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Araújo T, Khayat A, Quintana L, Calcagno D, Mourão R, Modesto A, Paiva J, Lima A, Moreira F, Oliveira E, Souza M, Othman M, Liehr T, Abdelhay E, Gomes R, Santos S, Assumpção P. Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer. World J Gastroenterol 2018; 24(47): 5338-5350
Paulo Assumpção, MD, MSc, PhD, Academic Research, Adjunct Professor, Surgical Oncologist, Núcleo de Pesquisas em Oncologia, Universidade Federal do Pará, Rua dos Mundurucus 4487, Belém 66073-000, Brazil. assumpcaopp@gmail.com
Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer
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World J Gastroenterol. Dec 21, 2018; 24(47): 5338-5350
Published online Dec 21, 2018. doi: 10.3748/wjg.v24.i47.5338
Taíssa Araújo, André Khayat, Luciana Quintana, Danielle Calcagno, Ronald Mourão, Antônio Modesto, Juliana Paiva, Adhara Lima, Fabiano Moreira, Edivaldo Oliveira, Michel Souza, Moneeb Othman, Thomas Liehr, Eliana Abdelhay, Renata Gomes, Sidney Santos, Paulo Assumpção
Taíssa Araújo, André Khayat, Luciana Quintana, Danielle Calcagno, Ronald Mourão, Antônio Modesto, Juliana Paiva, Adhara Lima, Fabiano Moreira, Sidney Santos, Paulo Assumpção, Núcleo de Pesquisas em Oncologia, Universidade Federal do Pará, Belém 66073-000, Brazil
Edivaldo Oliveira, Michel Souza, Laboratório de Cultura de Tecidos e Citogenética, Instituto Evandro Chagas, Belém 66087-082, Brazil
Moneeb Othman, Thomas Liehr, Institute of Human Genetics, Universitätsklinikum Jena, Jena 07747, Germany
Eliana Abdelhay, Renata Gomes, Laboratório de Célula Tronco, Centro de Transplante de Medula Óssea, Instituto Nacional de Câncer José Alencar Gomes da Silva, Rio de Janeiro 20230-130, Brazil
ORCID number: Taíssa Araújo (0000-0002-1716-4445); André Khayat (0000-0002-3451-6369); Luciana Quintana (0000-0003-3261-910X); Danielle Calcagno (0000-0002-4429-2573); Ronald Mourão (0000-0001-5582-4961); Antonio Modesto (0000-0003-1854-0358); Juliana Paiva (0000-0002-2082-2250); Adhara Lima (0000-0001-6315-2392); Fabiano Moreira (0000-0002-2799-3546); Edivaldo Oliveira (0000-0001-6315-3352); Michel Souza (0000-0003-1038-6448); Moneeb Othman (0000-0002-0743-1191); Thomas Liehr (0000-0003-1672-3054); Eliana Abdelhay (0000-0001-5166-0832); Renata Gomes (0000-0001-7590-0335); Sidney Santos (0000-0002-9417-8622); Paulo Assumpção (0000-0003-3846-8445).
Author contributions: Araújo T and Khayat A contributed equally to this work; Araújo T and Khayat A performed the majority of experiments; Moreira F, Oliveira E and Souza M analyzed the data; Modesto A, Paiva J and Lima A performed the molecular investigations; Quintana L, Calcagno D and Mourão R participated equally in cell culture management; Othman M and Liehr T performed molecular cytogenetics experiments; Abdelhay E and Gomes R performed proteomic assay; Santos S and Assumpção P designed and coordinated the research; Araújo T , Khayat A and Quintana L wrote the paper.
Supported by Fundação Amazônia de Amparo a Estudos e Pesquisa (FAPESPA), No. 174/2014.
Conflict-of-interest statement: The authors declare that they have no conflicts of interest.
Data sharing statement: No additional data are available.
Corresponding author to: Paulo Assumpção, MD, MSc, PhD, Academic Research, Adjunct Professor, Surgical Oncologist, Núcleo de Pesquisas em Oncologia, Universidade Federal do Pará, Rua dos Mundurucus 4487, Belém 66073-000, Brazil. assumpcaopp@gmail.com
Telephone: +55-91-984171112
First decision: August 31, 2018
Revised: September 7, 2018
Accepted: October 5, 2018
Article in press: October 5, 2018
Published online: December 21, 2018
To establish a permanent piwi like RNA-mediated gene silencing 1 (PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.
CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences (Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Wound-healing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit (Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer.
PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed mRNAs identified a total of 35 genes (5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes (DOCK2, ZNF503, PDE4D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed (being up- or down-regulated after knockout).
Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.
Key Words: Gastric cancer, Piwi like RNA-mediated gene silencing 1, CRISPR-Cas9, Migration, Invasion
Core tip:Piwi like RNA-mediated gene silencing 1 (PIWIL1) gene emerged as an interesting target for gastric cancer, as it is expressed in cancer, stem and germ cells, but it is absent in normal somatic tissue. Our results propose that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.
Citation: Araújo T, Khayat A, Quintana L, Calcagno D, Mourão R, Modesto A, Paiva J, Lima A, Moreira F, Oliveira E, Souza M, Othman M, Liehr T, Abdelhay E, Gomes R, Santos S, Assumpção P. Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer. World J Gastroenterol 2018; 24(47): 5338-5350
DOI: https://dx.doi.org/10.3748/wjg.v24.i47.5338
Gastric cancer is a major contributor to global cancer burden, being the third leading cause of cancer death worldwide and in both sexes[1]. This type of cancer is thought to be consequence of a multi-step process, resulting from different genetic and epigenetic changes. Specifically, dysfunction of oncogenes and tumor suppressor genes contributes to this malignant disease, and many candidate genes have been implicated to serve as gastric cancer biomarkers[2].
In this context, the piwi like RNA-mediated gene silencing 1 (PIWIL1) gene, located in 12q24.33 and having 22 exons, became an attractive target for gastric cancer treatment. PIWIL1 protein is expressed at increased levels in cancer tissues, stem cells and germ cells, but it has been shown to be absent in normal somatic tissues. This means that it could be a potential target for therapy, since most non-cancer cells would not be affected by cytotoxic effects[3-7].
PIWIL1 plays a key role in tumor cell viability, migration and invasion, and its expression is associated with the maintenance of stem-like characteristics of tumors, which in turn contribute to more severe histological grade, advanced stage and worse clinical outcome[8-10].
Wang et al[11] showed that expression of PIWIL1 in gastric cancer tissue was significantly higher than in adjacent-to-tumor tissue (tumor front). They also demonstrated that patients with a lower expression of PIWIL1 presented a significant better overall survival rate compared to patients with a higher expression levels. Additionally, the 5-year survival rate of patients with a higher expression level of PIWIL1 was significantly lower (36.5% vs 67.6%).
Liu et al[12] reported that expression of PIWIL1 progressively increases during cancer development. The expression ratio in normal gastric tissues, atrophic gastritis, intestinal metaplasia and gastric cancers varied from 10% to 76%.
To further investigate the potential functions of the PIWIL1 gene, Liu et al[12] also silenced PIWIL1 by antisense or short hairpin RNA and noted that suppression of this gene inhibited the growth of gastric cancer cells and induced G2/M arrest. Although relevant information regarding the possible role of PIWIL1 in gastric cancer carcinogenesis is provided by the current literature, the exact molecular mechanisms involved in this carcinogenic process remain unclear.
A recently introduced technology, based on the adaptive immune system of prokaryotes and known as type II clustered, regularly interspaced, short palindromic repeats (CRISPR)/associated protein (Cas), has been demonstrated to cleave double-stranded DNA and has emerged as a relevant genome editing tool[13-15].
This technology can be used both to perform permanent gene knockouts and the site-specific integration of a gene (knock-in)[16-19]. Importantly, it allows for the permanent silencing of the target gene, and it also creates a stable and permanent cell line with the desired modification[14,16].
Here we applied CRISPR/Cas9 technology for the first time to knockout PIWIL1 gene in a gastric cancer cell line and analyzed its phenotypic modifications.
The human gastric cancer cell line AGP01 was maintained in DMEM-F12 medium supplemented with 10% fetal bovine serum. The cell culture grew attached to a plastic flask in a monolayer in a humidified incubator maintained at 37 °C and 5% CO2.
