- Understanding Grails plug-ins (developerWorks)
- XML parsing with Groovy – developerWorks
HTML is the "lingua franca" of the world wide web.
Any foray into web design or web development should start with an understanding of HTML.
- The complete HTML Reference with CSS, color, character and font reference
- HTML Cheat Sheet (CMP)
- HTML Collections – List of helpful links on all things HTML (WebReference)
- Using the DOCTPE tag (WebReference)
- RGB Values (Web Design Group)
- XHTML (developerWorks)
- XRAY – Fantastic tool for web developers
CSS + – Cascading style sheets
- CSS2 Quick Reference (MeyerWeb)
- CSS2 Reference (W3C Schools)
- CSS 2.1 Properties (WebReference)
- CSS Quick Tutorial (Web Design Group)
- CSS Float Tutorial (BigBaer)
- Simple CSS Float Tutorial (maxdesign)
- Ten CSS Tricks (evolt.org)
- Hide CSS from buggy browsers (w3development)
- CSS Vault – Gallery of sites using CSS
- New Create animations using CSS3 transforms (MC Press)
HTML5 is is the latest revision of the HTML standard (originally created in 1990 and most recently standardized as HTML4 in 1997) and remains under development as of this writing.
It brings many capabilities and added structure, making coding web pages easier and more consistent for web developers.
- New Can I Use… – Compatibility matrix for HTML5 and CSS3
- HTML5 specification – a dry read from the W3C
- HTML5 Doctor – HTML5 blog + quick reference guide
- HTML5 Fundamentals (developerWorks)
- New HTML5 Please
- HTML5 Introduction (W3CSchools)
- New Elements in HTML5 (developerWorks)
- New HTML5 Structural Tags (developerWorks)
Java provides a "write once run anywhere" programming implementation on popular platforms.
- Java Home – All things Java start here at The Source for Java Developers.
- Java-net – The Java community's water cooler
- Java SE – Java Platform, Standard Edition
- J2SE End of Life (EOL) Versions
- Java EE Technologies (Sun)
- Code Samples Index (Sun)
- Java Collections – List of helpful links on all things Java (WebReference)
- Java Developers Almanac – Supplement to the book
- Java Forums – Ask questions, get answers (Sun)
- Java Ranch – Good place to get started with Java
- Tutorials and Online Training (Sun)
- Brewing Java – Comprehensive tutorial (Cafe au Lait)
- Java Tutorials and Documentation (WebReference)
- RMI Tutorial (Sun)
- EJB Tutorial (conceptGO)
- Java Code Conventions – The "Bible" on Java coding (Sun)
- How to Write Doc Comments for the Javadoc Tool (Sun)
- Advanced DAO Programming (developerWorks)
- Basics of Java class loaders (JavaWorld)
- Annotations (developerWorks)
- Compile time error messages – List of error messages and likely causes (mindprod)
- Covariant Return Types (Sun)
- Enumerated Types (developerWorks)
- Formatting numbers and currency (developerWorks)
- Garbage Collection (MCPress)
- High-Level Network Programming – Using the java.net package for network programming (Sun)
- I/O Streams (Sun)
- Introduction to Java Stack Traces (Sun)
- JAR Files Revealed (developerWorks)
- Java class file lifestyle – Introduction to the basic structure and lifestyle of the Java class file (JavaWorld)
- Java development with interfaces (JavaWorld)
- Java generics and bounded wildcards (developerWorks)
- Java FAQ and Tutorials – List of Java FAQ and tutorial links
- Java Naming and Directory Interface (JNDI) (Sun)
- Java Servlet Technology (Sun)
- Java Web Start (Oracle)
- Servlet Filters (developerWorks)
- Servlet Life Cycle (examulator)
- Synchronization (Oracle)
- Using the Java Regex Library (developerWorks)
- Using Java Reflection (Sun)
- Using Wildcards with Java Generics (developerWorks)
- Generate PDF files dynamically (developerWorks)
– Java Server Pages
- JavaServer Pages Fundamentals (Sun)
- JSP Tutorial (jsptut.com)
- Building Java Server Pages (Web Developer's Journal)
- JSP custom tags in five easy steps (developerWorks)
- Combine JavaBeans components and JSP (developerWorks)
- Create custom attributes in JSP (developerWorks)
- Error handling in custom tags (developerWorks)
- Introduction to taglibs (developerWorks)
Tools + – Handy tools to help with development and testing
- HttpUnit – Testing tool for web applications to emulate browser behavior