The AGP01 cell line was established by our research group in 2009[20] from cancer cells present in the ascitic fluid of a female individual with intestinal gastric cancer, located at the antrum and the body region of the stomach, and staged as T3N2M1. The cell line was tested and authenticated by conventional cytogenetics[20]. Recently, the AGP01 cell line was tested by multicolor-fluorescence in situ hybridization (FISH), and results are presented here.
24-color-FISH using all human whole chromosome painting probes
24-color-FISH using simultaneous all human whole chromosome painting (WCP) probes was done as previously reported[21,22]. A total of 20 metaphases was analyzed, using a fluorescence microscope (Axio Imager Z1 mot; Carl Zeiss AG, Oberkochen, Germany) equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain DAPI; the latter was used to induce a GTG-like banding pattern. Image capturing and processing were carried out using ISIS imaging system (MetaSystems, Altlussheim, Germany).
Targeted knockout of PIWIL1 using the CRISPR-Cas9 system
The CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences (Lafayette, CO, United States). First, 1 × 104 AGP01 cells/well were seeded in DMEM-F12 medium to a 96-well plate for 24 h. Subsequently, transfection was performed using CR-0046-03-005 (Dharmacon GE Life Sciences) for 48 h. For the transfection procedure, a solution containing 1 μL of the CRISPR RNAs (crRNAs) mixed with the trans-activating small RNA, 2 μL of Cas9 and 7 μL of DMEM-F12 medium (for each well) was prepared first. In another tube, 0.4 μL of DharmafecDUO and 9.6 μL of DMEM-F12 medium (to each well) were added.
The solutions were then incubated for 5 min at room temperature before being combined. After combining, the solution was incubated for 20 min at room temperature, and finally 80 μL of DMEM-F12 medium/10% fetal calf serum (FCS) (per well) was added. At the end of the transfection, all contents (from each well) were transferred to a 24-well plate containing DMEM-F12 medium/10% FCS/1% pen-strep.
After 24 h, samples were treated with 6 μg/mL of puromycin for 72 h to select the resistant clones. Next, 40 cells were plated per well in a 6-well plate to isolate the clones by the filter paper method.
This method consists of using a cut and autoclaved piece of filter paper so that it, after being soaked in trypsin, can be positioned above a single colony of cells that has grown from an isolated cell, allowing for the collection of this clone. Subsequently, each clone grew in a separate well in a 6-well plate, so it could reach the confluence needed to perform DNA extraction and sequencing.
DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, United States) according to the manufacturer's instructions. For PCR, specific primers targeting the binding region of the purchased crRNA were constructed using the online program Primer3 (Supplementary Table 1).
The quantities of the reagents used in the PCR for a final volume of 12 μL were as follows: 6.25 μL of nuclease-free H2O, 0.5 μL of forward primer (10 ng/μL), 0.5 μL of reverse primer (ng/μL), 4.25 μL of GoTaq Colorless Master Mix 2× (Promega Corporation) and 1 μL of DNA (10 ng/μL).
The conditions using the MasterCycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) were 1 cycle at 95 °C for 3 min for initial denaturation followed by 35 cycles consisting of denaturation at 94 °C for 2 min, primer annealing at 59 °C for 1 min and extension at 70 °C for 2 min, ending with 1 cycle at 70 °C for a final extension for 30 min.
For direct sequencing of the PCR product, the quantities of the reagents used for a final volume of 20 μL were as follows: 15 μL of nuclease-free water, 0.5 μL of forward or reverse primer (10 ng/μL), 0.5 μL of Big Dye, 3 μL of Save Money and 1 μL of the PCR reaction. For this reaction, the ABI PRISM Big Dye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Hercules, CA, United States) was used.
The sequencing was performed using the MasterCycler Gradient (Eppendorf) thermal cycler according to the following 25-cycle thermocycling conditions: denaturation at 96 °C for 50 s, primer annealing at 59 °C for 1 min and extension at 60 °C for 4 min, ending with 1 cycle at 4 °C for 5 min.
After this procedure, a precipitation step was carried out in order to purify the product of the reaction before continuing. For this step, samples were washed with 70% isopropanol and 70% ethanol. Subsequently, sequencing was performed using the ABI Prism 3500 DNA Sequencer (Applied Biosystems). The methodology used was based on the biochemical synthesis of the DNA strand by the Sanger method.
Sequencing analysis
Reference sequence for exon 15 in the PIWIL1 gene was obtained from the National Center for Biotechnology Information (NCBI) and compared with the DNA sequence of the modified cell line as well as the negative control (cell line without the gene knockout). To infer the effect of changes in protein synthesis, we applied Gene Runner v.3.05 (Hastings Software Inc., Hastings, NY, United States; http://www.generunner.com ).
Wound-healing assay
Cells were grown in 12-well plates at a density of 2 × 105 cells/well and maintained for 24 h in 5% CO2 at 37 °C. After this period, cells were injured with a 10 μL tip in the center of each well. The medium was then removed to eliminate suspended cells, and wells were washed with 1 × phosphate buffered saline before fresh DMEM-F12 medium/10% FCS/1% pen-strep was added again.
The behavior of cells was observed and photographed immediately after injury and at 6 h, 12 h and 24 h after injury. All experiments were performed in triplicate.
Boyden chamber invasion assay
Boyden inserts (8 μm pores) (BD BiosciencesTM, Franklin Lakes, NJ, United States) were coated with 200 μL of Matrigel (10-13 mg/mL) in 12-well plates. Cells (2 × 105) were seeded in the upper chamber in 1 mL of DMEM without fetal bovine serum. In the lower chamber, DMEM-F12 medium/10% FCS/1% pen-strep was added, functioning as a chemoattractant for the cells present in the upper chamber.
After 48 h, the remaining cells above the filter were removed by scraping with a sterile swab. The cells at the bottom of the filter were fixed with 4% paraformaldehyde and stained with Giemsa. Cells were photographed and analyzed using a light microscope and counted in optical fields (100 ×). All experiments were performed in triplicate.
Total RNA extraction
The mRNA extraction was performed using Promega's Total RNA Isolation System kit, according to the manufacturer's specifications. AGP01 and AGP01 PIWIL1 knockout cells were prepared for mRNA extraction. Samples were lysed with lysis buffer containing beta-mercaptoethanol and then diluted in RNA dilution buffer. The samples were centrifuged for 10 min at maximum speed. Then, 95% ethanol was added to ensure adequate membrane binding conditions.
The samples were then transferred to centrifuge columns where the RNA could bind the membrane of the column, facilitating washing to eliminate possible contaminants as well as favoring the extraction of high quality of total RNA.
At the end of the procedure, the RNA was diluted in 60 μL of nuclease-free water. The total RNA was quantified using a Nanodrop spectrophotometer ND-1000 UV-VIS version 3.2.1 (Nanodrop Technologies, Wilmington, DE, United States). The RNA quality was also evaluated by analyzing the A260/A280 ratio according to the manufacturer's specifications. Purified RNA was stored at -80 °C for the microarray expression assay.
Microarray expression
For the microarray assay, a one-color microarray-based gene expression analysis kit (Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. The gene expression profile was evaluated in both cell lines (AGP01 with and without PIWIL1 knockout).
The total RNA obtained during the extraction phase was used as the template for the synthesis of the first cDNA strand by reverse transcription using T7 RNA polymerase. Synthesis of the second cDNA strand was used as the template for the in vitro transcription reaction for cRNA production. The cRNA was then incorporated into the fluorochrome 3-cyanine (Cy-3) using the Low Input Quick Amp Labeling kit (Agilent Technologies) according to the protocol provided by the manufacturer. Thereafter, the cRNA purification process was carried out.
The cRNA was quantified by a spectrophotometer (pmol/L), by which it was possible to analyze the absorbance ratio (260 nm/280 nm) and the cRNA (ng/μL) concentration in each sample. After, hybridization was performed for 17 h in a hybridization chamber at 65 °C at 10 rpm. After this period, the slide was washed and immediately scanned in the Agilent G4900DA SureScan Microarray System.
The following setup was used to scan the microarray slides for one color: scan region of 61 mm × 21.6 mm, 5 μm scan resolution, dye channel of green. Next, the images were obtained by using Feature Extraction v10.10 software, and the data were analyzed with GeneSpring GX 9.0 and IPathwayguide (Advaita Bioinformatics Company, Plymouth, MI, United States) programs. Gene identification followed a restriction criterion with a fold-change of > 2.
Differential expression and gene ontology enrichment analysis
To identify differentially expressed (DE) mRNAs, we compared the probes' expression profiles before and after PIWIL1 knockout. Probes with a mean fold-change < 0.5 and a mean fold-change of > 2 [|Log2(fold-change)| > 1] were selected for differential analysis. Student's t test was performed in 1222 selected probes, false discovery rate adjustment[23] was performed, and genes with an adjusted P-value of < 0.05 were tagged as DE. For the volcano plot, the fold-change and the P-value of all probes were used, and probes were tagged as DE following the previous criteria. Gene ontology enrichment was performed using org.Hs.eg.db[24] and Gostats[25] R libraries. All graphical and statistical analyses were performed in the R platform (R Core Team, 2017, Vienna, Austria; https://http://www.r-project.org/ ).
24-color-FISH using all human WCP probes
24-color-FISH using all human WCP probes revealed in AGP01 cell line a complex karyotype as follows: 63,XX,inv(1)(p12q43),der(1)(1pter->1p12::1q43->1p12::9p12->9pter),+der(1)t(1;12)(q21;q12),+del(1)(p12),+del(2)(p12),+der(2)t(2;8)(p12;q11.2),inv(3)(p21q13)x2,+inv(3)(p21q13),+der(3)t(3;5)(p14;q13),t(4;14)(p12;q11.2),dic(4;12)(p15;q12),+der(4)t(2;4)(p or q?;q12),del(5)(p13)x2,+der(5)x2,del(6)(q12)x2,+del(6)(p21),inv(7)(p12;q11.2),der(7),der(9)t(9;acro)(p21;p12),der(9)t(6;9)(p12;p12),t(13;13)(p10),der(15)(:q13->p11::p11->qter),-15,der(16)t(X;16)(q or p?;q23),+der(16)t(12;16)(q12;q23),21p+,t(22;22)(p10) (Figure 1).
Figure 1 AGP01 cell line multicolor-FISH showing several chromosomal changes, including the monosomy of chromosome 12, where the PIWIL1 gene is located.
From the analysis, we verified that the unique chromosome 12 (where PIWIL1 gene is located) remains intact, without translocations or derivative chromosomes. It is important to note that the monosomy of chromosome 12 agrees with the sequencing result, since the 7 bp insert sequence was observed in hemizygous status.
Targeted knockout of the PIWIL1 gene using the CRISPR-Cas9 system
PIWIL1 gene knockout was successful, as determined by Sanger sequencing. The latter revealed an insertion of 7 adenines in the PIWIL1 gene sequence (Figure 2), which caused a frameshift mutation that impaired protein synthesis.
Figure 2 Electropherograms showing the insertion of seven adenines in the PIWIL1 gene sequence after using the CRISPR-Cas9 system (B) in comparison with the reference sequence of the negative control (A).
Prediction of the encoded protein indicated a premature stop codon (Figure 3), suggesting that this insertion generates a truncated protein consisting of 573 amino acids (the wild-type contains 861) with a loss-of-function phenotype, which means that knockout was efficient.
Figure 3 Amino acid sequences of the wild-type PIWIL1 protein and the PIWIL1 protein after the insertion of seven adenines by the CRISPR-Cas9 system.
Notably, the PIWIL1 knockout cell line remained viable and could be used for further experiments.
The PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity after 24 h (P < 0.01; Figure 4), which is consistent with the fact that this protein is related to various pathways that regulate cell motility.
Figure 4 Analysis of the migration capacity of the AGP01 cell line with and without PIWIL1 gene knockout. T0: Immediately after injury; T6: 6 h after injury; T12: 12 h after injury; T24: 24 h after injury. NC: Negative control. aP < 0.01. Two-way ANOVA, Bonferroni post-test. Photomicrography of AGP01 cell migration. A: Immediately after injury; B: 6 h after injury; C: 12 h after injury; D: 24 h after injury. The black lines represent approximation of the edges over time, demonstrating the migration capacity of the cells.
Also, PIWIL1 gene knockout caused a significant decrease in AGP01 invasiveness (P < 0.001; Figure 5), which is also consistent with the fact that this protein is related to various pathways that regulate cell motility.
Figure 5 Analysis of the invasion capacity of the AGP01 cell line with and without the PIWIL1 gene knockout. Statistically significant difference between groups was shown by the Student's t test (cP < 0.001). Photomicrography of the cell invasion assay demonstrating the decrease in the number of cells that invaded when PIWIL1 was knocked out. NC: Negative control.
mRNA array and gene ontology enrichment
Differential analysis: By comparing expression profiles after PIWIL1 permanent knockout in the AGP01 cell line, a total of 251 mRNA were found to be DE [adjusted P-value of < 0.05 and |Log2(Fold-Change)| > 1]. A total of 43 mRNA probes were up-regulated and 208 were down-regulated (Figure 6). The DE genes are described in Supplementary Table 2.
Figure 6 Volcano plot comparing gene expression after PIWIL1 permanent knockout in the gastric cancer AGP01 cell line. Differentially expressed probes [adjusted P-value of < 0.05 and |Log2(Fold-Change)| > 1] are on superior left and right areas (red). mRNAs involved in invasion and migration processes are in green.
We performed a functional analysis by grouping all DE mRNAs. This approach revealed that a total of 35 genes (five up-regulated and 30 down-regulated) encoded proteins involved in invasion and migration cellular processes (Supplementary Figure 1; Supplementary Tables 3 and 4).
According to the current literature, 9 of these 35 genes (DOCK2, ZNF503, PDE4D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed (being up- or down-regulated after knockout).
Evidence indicating that PIWIL1 is an oncogene that regulates several cellular mechanisms important for the carcinogenic process of many types of cancers is rising in recent years. PIWIL1 seems to be especially implicated in proliferating activity of cancer cells[8,10,26,27].
Here, we introduce a new gastric cell line, i.e., AGP01, and provide a first description of its karyotype. As is usual for cancer cell lines, there were slight changes from cell to cell concerning number of chromosomes or single cell chromosomal rearrangements[28]. The here-given karyotype was the most frequently observed one. Interestingly, inversions, dicentrics and reciprocal translocations of homologous acrocentric (#13, #15, #22) was observed. Overall, gain of the following regions was present: #1, #3, large parts of #2 and #5, 8q11.2 to 8qter, 12pter to 12q12. Besides, the following regions were under-represented: 2pter to 2p12, 9pter to 9p12, 15q12 to 15qter. These imbalances are in concordance with the literature[29].
In AGP01, we performed for the first time an in vitro knockout experiment of PIWIL1 gene using the CRISPR-Cas9 system. It could be shown that absence of this gene significantly impairs migration and invasion capacity of AGP01 cells. Thus, the AGP01 cell line behaves like that previously reported for gastric cancer cells[12] or lung adenocarcinoma[26]. Together, these studies suggest that PIWIL1 expression is strongly associated with an increased aggressiveness of cancer cells.
According to Wang et al[30], one of the mechanisms by which PIWIL1 regulates the migration and invasion of cancer cells is by promoting the expression of MMP2 and MMP9, two important metalloproteinases involved in the degradation of the extracellular matrix, thereby creating paths for the locomotion of cancer cells[31].
Additionally, Amaar et al[32] demonstrated that the over-expression of PIWIL1 down-regulates the tumor suppressor gene IGFBP5, a member of the insulin-like growth factor binding protein family and whose expression is implicated in suppressing epithelial-mesenchymal transition and reducing the expression of E-cadherin and HIF1α, indicating that is it critically related to cancer progression[33,34].