- JGoodies Looks – Java User Interface Design
- Java Tester – Verify functionality of Java applets in your web browser
- JUnit – Widely-used testing tool for Java applications
- JWebUnit – Testing tool for Java web applications
Client-side scripting is a necessity for today's web development and JavaScript is the tool to bring your web pages to life.
- Conditional Compilation of JScript/JavaScript in IE (JavaScript Kit)
- Demos & Applications (cross-browser.com)
- Introduction to the Document Object Model (BrainJar)
- Introduction to JavaScript (W3CSchools)
- JavaScript Reference (JavaScript Kit)
- JavaScript 1.5 Core Guide (WebReference)
- JavaScript Samples (JavaScript Reference)
- JavaScript Reserved Words (quackit.com)
- JavaScript Library – Thousands of searchable scripts
- JavaScript Tutorial for Programmers (WDVL)
- JavaScript Tools (WebReference)
- jQuery – JavaScript library
- New Intro to jQuery Mobile with examples (MC Press)
- List of major JavaScript frameworks with descriptions and links to each (InfoQ)
- Microsoft JScript Reference
- MooTools – JavaScript framework
- Tutorials, scripts & answers (about.com)
PHP is a widely used general purpose scripting language that is especially suited for Web development.
- New Date arithmetic in PHP (MC Press)
- The Future of PHP – PHP V6 Changes (developerWorks)
- PHP – The definitive source for PHP information
- PHP Builder – PHP news, articles and forums
- Practical PHP Programming – Great reference source from the author of "PHP in a Nutshell" (hudzilla)
- PHP Pocket Reference (O'Reilly)
- CVS parsing in PHP (MC Press)
- PHP developer resources (Zend)
- PHP frameworks – List of PHP frameworks and their features
- Simple PHP tutorial (php.net)
- Introduction to PHP (WebReference)
- PHP database access (Intranet Journal)
- PHP Security Techniques (WebReference)
- PHP Eclipse-Plugin – This is a must-have if using Eclipse as your IDE
- PHP Interactive – Interactive shell for debugging and testing PHP code
- PHP Code Beautifier – Tool for cleaning up PHP code
- Validating XML in PHP (developerWorks)
Schema is a type of microdata that makes it easier for search engines to parse and interpret the information on your web pages more effectively.
- schema.org home
- Using schema markup for local SEO (Search Engine Journal)
- Structured Data Testing Tool (Google)
SQL (Structured Query Language) is the most commonly used programming language for querying and modifying data and managing databases.
- Fun with Dates and Times (developerWorks)
- New How to Prevent SQL Injection Attacks (Code Project)
UML is an acronym for unified modeling language.
It is a standardized visual specification language for object modeling.
- UML Specifications (Object Management Group)
- UML Hands-On Introduction (Code Gear)
- ArgoUML – UML design tool
Though there are many ways to define Web Services, the most essential aspect is machine to machine interaction over a network.
- WS-I – Standards organization for web services interoperability
- SOA and Web services (developerWorks)
- WSDL Tutorial (W3CSchools)
- WSDL Essentials (Developer)
- Which style of WSDL should I use? (developerWorks)
- Loosely typed versus strongly typed Web services (developerWorks)
XML stands for extensible markup language.
The key word is "extensible" as it allows users to define their own elements.
Its primary purpose is to facilitate the sharing of structured data across different information systems, making it ideal for web services.
- Extensible Markup Language (XML) – Information from the W3C
- Extensible Markup Language (XML) 1.0 – Specification from the W3C
- Extend Enumerated Types (developerWorks)
- JDOM – Java-based solution for accessing, manipulating, and outputting XML data
- xml.com – Information and services for the XML community
- XML.org – Online community for those interested and involved in XML-related standards and specifications
- XML for the absolute beginner – A guided tour from HTML to processing XML with Java (JavaWorld)
- XML Schema Part 0 – Essential primer for XML schemas (W3C)
- XML Schema Part 1 – Essential reference for XML schema structures (W3C)
- XML Schema Validator (W3C)
- XML Tutorials, Code and Forums (developerWorks)