Data obtained from our gene expression experiments also provided corroborating evidence that the PIWIL1 gene plays a key role in cancer cell migration and invasion because several genes involved in these cellular processes were observed as DE when the cell lines were compared before and after PIWIL1 knockout.
Many studies have demonstrated the oncogenic activities of the DOCK2, ZNF503, PDE4D, ABL1, ABL2, LPAR1, SMAD2 and WASF3 genes and their relation to tumor aggressiveness in several types of cancer, including gastric cancer[35-58]. Interestingly, PIWIL1 knockout led to a decreased expression of these genes as well as an increased expression of the tumor suppressor gene DACH1, demonstrating that PIWIL1 plays a crucial role in the pathway of development and progression of gastric cancer, and is likely a promising candidate for therapeutic intervention.
Zhu et al[53] reported that the over-expression of DACH1 impaired the proliferation and invasion ability of lung adenocarcinoma cells in vitro via the down-regulation of PRX3, an oncoprotein required for the maintenance of mitochondrial function and tumorigenesis[59]. DACH1 expression also inhibited epithelial-mesenchymal transition and metastasis by affecting TGF-β signaling and decreased proliferation of cancer cells by inducing cell cycle arrest at the G2/M phase[60,61].
Regarding the reported oncogenes, Rahrmann et al[37] observed that PDE4D is over-expressed in human prostate cancer and demonstrated that the knockdown of this gene reduced the growth and migration of prostate cancer cells in vitro as well as the growth and proliferation rate of prostate cancer xenografts in vivo[38]. Delyon et al[39] demonstrated that PDE4D is also over-expressed in melanoma cell lines and pinpointed this gene as a regulator of cell invasion by interacting with FAK through RACK1, constituting a signaling pathway that when activated promotes tumor progression and metastasis[43].
Recent studies have determined the role of ABL members from the tyrosine kinase family, ABL1 and ABL2, in the development of many types of solid tumors. These proteins induce the activation of actin polymerization machinery by modulating the expression of several MMPs to promote morphological changes, including the formation of membrane protrusions and altered cell adhesion. Consequently, activation of ABL1 and ABL2 in cancer cells promote enhanced proliferation, migration and invasion, as well as drug resistance[39-42].
DOCK2 (dedicator of cytokinesis) belongs to the DOCK family of proteins and is expressed in hematopoietic cells[44]. According to Kulkarni et al[45], DOCK2 has been reported to activate Rac, which is known to regulate several crucial processes, including lymphocyte migration, activation and differentiation of T cells[46]. Wang et al[47] knocked out DOCK2 in a B-cell lymphoma cell line and observed a decrease in Rac1 expression. Additionally, analysis of the growth curves of both cell lines demonstrated that the DOCK2 knockout grew less than DOCK2, as evidenced by the lower cell proliferation.
ZNF503 is expressed in the mammary gland and other tissues, and there is a high incidence of association between this gene deregulation and tumor aggressiveness in several kinds of tissues, such as lung, kidney and intestine[49]. Shahi et al[48] performed scratch and 3D Matrigel culture assays in two mammary epithelial cell lines to analyze the cell motility and migration. Both assays demonstrated that cell lines with ZNF503 knockout migrated less, did not close the gaps, and inhibited invasiveness when compared to the control cells. These data indicate that ZNF503 promotes cellular invasion and migration, and high levels of this gene are closely related with poor patient survival, breast cancer progression and increased metastasis.
Yu et al[50] demonstrated that the lysophosphatidic acid receptor 1 (LPAR1) gene is related to migration and invasion in ascites from ovarian cancer and is expressed at higher levels in metastatic cell lines, when compared to non-metastatic cell lines. They also observed that the presence of high levels of lysophosphatidic acid are directly connected to cell migration stimulation, and LPAR1 silencing reduced lysophosphatidic acid-induced invasion. Additionally, in breast tumors, a higher expression of LPAR1 is related to a worse lung metastasis-free survival rate[51].
Wiskott-Aldrich syndrome protein family 3 (WASF3) is an important gene, which has C-terminal domains that are responsible for actin polymerization activation, playing a role in cell proliferation and migration. The WASF3 gene is normally over-expressed in several types of tumors, such as breast cancer, osteosarcoma and prostate cancer[52,53]. In gastric cancer, little is known about this gene; however, since micro (mi)RNAs and their targets are considered potential biomarkers for gastric cancer, Wang et al[54] performed a luciferase assay and western blotting to investigate the relationship between miR-218 and WASF3. Their results demonstrated that over-expression of WASF3 harms miR-218 and results in the inhibition of cell proliferation and migration, suggesting that WASF3 is over-expressed in gastric cancer and induces cell proliferation and migration. Additionally, in qRT-PCR, WASF3 mRNA expression levels were higher when compared to normal gastric cell lines.
Smad2 is the first intracellular protein in the signaling cascade of the TGF-β1 signaling pathway, which is involved in the progression of gastric cancer. In advanced stages of cancer, TGF-β1 acts as an oncogene, regulating multiple cellular functions, including stimulation of proliferation, differentiation and the inhibition of apoptosis[55,57].
Interestingly, Lv et al[56] observed that TGF-β1 levels in peritoneal lavage fluid are directly connected to peritoneal metastasis. Corroborating evidence was provided by Shinto et al[58], whose experiments demonstrated that p-Smad2 expression was higher in diffuse-type tumors and in peritoneal metastasis cases. Notably, the AGP01 cell line used to perform the PIWIL1 knockout in our study was obtained from a patient with peritoneal metastasis, and we found SMAD2 was over-expressed.
Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer, mainly in patients with peritoneal carcinomatosis, which is a condition associated with poor prognosis and a decreased overall survival.
Gastric cancer (GC) remains a major public health problem, having the third highest incidence of death worldwide. Piwi like RNA-mediated gene silencing 1 (PIWIL1) is involved in regulation of widespread biological processes, including stem cell proliferation, embryogenesis, growth, and development, and has been found to be frequently over-expressed in various tumor types, including GC. Previous studies have demonstrated that PIWIL1 is implicated in improving tumor malignant behavior. PIWIL1 expression has been shown to be absent in normal somatic tissues, making it a very intriguing target for therapy. We attempted to investigate the role of PIWIL1 on the migration and invasion capacity of metastatic GC cells, using the AGP01 cell line, as well as checking the expression status of genes and proteins involved in these cellular processes, in order to elucidate the mechanisms by which PIWIL1 provokes tumorigenic effects and to shed light on potential new strategies to target PIWIL1
Research motivation
Many aspects of gastric carcinogenesis remain elusive, and much effort has been made to improve patient prognosis. The PIWIL1 has been identified as a novel extremely highly expressed gene in many types of cancer and its expression in GC tissue is related to poorer overall survival, suggesting that high expression of PIWIL1 is associated with poor prognosis and that it could be used as a predictive marker or even a target for therapy. Although PIWIL1 has been correlated with worse outcome, the involved mechanisms remain unclear, and many hypotheses are being tested. Once the upstream and downstream signaling pathways of PIWIL1 are elucidated, it will be possible to create new therapeutic strategies for gastric carcinogenesis, in order to improve the overall health of patients affected by this disease.