subset: commoncrawl


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Working in a joined up way
Making the move into work easier
Helping with the cost of raising a child
Posted on 1st December, 2014 by Lincoln Against Poverty
People struggling financially in the run-up to Christmas are being urged not to panic by turning to high interest lenders.
Lincoln Against Poverty, a partnership between City of Lincoln Council and several city organisations, is signposting anyone needing debt advice to Christians Against Poverty in the third phase of its 'Say no to high cost borrowing' campaign taking place in the lead-up to the festive season.
Christians Against Poverty offers help to people struggling with debt, as well as running job clubs and giving assistance to those who need support with their budgeting.
The organisation has visited more than 500 people in the city since launching in 2006.
Simon Hoare, Manager for Christians Against Poverty (CAP) in Lincoln, said: "For many of us, it's not just Christmas cards we can expect through the door but payment demands and credit card statements too.
"Talking about money doesn't come easily but that doesn't mean anyone should have to carry the burden of debt alone, especially at this time of year.
"CAP exists to support people to feel back in control of their finances and to succeed in becoming debt free. A free, confidential home visit is only one phone call away – 0800 328 0006."
The city council also has a range of benefits advice it can give to those who are worried about their money.
Martin Walmsley, Head of the Shared Revenues and Benefits Service, said: "Many people do not realise that they are entitled to benefits.
"Also, people sometimes think because they are working they will not be entitled – but many customers are entitled to benefits as well as their wage.
"In fact, many benefits are paid no matter how much you have coming in (not 'means-tested'). Also, means-tested benefits increase their rates regularly, and people who didn't qualify for benefits such as Council Tax Benefit in the past, may now qualify. For further information contact us by e-mail email@example.com or by telephone on 01522 873382."
The 'Say no to high cost borrowing' campaign got under way in October and runs until January 2015 with the aim of showing where people can access affordable credit or debt advice if they are worried about their finances.
A range of services offering help to people who are living in poverty can be found on the Lincoln Against Poverty website, www.lincolnagainstpoverty.co.uk/help