We performed permanent knockout of the PIWL1 gene to verify phenotypic modifications in the AGP01 metastatic GC cell line, as well as alterations in expression level of mRNA and protein, in an attempt to better understand the mechanisms by which PIWIL1 promotes tumor malignant behavior. This research demonstrates the importance of studying PIWIL1 in GC, since data obtained through the achievement of our objectives showed that this protein has a crucial role in gastric carcinogenesis, promoting molecular and phenotypic alterations compatible with enhanced tumor aggressiveness. The elucidation of the role of PIWIL1 protein in cancer cell invasion and migration will pave the way for developing potential clinical interventions, aiming to control GC dissemination.
We applied CRISPR/Cas9 technology to knockout the PIWIL1 gene in a metastatic GC cell line, and analyzed its phenotypic modifications, as well as alterations in gene and protein expression. CRISPR-Cas9 technology was considered in 2015 as one of the most important technological advances of science. Mainly, it allows permanent silencing of the target gene and also creates a stable and permanent cell line with the desired modification. By this way, multiple experiments can be carried on, including long term evaluation of the downstream events caused by the molecular alteration, as well as discovering potential pathways influenced by the studied gene. Therefore, after permanent knockout of PIWIL1 in the AGP01 cell line, we analyzed phenotypic modifications by performing wound-healing and Boyden chamber invasion assays, to assess migration and invasion, respectively. Moreover, aiming to shed light on the molecular mechanisms used by PIWIL1 to make changes in the migration and invasion capability of cells, we carried out proteomic and microarray assays, using multidimensional protein identification technology (commonly known as MudPIT) and a one-color microarray-based gene expression analysis kit, respectively.
PIWIL1 gene knockout was successfully performed and confirmed by Sanger sequencing, which revealed an insertion of seven adenines in the PIWIL1 gene sequence. In silico prediction of the encoded protein pointed to the appearance of a premature termination codon, suggesting that this insertion generates a truncated protein with a loss-of-function phenotype. PIWIL1 knockout promoted a significant decrease in cell migration and invasion capacity (P < 0.01 and P < 0.001, respectively), which is consistent with data present in the literature demonstrating that this protein is implicated in several signaling pathways that regulate cell motility. By comparing expression profiles after PIWIL1 knockout, a total of 251 mRNA were found to be differentially expressed, with 43 up-regulated and 208 down-regulated mRNA. A functional analysis grouping all differentially expressed mRNAs demonstrated that 35 genes encoded proteins were involved in invasion and migration cellular processes. After extensive review of data presented in the literature, we selected 9 of these 35 genes (DOCK2, ZNF503, PDE4D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) as possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed (being up- or down-regulated after knockout). Additionally, the analysis of proteomic data revealed that PIWIL1 knockout caused modification in the expression of 27 proteins involved in epithelial-mesenchymal transition (EMT). Twenty-two oncoproteins related to EMT promotion, including FGFR1, PCNA, ACTN4, GSN and TUBB3, were expressed in the AGP01 cell line before knockout and reduced to a level that were not detectable by the technique after knockout. On the other hand, PIWIL1 knockout caused an increase in the expression of six proteins implicated in EMT suppression, such as ACSM3, ADGRG1 and ANPEP, that were absent in AGP01 before knockout. To the best of our knowledge, this is the first report describing molecular alteration compatible with phenotypic alterations after permanent knockout of PIWL1 in GC. Detailed mechanisms leading to PIWIL1 over-expression in cancer as well as the pathways by which this protein improves the malignant phenotype should be further investigated.
Research conclusions
In the current study, we pioneered the performance of an in vitro knockout of the PIWIL1 gene by using the CRISPR-Cas9 system, and found that absence of this gene significantly impaired the migration and invasion capacity of the AGP01 cell line, besides modifying mRNA and protein expression of potential molecular targets involved in the EMT process. The results of such experiments contributed to understanding of the mechanisms used by PIWIL1 to promote alteration in migration and invasion capacity of gastric cells during tumorigenesis, and also revealed the participation of new players related to PIWIL1 expression, such as FGFR1, PCNA, ACTN4, PDE4D and SMAD2. Our results demonstrated that knockout of PIWIL1 promotes several changes in cell phenotype, suggesting the critical role of the PIWIL1 oncogene in GC, and confirmed the hypothesis that PIWIL1 expression provokes migration, invasiveness and EMT as potential mechanisms of improved tumor aggressiveness. The presented findings open new perspectives for molecular interventions in GC
Research perspectives
Definite silencing of PIWIL1 by the CRISPR-Cas9 system resulted in robust findings favoring the discovery of new mechanisms involved in gastric carcinogenesis. The presented results must be validated by other researchers, and if confirmed, might lead to innovative interventions aiming to treat GC.
Manuscript source: Unsolicited manuscript
Specialty type: Gastroenterology and hepatology
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P- Reviewer: Chen Z, Kimura A, Matowicka-Karna J, Park WS S- Editor: Wang XJ L- Editor: Filipodia E- Editor: Huang Y
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Q: Cucumber error when compiling HTML reports I'm using Cucumber for the Java implementation and after running my test suite on Jenkins I'm getting a Java heap space error. I've tried to raise the memory for the process but still get the error. I've also tried to raise the memory for the surefire and failsafe maven plugins but still doesn't work as the problem is not taking place during test/integration phases.
It happens when compiling and generating the JSON reports but also sometimes when running the tests. Any clues about it?
The error trace is:
[CucumberReportPublisher] Compiling Cucumber Html Reports ...
[CucumberReportPublisher] copying all json files from slave: /apps/hudson/workspace/cbs-billing-web-nightly1/trunk/cbs-billing-web-qa-test/target/cucumberReport to master reports directory: /apps/hudson/jobs/cbs-billing-web-nightly1/builds/2014-05-15_02-01-24/cucumber-html-reports
[CucumberReportPublisher] Found the following number of json files: 1
[CucumberReportPublisher] 0. Found a json file: cucumber.json
[CucumberReportPublisher] Generating HTML reports
FATAL: Java heap space
java.lang.OutOfMemoryError: Java heap space
at java.util.Arrays.copyOf(Arrays.java:2882)
at java.lang.AbstractStringBuilder.expandCapacity(AbstractStringBuilder.java:100)
at java.lang.AbstractStringBuilder.append(AbstractStringBuilder.java:390)
at java.lang.StringBuffer.append(StringBuffer.java:224)
at java.lang.StringBuffer.append(StringBuffer.java:284)
at java.util.regex.Matcher.appendReplacement(Matcher.java:746)
at java.util.regex.Matcher.replaceAll(Matcher.java:813)
at java.lang.String.replaceAll(String.java:2189)
at net.masterthought.cucumber.util.Util.U2U(Util.java:107)
at net.masterthought.cucumber.ReportParser.parseJsonResults(ReportParser.java:30)
at net.masterthought.cucumber.ReportParser.(ReportParser.java:18)
at net.masterthought.cucumber.ReportBuilder.(ReportBuilder.java:65)
at net.masterthought.jenkins.CucumberReportPublisher.perform(CucumberReportPublisher.java:97)
at hudson.tasks.BuildStepMonitor$1.perform(BuildStepMonitor.java:20)
at hudson.model.AbstractBuild$AbstractBuildExecution.perform(AbstractBuild.java:804)
at hudson.model.AbstractBuild$AbstractBuildExecution.performAllBuildSteps(AbstractBuild.java:776)
at hudson.maven.MavenModuleSetBuild$MavenModuleSetBuildExecution.post2(MavenModuleSetBuild.java:1030)
at hudson.model.AbstractBuild$AbstractBuildExecution.post(AbstractBuild.java:725)
at hudson.model.Run.execute(Run.java:1695)
at hudson.maven.MavenModuleSetBuild.run(MavenModuleSetBuild.java:519)
at hudson.model.ResourceController.execute(ResourceController.java:88)
at hudson.model.Executor.run(Executor.java:231)

A: I have usually seen this happening when you embed your own text in cucumber HTML reports using the Scenario object. I used to embed whole JSON objects in my cucumber reports using scenario.write(myObject.toString()), this caused the Cucumber Jenkins plugin to crash with Java heap space error that you mention. After I limited such writes to the report, everything started working fine.

redpajama_set_name: RedPajamaStackExchange


News in Europe: Camel Wrestling?
Yes. There will be something about camel wrestling later, but let's have some news first.
The fallout from Charlie Hebdo continues: Two terrorists were killed by police in Belgium, the weekly Pegida rally in Dresden had to be cancelled (muhaha), and security measures have been upped all over the place.
Speaking of Pegida: Amidst reports of racially motivated crimes, an Eritrean refugee was killed in Dresden, and some Pegida supporters' comments are enough to make you sick. For shame!
In other news, tensions are flaring up in Ukraine again, with several deaths and new attacks on Donetsk airport. The Russian threat is making a lot of people uncomfortable, so much so that Lithuania is now issuing survival leaflets.
The Greek election is looming amidst discussions about the possibility of forgiving the country's debts.
Polish prosecutors have requested Roman Polanski's extradition. About time.
In not exactly good, but Schadenfreude news, it has been revealed that the number of British benefit claimants in some European countries is even higher than their counterparts in Britain. Take that, Ukip! (But, fear not, those data can be interpreted to show the exact opposite.) Here's an interesting article about Poland's only British benefits claimant. What a brave man, trying to get by in a country with such a completely impossible language… But, really, it just goes to show that life is hard.
Are we uncomfortable hearing foreign languages spoken in the street? I'm not, but it looks like some people are. A lovely article there by AL Kennedy.
In exciting news, this: It turns out that modern technology has made it possible to partially read burnt documents buried during the eruption of Mount Vesuvius that destroyed Herculaneum. This was almost 2000 years ago — which makes the whole thing pretty awesome.
Also, this guy is ace. Poles for the win!
And here we have it: A wrestling camel. It's a thing. Are we intrigued? I can't even imagine how this would work, but I'm more than a bit too scared to google it.
The article on hearing foreign languages was really interesting. It's amazing how we perceive and react to different languages.
The Vesuvius news is epic! Seriously, love it!


11.2 : ORA-12518 Listener Hangs and Reports "Too Many Open Files"
These WARNINGS appear every minute or so, showing that the listener cannot connect to ONS server.
The number of sockets opened by listener process is increasing in time.
Listener is leaking sockets and these file descriptors will increase in number exhausting the server resource.
No changes to the system.