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I just found out that Al Lehman died on Monday. It's been a long journey, this death.
Al and Lois have been part of North Valley Friends since before its founding. I remember when Hal and I were newly married and began attending Springbrook Friends, one of the meetings that merged to form North Valley. Al was teacher of the adult Sunday school class. Newly graduated from college, as well as newly married, I was a bit of a rebel at the time, highly critical of anything to do with church. But I loved that class. More to the point, I loved Al and his gentle way of opening the Scriptures and of encouraging us to engage with them and with each other.
In the following years, each time Hal and I would return from Bolivia for our furlough year in Oregon, Al and Lois were a stabilizing factor for us in the church. They were like parents in the faith and never seemed to change, were always part of the life and health of the community we came home to.
I was with the elders visiting Al and Lois in their home on Sunday afternoon. Al never woke up during that time, and his labored breathing formed a sort of background music to our visit. We sat with Lois and Bev, sang some old hymns, with Hal's harmonica accompaniment—songs like "Blessed Assurance," "Great Is Thy Faithfulness," "When the Roll Is Called up Yonder." We then all talked about what Al had meant to us, and the strong testimony of a consistent, faithful, gentle life unfolded.
Lois shared that the day before God had given her an unexpected gift. Al woke up and was conscious for about an hour. During that time they again expressed their love to each other. Lois was smiling as she told us, "I didn't think I get another chance to tell him I loved him." Bev shared about how her father never wanted his last months to be like this, did not want to be so dependent on family for every need, but that as his condition gradually worsened, he just seemed to accept that this was how it was to be. He walked gently and submissively through the whole experience.
We prayed for Al and the family, sat around for a short while longer and left, not realizing this was his last day with us.
I'm remembering reading about the spiritual discipline of the "good death," a practice in years gone by, not spoken of much anymore. For the life of me (interesting phrase), I can't find the source of my reading. I've googled it and find thousands of references to a "good death," all contemporary. Today the phrase pertains more to the medical profession than to Christianity, and is linked with practices such as hospice care. It basically means a death with as little physical and spiritual pain as possible. That's good. Al and the family benefitted from hospice care during the last several months.
But the "good death" as a spiritual discipline has another sense entirely. Rather than something the dying person receives at the hand of others, it is a gift that person gives to others. It refers to letting one's death be as full of Jesus and of the fruits of the Spirit as one's life was. It means letting the way the person handles death become a ministry in itself, a blessing to the community. It results in a deep joy that mingles with sorrow as the person finally slips over the edge and into Life.
Of course, for that to even be possible, a person would have to have lived a consistently Spirit-filled life. Over a long period of time. Al Lehman was such a person, and while we will all miss him, I smile as I imagine him now in the presence of the One he loved. And I thank him for giving us the gift of a good death.
Wednesday, October 26, 2011
The good death
Posted by Nancy Thomas at 8:27 AM
Labels: Writing death, spirituality
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General Terms and Conditions
Article 1: Definition of terms
1.1. In these General Terms and Conditions the terms listed below are defined as indicated:
a. Event: The annual LBL Ardennen Classic cycling event organised by Vereniging Le Champion.
b. Participant: A natural person who has registered to take part in the Event in accordance with the registration procedure required by the Organizer.
c. Agreement: The Agreement in accordance with which the Participant is entitled to take part in the Event.
d. Organizer: The legal entity (Vereniging Le Champion) with which the Participant has entered into an Agreement.
1.2. Every Agreement is subject to these General Terms and Conditions.
Article 2: Participation
2.1. The Participant must have reached the age of 14 on the day of the Event.
2.2. The Participant has to wear a helmet.
2.3. The Participant may only participate in the Event if he has filled in the Registration Form provided for this purpose fully and truthfully, paid the registration fee, and agreed to the General Terms and Conditions.
2.4. Cancellation (registration fee, payment for hotel and any other facilities or services ) is possible in writing for members of Le Champion until 10 july. For other participants no refunds or whatsoever are possible.
2.5. The Organizer may decide to cancel the Event at a late stage on the grounds of force majeure, in which case the Event will not be rescheduled. The next edition of the Event will be held a year later. In such a situation registration fees and payment for any other facilities or services (such as hotel over night costs e.g.) are non-refundable.
2.6. The Organizer is not liable for any costs incurred by the Participant if the Event has to be cancelled due to force majeure.
Article 3: Liability
3.1. The Participant participates in the Event at their own risk. The Organizer is not liable for losses of any kind, however they may be termed, that may be incurred by the Participant as a result of participation in the Event, unless the losses in question are the direct result of wilful intent or gross negligence on the part of the Organizer. This exclusion of liability also applies to serious losses, such as losses due to injury or death.
3.2. If, despite the provision of the first clause of this article, the Organizer is obliged to accept liability for losses incurred by the Participant, the obligation on the part of the Organisers is limited to reimbursement of losses up to the maximum sum paid out by the Organisers' insurers in the case in question.
3.3. The Participant must be adequately insured against the risk of losses that they or a dependent may incur due to illness, injury or death as a result of participation in the Event.
3.4. The Participant declares that they are aware of the fact that participation in the Event requires sound physical and mental health and that they meet this requirement and that they have undertaken sufficient preparation for the Event in the form of training and other activities. The Organisers expressly advise and urge Participants to undergo a sports medical examination in preparation for their participation in the Event.
3.5. The Participant indemnifies the Organizer against all liability for losses incurred by third parties as a result of actions or omissions attributed to a Participant in connection with the Event. The Participant must be adequately insured against the risk of liability for such losses.
3.6. The Participant indemnifies the Organisers against all liability for losses incurred by the Participant as a result of participation in the Event, unless the losses in question are the direct result of wilful intent or gross negligence on the part of the Organisers.
Article 4: Portrait rights
The Participant gives the Organizer advance permission to publish photographs and other visual materials on which the Participant is visible taken or created during or in connection with the Event.
Article 5: Personal details
The Organizer will keep the personal details provided by the Participant on file. In participating in an Event, a Participant authorises the Organizers to use their personal details to send them information and (provided that the Participant has given permission on the Registration Form) to pass on their personal details to third parties so they can send the Participant information. In entering into the Agreement the Participant gives the Organizer permission to publish their name and the results of the Event by means of publication in newspapers and on the internet for example.
Article 6: Rules and Regulations
The Participants participation in the Event is subject to the Rules and Regulations of the LBL Ardennen Classic which form an integral part of these General Terms and Conditions.


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|Skilled visas
|Business visas
|Temporary visas
|Free assessments
|About us
|Contact us
|Immigration websites
|Career websites
Workpermit.com is registered with the Office of the Immigration Services Commissioner and offers immigration and work permit services for entry to the UK, Australia, USA, New Zealand, Canada and most Western European Countries.
Workpermit.com has one of the top immigration related web-sites in the World as rated by Yahoo and has been on the internet since 1995.
Workpermit.com is one of the oldest and largest UK immigration consultancy firms having been founded in 1988 (formerly BCL Immigration Services)
Workpermit.com employs top consultants and lawyers a number of whom have over ten years of experience in immigration law.
Workpermit.com is an Associate Member of Intellect the UK industry association for the IT and Electronics industry.
Call our Fleet Street, Central London Office on +44 (0) 20 7842 0800 for an appointment or a telephone consultation.