Welcome to Creekside where you'll find Bosa quality at its finest in the heart of the False Creek community. Ticking all the boxes, this spacious 1 Bed + Den/1 Bath offers an open concept layout including a large, multipurpose Den space has been beautifully redesigned into a custom walk-in dressing room that offers amazing storage/closet organization but could easily be converted back to an office or nursery space if desired. Other interior highlights include SS kitchen appliances, granite counters, U shaped kitchen w/ breakfast bar + ample storage, fresh paint and much more including 1 pkg + 1 locker. Exceptional value in this rapidly evolving area where you will find yourself steps to the best Vancouver has to offer including trendy restaurants, breweries, Seawall and Skytrain.


text:
NATO Secretary General spoke with U.S. President-elect Biden
NATO Secretary General Jens Stoltenberg spoke with Joe Biden on Monday (23 November 2020) to congratulate him on his election as the next President of the United States and Kamala Harris as the next Vice-President. Mr. Stoltenberg thanked President-elect Biden for being a long-standing supporter of NATO and the transatlantic relationship.
They discussed the importance of our transatlantic Alliance as the cornerstone of our collective security. The Secretary General told President-elect Biden that he looked forward to working very closely with him to further strengthen the bond between North America and Europe and to prepare next year's meeting of NATO leaders.
NATO Secretary General visits the United States 04 Apr. 2016 NATO Secretary General Jens Stoltenberg and US Vice President Joe Biden
NATO Secretary General attends Munich Security Conference 07 Feb. 2015 Bilateral meeting between NATO Secretary General Jens Stoltenberg and US Vice President Joe Biden
NATO Secretary General congratulates US President-elect Joe Biden and Vice President-elect Kamala Harris 07 Nov. 2020


Weyfringe is part of Cleveland Technologies Group.
One of three divisions, Weyfringe is joined by printed circuit board manufacturer Cleveland Circuits and interconnection and electronics contract manufacturer Connect-2 Technology who make up the manufacturing companies within our organisation. If your manufacturing process uses printed circuit boards or wires, cables, looms and harnesses then perhaps we can further assist you in addition to providing your barcode and labelling requirements.


For Osteoarthritis: I don't like using medicines at all, especially long-term, but Celebrex did tame my symptoms enough for me to be able to pursue other therapies. It wasn't perfect. I still limped and had some pain and stiffness issues but it made a world of difference. I am now off of it, and have taken a big hit in my flexibility and mobility.
For Osteoarthritis: For pain and swelling of my joints it worked the best, but my blood pressure went up and heart disease runs in my family. I wish I could have continued taking it.
Common side effects include: diarrhea, hypertension, and abnormal hepatic function tests.
See the full Celebrex side effects document.


text:
The Rugby Championship 2015 war die vierte Ausgabe des jährlich stattfindenden Rugby-Union-Turniers The Rugby Championship, Nachfolger des seit 1996 bestehenden Wettbewerbs Tri Nations. Im Unterschied zu früheren Saisons wurde der Wettbewerb dieses Jahr nur an drei Wochenenden zwischen dem 17. Juli und dem 8. August 2015 ausgetragen. Jede der vier Nationalmannschaften spielte nur einmal gegen die jeweils anderen drei Teams. Durch diese Verkürzung erhielten die Mannschaften mehr Vorbereitungszeit für die Weltmeisterschaft in England.

Zum ersten Mal gewann Australien das Turnier; den Vorgängerwettbewerb mit eingerechnet, war es der erste Turniersieg seit 2011. Südafrika platzierte sich zum ersten Mal am Tabellenende. Dennoch verteidigte Neuseeland den Bledisloe Cup und den Freedom Cup, während sich Australien die Mandela Challenge Plate und die Puma Trophy sicherte.

Tabelle

Spiele und Ergebnisse

Erste Runde

Zweite Runde

Dritte Runde

Statistik

Meiste erzielte Versuche

Meiste erzielte Punkte

Weblinks
 The Rugby Championship 2015 auf ESPN (englisch)
 Turnier-Website

Rugby 2015
2015

redpajama_set_name: RedPajamaWikipedia


text:
Q: MySQL: How to select rows from a Child tablename composed of preset prefix and parent table field value without running multiple queries Assume I have three tables like so (the latter two are similar for the sake of clarity, but in reality, are not similar at all):
events
-------------------------------
| id | short | event          |
-------------------------------
|  1 | moo   | Cow Tipping    |
-------------------------------
|  2 | baa   | Sheep Shearing |
-------------------------------

registrants__moo
-----------------------
| id | name           |
-----------------------
|  1 | Dave           |
-----------------------
|  2 | John           |
-----------------------

registrants__baa
-----------------------
| id | name           |
-----------------------
|  1 | Carol          |
-----------------------

How would I execute the following query, such that the ??? in the subselect would be replaced w/ short such as 'moo' or 'baa' depending.
SELECT
    E.*
    (SELECT count(*) FROM `registrants_???`) AS `registrants`
FROM
    `events`

With the expected result of the final query being:
---------------------------------------------
| id | short | event          | registrants |
---------------------------------------------
|  1 | moo   | Cow Tipping    | 2           |
---------------------------------------------
|  2 | baa   | Sheep Shearing | 1           |
---------------------------------------------

I've tried variations on CONCAT [Like: SELECT count(*) FROM CONCAT(registrants__, short)) AS registrants) ] , but haven't had any luck. I'm assuming I'm missing something really simple and just can't see the solution. =\

A: If you can only use a single query and changing the schema is out, your only option is something like this:
SELECT
  e.id,
  e.short,
  e.event,
  COUNT(e.id) AS registrants
FROM
  events AS e
  LEFT JOIN registrants__moo AS moo
    ON e.short = "moo"
  LEFT JOIN registrants__baa AS baa
    ON e.short = "baa"
GROUP BY e.id;

If you have a ton of registrants__ tables you're in for a very long query, but there's nothing you can do about it.  At least this way is shorter than using a bunch of UNIONs.


This chapter turns to the right to carry arms. The matter of whether foreign forces can carry weapons in the execution of their duties touches upon the core of State sovereignty. Governments have the monopoly of the legitimate use of physical force within their territory. On the other hand, the right to carry arms is essential to armed forces in order to successfully execute their mission. While the Receiving State can be deemed to have consented to such right when agreeing to a military force on its territory, matters such as the scope of the right to carry arms remain subject to agreement between the States. Force security aspects deserve some discussion as to the responsibilities of the Receiving State and Sending States. Also the legal limitations on the use of force require specific attention, as well as the rules of the Receiving State regarding the importation of arms.