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INTORQ: Exceed expectations – convince customers
Sybit implements SAP Hybris Cloud for Sales in just three months
INTORQ develops and sells electromagnetic brakes and clutches with the highest production standards. Be it for engines or geared motors, in cranes, wind turbines, or lift systems – the company headquartered in Aerzen offers both configurable standard products and customer-specific solutions. Major, globally operating plant manufacturers rely on the company's quality. To be able to respond to the increasing demand for tailor-made solutions, all the employees involved – from Product Development to Sales – were to have access to all data. It took Sybit just three months to implement the SAP Hybris Cloud for Sales solution. The result: INTORQ can now respond faster to customer requests and process inquiries more efficiently.
The Project
- The customer data was stored in local folders and e-mail systems and not accessible to all involved employees
- In the case of individuals' absences or holidays, the information flow was not fully guaranteed
- Increased communication volumes due to expanding project business and therefore increasing demand regarding customized solutions
- Collaboration with globally operating large customers requires a clear overview of scheduling and communication
- One central system for the entire customer relationship management
- Data accessible any time and from any device – with offline capability
- Central data source for lead and account management thanks to implementation of SAP Hybris Cloud for Sales
- Comprehensive consulting and very speedy system implementation by Sybit
The Company
The INTORQ brand stands for reliable brake solutions of the highest standard. Whether in cranes, hoists, electric vehicles, wind turbines or lift systems – INTORQ products are used in the most diverse of applications. Rely on Intorq to create the right solution for your application. Our modular system of INTORQ products are used worldwide in motors and geared motors and has set the standards worldwide. The company, which is headquartered in Aerzen and has subsidiaries in Shanghai, Atlanta and Pune, offers international sales and service around the globe.
"We offer our customers customized solutions. That requires extensive communication. With SAP Hybris Cloud for Sales, we have improved collaboration and can focus more strongly on our customers."
Alexander Patzig, Head of Marketing and Sales, INTORQ GmbH & Co. KG


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Part 7: Setting Up Worklists for Smart Goals
(If you missed the beginning of this series, click here)
Now that we have our framework for our SMART collection, we need to add some sub-tools to get the work done. For this, we will be adding three tools within the collection: A Scope Check tool, an Action Plan, and a Metrics table to capture the before/after measurements.
First, we will add a Checklist tool, to let us list the initiatives or corrections we could put in place, then utilize the checkmarks to define which are in-scope, and more importantly, out-of-scope.
Add a new Checklist tool by using the "Add Tool – Browse Tool Catalog…", then on the "Basic" list, select "Checklist".
This time, instead of "moving it to the bottom" of the canvas, we are going to add it to the collection instead. To do this, each tool has a "More Actions" option. In that menu, you should see "Move To Collection…"
Here, we will move it to our SMART Goal #1 collection.
At this point, your canvas for your template is getting fairly complicated. Please note that on the left-side of your template, you will find the "Work Items" section. This section essentially lists out your tools in the template (or when live, an actual activity). It is simple to just navigate down the tool list by clicking on the focus item you wish to review. For example, if we click on the SMART Goal #1 collection, we will see the list of sub-tools within the collection. Here we see our newly added (unnamed) checklist.
Click on the Checklist in the canvas, and rename it to "#1 – Brainstorming / Scope Check". Then add 2 Action Items to the tool: one for Brainstorming, and another for scope check. Your final tool will look like this.
Next, we want to add a Timeline tool to the collection. Again we use "Add Tool – Browse Tool Catalog…". The Timeline tool is located in the Coordinating section.
Remember to move it to the collection:
Rename the Timeline to "#1 – Action Plan", and add an Action Item to prompt the team to create the action plan.
The final tool we will add is a simple table to keep track of our metrics. Again, we use the "Add Tool – Browse Tool Collection…". Tables are located on the "Basic" list.
Move your table to the Collection.
Finally, setup your table with 3 columns (Metric, Before, After), and delete the 4th through 8th rows (3 metrics are usually fine). Rename your table, and create an Action Item for the team to fill it in. Your SMART Goal #1 should now be complete.
Finally, we need to duplicate our efforts for SMART Goal #1 to create SMART Goal #2 and SMART Goal #3. There is a "copy" function of each collection that will help us achieve this. The Copy function is located under the "More Actions" menu of the collection.
This will copy the items within the Collection to a new collection within a template. Be sure to select your Six Sigma template. Now, you simply have to rename all of the items within the collection. (NOTE: You may need to refresh your browser window in order to see the new collection within your template)
You can move the new collection to the bottom of the list by selecting the grey-bar next to the collection when you hover over it. When you do this, you can click-and-drag the collection down to the bottom of the canvas (after the #1 goal).
Now, rename the collection and the tools to represent SMART Goal #2.
IMPORTANT: There currently is an issue (or feature?) with the Copy function. It DOES NOT copy any action items. So, this means that you will have to go back through every tool within SMART Goal #2, and re-create those action items within the template. Kinda a bummer!
Finally, copy the SMART Goal #1 one more final time, and make it SMART Goal #3. Remember to rename all tools, and re-create your Action Items as above. You should end up with a template and a Canvas that looks very much like this:
That's it. Your StreamWork Six Sigma template is now complete. This template should help define a winning process to focus teams, and tackle any form of challenge within your company. In the next section, we will walk you through an example project, to see how all the pieces come in to play.


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Yarra City Council
The City of Yarra is an inner metropolitan municipality which is home to a diverse community of about 80,600 people.
We'd love to hear your feedback on our services
We have a small Occasional Care child minding service at Fitzroy Swimming Pool and Richmond Recreation Centre.
Available 9am to 12 noon.Read more
We offer massage and myotherapy on Tuesdays and Thursdays at Richmond Recreation Centre.
To book call 9205 5032.Read more
We have 2 outdoor courts for hire at Richmond Recreation Centre. Our courts are available outside of school hours and school holidays.
To book call 9205 5032.Read more
Our pools, rooms and spin studio can be hired for your private events and activities.
You must have Public Liability Insurance and appropriate qualifications to hire our spaces and staff.Read more


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OnTheThrottle.TV recently spoke with Costilla about his move to the BSB series. "I still can't believe I will be leaving for England in a few days," says Costilla, "I'm so grateful to Cresswell Racing for this opportunity!"
Costilla, who began his racing career with M1GP in southern California, will join riders Jayson Uribe and Ricky Tarren to complete Cresswell Racing's 2013 team.
The team will compete in the BSB Motostar series with two 2004 RS 125 Hondas, prepared by Ian Emberton of IAN-E Racing, and a KTM RC125, prepared by Redline KTM from Loughborough, Leicestershire.
"Daniel will be competing in all 12 BSB Motostar 125/Moto3 Championship rounds sponsored by Monster Energy in 2013 at all UK premier circuits including Brands Hatch, Silverstone and our team's home round of Donington Park," explains John Cresswell, Team Manager.
Last year, Costilla competed in the US WERA West Series, where he honed his skills in the Honda 80 and Honda 125 classes. This year, he joins Cresswell Racing, a move he hopes puts him one-step closer to his ultimate goal— racing MotoGP.
Costilla was discovered, of all places, on Facebook. "I put word out on Facebook and was given Daniel's name. I did a bit of research on Daniel through various sources, then contacted Daniel and told him about the ride available. That's how we have come to today's present situation," says Cresswell.
Watch the latest video of Daniel Costilla, available here from OnTheThrottle.TV: http://www.onthethrottle.com/races/american-racer-daniel-costilla-headed-for-bsb-motostar-in-2013/.
For more updates on Daniel "Danger" Costilla and to cheer him on, be sure to follow him on Facebook, https://www.facebook.com/daniel.d.costilla. Additional 2013 sponsorship opportunities are still available. Find out more at email@example.com or call (760)535-4664. To learn more about Cresswell Racing visit www.cresswellracing.co.uk.
Founded in 2006, OnTheThrottle.TV provides teams, riders, sponsors, and manufacturers with dedicated coverage to help boost brand awareness, promote audience engagement, and increase sales. Having worked with dozens of partners in creating thousands of videos, OnTheThrottle.TV is an established leader in creating specialized, motorcycle-related content that entertains and educates viewers. For more information about OnTheThrottle.TV and its video production services, visit www.onthethrottle.tv or call (650) 479-4688.


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NEW YORK, Feb 25 (Reuters) - Berkshire Hathaway Inc.'s gain on its investment in Apple Inc. stands at more than $1.6 billion after shares of the iPhone maker surged.
The stake of 61.2 million shares was acquired last year for $6.75 billion, an average of about $110.17 apiece, according to the annual report Saturday from Berkshire, which is led by billionaire chairman Warren Buffett.
The holding was valued at more than $8.3 billion as of Friday's $136.66 closing price.
Berkshire became one of the top 10 Apple investors in 2016, taking a stake of more than 9 million shares in the first quarter and then accelerating purchases in the last three months of the year.
The Apple investment appears to reflect much of the $12 billion of stock that Buffett said he had bought between the Nov. 8 Presidential election and the end of January. (Reporting By Jennifer Ablan; Editing by David Gregorio)


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The Following Items are Old Inventory from Francis Bannerman and Sons. This is what remains of what was once the largest and oldest military surplus dealer. The unfinished Krag parts represent different steps in the manufacturing process. They provide a unique insight into the production of these small arms.
Store Specials


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IGN: Nikita's emotionally complicated second season continued in fine style with "Partners", a well-crafted episode about shifting relationships and unexpected consequences that gave fans a much needed thaw in Nikita and Alex's cold war.
FilmWatch is a community of awesome people posting and discussing the latest movie news. It's part of NewsBoiler, a network of social news sites covering today's pop culture.


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Marketing managers play a key role in any business -- developing a detailed marketing strategy to help maximize profits and increase customer return and loyalty.
Marketing managers work closely with market researchers to determine the demand for a company's products and services, track competitor products, and identify potential customers.
They develop pricing strategies to increase the firm's profits or share of the market while also ensuring customer satisfaction. They work with sales and product managers to oversee product development and monitor trends that suggest the need for new products and services. Lastly, marketing managers coordinate with advertising and promotion managers to promote the firm's products and services and to attract potential customers.
Education and Training
Most marketing managers have at least a bachelor's degree in business administration, with a concentration in marketing. Many marketing specialists earn an MBA in order to become a marketing manager.


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We're SO excited to be hosting a Twitter Party on Monday, July 30th with Naturalizer Shoes!
We'll be chatting about Fall shoe trends and why Naturalizer Shoes are a PERFECT fit for your feet! We're also excited to share that Naturalizer Shoes is our footwear sponsor at our Getting Gorgeous Event being held on Saturday, August 4th!
We simply CANNOT wait!
We'll be dishing about our FAVE Naturalizer Shoes!!
When: Monday, July 30th at 8PM (ET)
Where: Twitter
Hashtag: #Naturalizer
Hosts: @AudreyMcClellan @VeraSweeney
Tweetgrid: http://bit.ly/NC0Gbc
See you there!!


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If you're curious to what are the health benefits of hibiscus tea, then this guide is perfect for you. In this guide we take a look at the wide range of health benefits and side effects you may get from drinking this tea.
What is Hibiscus Tea?
Hibiscus tea is an herbal tea that is made by infusing the Roselle flower in water. The Roselle flower is a species of hibiscus that is native to West Africa. Once brewed this tea has a tart cranberry like flavour.
You are able to add sugar to the tea to help remove the some of the tartness and make it a bit sweeter. cup of hibiscus tea contains between 15-30% of a range of organic acids, which include, citric acid, malic acid, and tartaric acid.
If you're asking yourself does hibiscus tea have caffeine, then the answer is no. This is because it is an herbal tisane therefore doesn't contain any stimulate to keep you awake.
Hibiscus tea goes by a variety of different names around the world the following is a few examples:
- Latin America: Arthul Ka Phool
- Australia: Rosella
- India: De Jamaica
- Egypt, Italy, Sudan and most post-soviet states: Karkadé
- Iran: Chai Torsh
- Iraq: Chai Kujarat
- Philippines: Gumamela
- West Africa: Bissap
- Wider Caribbean: Red Sorrel
- Barbados, Trinidad and Jamica: Sorrel
- US & Most other regions: Jamaica
The Health Benefits of Hibiscus Tea
The following are the some of the large health benefits of hibiscus tea that you may get from drinking it. There are lots of smaller health benefits and side benefits that we might not have touched on in this guide.
Hibiscus Tea Helps with Blood Pressure Maintenance
High blood pressure is a major problem for many people today, thanks to the modern-day stressful environment. High blood pressure increases the risk of heart disease, stroke, and death from cardiovascular disease.
As a lot of people know, one way to manage high blood pressure is through a diet change. Hibiscus tea is well-known for its ability to lower high blood pressure, providing a natural, risk-free and tasty way to help lower your blood pressure.
A review done by the well trusted Cochrane Collaboration on the effects of hibiscus found that it has a positive blood pressure maintenance effect. Just having as little as 2 to 3 cups of hibiscus tea every day helps reduces the need for the use of anti-hypertensive prescription medicines. The anti-hypertensive medicines can have some potentially bad side effects.
The tea is also recommended for many who are suffering from type 2 hypertension to manage or even prevent having high blood pressure.
Hibiscus Tea is Naturally High in Antioxidants
One of the many health benefits of Hibiscus tea is that it is made from the dried calyces of hibiscus flowers. Calyces are a well-known natural source of antioxidants. Antioxidants are an essential part of our everyday diet.
The antioxidants help to rid our bodies of potential free radicals and help protect against a variety of chronic diseases such as, heart disease, diabetes and cancer.
Antioxidants are also known to fight and prevent low-grade chronic inflammation which is linked to a variety of cancers and other illnesses.
Consuming Hibiscus tea helps reduce this inflammation, which reduces the risk of developing various chronic diseases. Another benefit is that the antioxidants also helps reverse the effects of ageing on our bodies.
Hibiscus Tea Helps Lower Cholesterol
Another well researched and documented health benefit of hibiscus tea is that it helps reduce the amount of the bad cholesterol (LDL) and even provide an increase in the amount of good cholesterol (HDL).
Studies published in 2003 in the "Journal of Agricultural and Food Chemistry", and in 2009 in the "Journal of Alternative and Complementary Medicine" both back up these findings. Tests performed by these two studies showed that when someone was given Hibiscus tea twice a day for one month there was a noticeable increase in their HDL cholesterol and an obvious drop in their levels of LDL cholesterol.
Hibiscus tea is a tart, but tasty drink that brings with it a multitude of health benefits that anyone can enjoy without risk of any side effects. With just drinking two cups a day you can help maintain your blood pressure, give yourself a good dose of helpful antioxidants while also managing to lower your bad cholesterol. Proving a guilt free, side effect free way of keeping yourself healthy.
Our hibiscus tea recipe is a great way to learn how to make hibiscus tea. It will take you through all the steps to getting the perfect tasting tea that everyone will love.
If you're interested in picking up some tea you can find both tea bags and loose leaf tea over at amazon. You can also find it for sale most supermarkets or any specialised tea shop.
I hope this guide has helped you learn more about the health benefits of hibiscus tea and its side effects. If you have something to share on this amazing tea, feel free to leave a comment below.


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We are passionate about ceramics
State-of-the-art solutions of high-performance ceramics by 3M Technical Ceramics, a branch of 3M Deutschland GmbH.
Founded in Kempten, Bavaria, in 1922 as Elektroschmelzwerk Kempten (ESK), 3M Technical Ceramics develops and produces technical ceramics and coatings for customers in virtually all industries. On this background, the company has established itself as a world leader in high-performance ceramics. Its long-term success story is based on a wide knowledge of materials, as well as the technical, scientific and use-specific passion for these materials. This know-how has enabled the company to play a key role in the development of high-performance ceramics in the 20th century. The extensive range of materials comprises borides, carbides and nitrides, such as boron carbide, boron nitride, silicon carbide, silicon nitride or titanium diboride. The product portfolio consists of ten specialized, application-oriented ceramics, which are used by customers from over 30 industries around the world. These include the automotive and electronics industries, the oil & gas industry, machinery and plant engineering, metallurgy, fluid handling, as well as the military technology. In cooperation with customers and research institutes 3M Technical Ceramics continuously develops innovative product solutions that enable new production processes.
For this purpose, the company relies on cost-optimized manufacturing processes through extensive productivity management. With its intelligent innovation management, 3M Technical Ceramics permanently develops new products, thus opening up new fields of application. Our high demands in terms of quality improvement and consulting competency make us the optimum development partner for market-oriented innovations.
Sustainability is an essential part of our values. In addition to high quality standards, these include the efficient use of resources as well as safe and environmentally-friendly products and manufacturing processes.
Our quality and environmental management are certified to ISO 9001 and ISO 14001; our power management to ISO 50001; and our surface engineering to ISO 16949. 3M Deutschland GmbH's quality and environmental management is certified to ISO 9001. The environmental management system is based on the internationally recognized ISO 14001 standard, to which almost all of the company's production facilities are certified. 3M Deutschland GmbH's power management system is certified to ISO 50001, and its surface engineering to ISO 16949.


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Inside the Caves of Rotten Teeth - A Review by Ivor W. Hartmann at AfricanWriter.com
...There is a wonderful quality to Barrett's style of writing evident in From Caves of Rotten Teeth, from vivid descriptions and dialogue, to heart wrenching circumstance and fiendish plots. It is I feel written with a bold and noble spirit, one that shines to illuminate the good, bad, beautiful, grotesque, brave, cowardly, happy and insane. In this Barrett almost leaves no stone unturned, no matter what may crawl out into the light... Full Review at African Writer


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|Jeppesen - Honolulu, Hawaii
Jeppesen offers Dispatcher Certification training which is approved by the Federal Aviation Administration (FAA) and several other National Aviation Administrations (NAA) worldwide. Jeppesen is approved for VA benefits and Post-911 GI Bill benefits for our dispatcher program. Obtain your dispatcher training with the trusted company that also provides worldwide airlines with a wide variety of Flight Operations and Airline Operations Control (AOC) based products and training.
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