text:
Features & Commentary
Italian Police confiscated an arsenal of weapons from right-wing extremists that included an air-to-air missile made in France and originally owned by the Qatari military. (PHOTO: YouTube/Vista Agenzia Televisiva Nazionale)
Italian Police raid ultra-right extremists, confiscate air-to-air missile from Qatar
20.7.2019 10:26 full story
videoItalian PM wants to deport Roma, MEP Romeo Franz calls his behavior racism and the shame of Europe
Pope stands up for Romani people attacked by (neo)Fascists in Rome who threatened rape
13.5.2019 8:53 full story
Italian theater cancels neo-Fascist concert co-opting Czechoslovak historical figure, so it moves from Verona to Lazis
Czech Embassy in Italy trying to prevent neo-Fascist co-optation of Czechoslovak historical figure Jan Palach there
Czech students protest co-optation of Czechoslovak historical figure in Italy
8.1.2019 11:25 full story
Italian neo-Nazis want to co-opt the legacy of Czechoslovak who set himself on fire to protest the '68 invasion
Italian Deputy Prime Minister says EU Justice Commissioner's critique of Italy's treatment of Roma is "nonsense"
31.10.2018 11:44 full story
Italian Deputy PM Salvini is behaving like a neo-Fascist, former Italian Labor Minister says
28.10.2018 13:43 Former Italian Labor Minister Elsa Forner, speaking in a recent radio interview, said the current Italian Deputy PM and Interior Minister Matteo Salvini is behaving like a neo-Fascist. Salvini, who heads the anti-immigration party Lega ("The League"), and wasted no time in responding bluntly. full story
Italy: Moroccan beaten to death, athlete of Nigerian origin attacked as fascists in the Govt spread racism
1.8.2018 11:29 Italian Interior Minister Matteo Salvini, the boss of the anti-immigrant Lega party, is facing criticism from opposition politicians for creating a hateful atmosphere in the country. In recent days Italy has witnessed a series of attacks with apparently racist motivations including Monday's attack on Daisy Osakue, an Italian athlete of Nigerian origin. full story
videoLIVE BROADCAST TODAY AT 16:00 in Prague: RomanoNet protests discrimination and racism against Roma in Europe, publishes manifesto Dost! Basta! Enough!
31.7.2018 13:53 Today at 16:00 a march will take place in Prague, Czech Republic to support the Roma of Ukraine. The event is part of a larger series of protests taking place in several European countries full story
LIVE BROADCAST: RomanoNet delivers protest manifesto to the Italian Embassy about Italy's racist measures against Roma
30.7.2018 13:52 Representatives of RomanoNet will deliver a protest manifesto to the Ambassador of Italy to the Czech Republic, Aldo Amati, very shortly. The organizations are protesting against the situation of Romani in Italy, where the Italian Interior Minister and leader of the ultra-right Lega group, Matteo Salvini, is demanding a census of Romani people and the application of racist policies against them.
Romania: 900 protest recent violence against Roma in Italy and Ukraine, more demonstrations in other European countries to follow
29.7.2018 13:34 As many as 900 people assembled on Saturday, 28 July 2018 in Bucharest, Romani in front of the Italian and Ukrainian embassies to express their disagreement with many violent anti-Romani crimes and to protest antigypsyism in Europe. The assemblies were the first in a series of protests that are being realized in nine European countries. full story
Italy: Romani infant hospitalized after being shot with pellet gun
21.7.2018 8:55 On 19 July Italian media reported that a pellet fired by an air gun struck a Romani baby as she was being held in her mothers arms. The child is now in intensive care in a hospital in Rome. full story
Italy: Court in Rome convicts four perpetrators of 2011 pogrom on Romani camp in Turin
20.7.2018 9:45 The ANSA wire service reported last week that a court in Rome, Italy found four people guilty of ethnic and racial hatred when they set fire to a Romani camp in the Vallette suburb of Turin in 2011. The appeals court in Turnin had upheld the first-instance conviction but reduced the sentences to between two and four years in prison, according to court sources. full story
Italy asks Czech Republic to receive asylum-seekers, Czech PM refuses
16.7.2018 10:13 Responding to a question from the Czech News Agency (ČTK) about the current Italian request for aid with refugees arriving on Italian territory, Czech Prime Minister Andrej Babiš (ANO) said the Czech Republic will not be accepting any migrants and that he is insisting the reception of migrants be governed by the principle of voluntary action by the EU Member States. Italian PM Giuseppe Conte has asked the EU Member States for aid with redistributing 450 such people who recently ended up lost at sea. full story
European Parliament: Shouting matches over Italian "census" of Roma - Járóka finds it reasonable, Post calls for a halt
8.7.2018 22:22 The European Parliament experienced a stormy debate last week over the most recent anti-Romani statements by Italian Interior Minister Matteo Salvini. The minister announced that he is planning a census of Romani people in the country and the deportation of all who do not have documents. full story
Italian Interior Minister threatens to cancel police protection for crimefighting author who criticizes his anti-Roma attitude
27.6.2018 9:07 Roberto Saviano, the Italian author and journalist whose work has been key to combating the mafia, is criticizing Interior Minister Matteo Salvini's harsh stance against refugees and Romani people. The author has faced death threats for years because of his book about the Camorra mafia in Italy - and last week the Interior Minister said he could lose his police protection. full story
Italian Interior Minister insists on "census" of Roma despite criticism
20.6.2018 12:13 Italian Interior Minister Matteo Salvini, despite harsh criticism of his announcement Monday that he is planning to conduct a census of Romani people and to deport those without documents, is insisting on his plans. Yesterday he tweeted his defiance of the criticism. full story
Italian Interior Minister plans to deport Romani people, opposition calls the plan repugnant
19.6.2018 17:39 Developing an "inventory" of Romani people and deporting those who do not have proper documentation is a plan being developed by Italian Interior Minister Matteo Salvini. Last week he also advocated for taking a hardline approach against boats in the Mediterranean Sea that rescued several hundred migrants. full story
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I look between the two men, absolutely bewildered, and really unsure what to say. "Well, ... I wasn't sent on some holy mission. I was busy, caught in a battle between two evil beings, and one of them found my presence to be a nuisance and while I was distracted, banished me from his world.
I fell for a long while, until this world ... reached out and grabbed me. Swallowed, really. I saw a great mouth open, and I fell in, and now I'm here. My arrival here is simply because I was unprepared for the power Lord Godfrey had to expel me before I destroyed him.
My memories are in a straight line. There are times I don't remember much, but that is the haze of memory, not confusion as to what was reality or dreams. I wonder, is it this place that has done this to you? Or the journey here?"


text:
Q: Extract text from URL using javascript and display on php file I have an error message on the URL when wrong password or captcha is added. Error looks like this on the url:
www.domain.com/signup.php?error_msg=The+characters+you+entered+did+not+match+the+word+verification
How to extract this from URL and display on the PHP file?

A: Just use the global $_GET array to retrieve the value:
echo $_GET['error_msg'];

More about this array can be found in the official documentation:
http://php.net/manual/en/reserved.variables.get.php


text:
Kəramət Hüseynov (* 30. August 1998 in Baku) ist ein aserbaidschanischer Judoka, der 2021 eine Bronzemedaille bei den Europa- und den Weltmeisterschaften gewann.

Sportliche Karriere
Hüseynov kämpft im Superleichtgewicht, der Gewichtsklasse bis 60 Kilogramm. 2016 belegte er den fünften Platz bei den Junioreneuropameisterschaften. Im Jahr darauf gewann er den Titel und wurde Zweiter der Juniorenweltmeisterschaften. Bei den U23-Europameisterschaften gewann Hüseynov 2017 eine Bronzemedaille. Bei den Weltmeisterschaften 2018 in seiner Heimatstadt Baku unterlag er im Viertelfinale dem Japaner Naohisa Takato. Nach einem Sieg in der Hoffnungsrunde verlor er den Kampf um Bronze gegen den Georgier Amiran Papinaschwili. 2019 fanden die Europameisterschaften im Rahmen der Europaspiele 2019 in Minsk statt. Hüseynov schied im Achtelfinale gegen den Niederländer Tornike Tsjkadoea aus.

2021 verlor Hüseynov im Viertelfinale der Europameisterschaften in Lissabon gegen den Spanier Francisco Garrigós. Mit Siegen über den Italiener Angelo Pantano und den Bulgaren Janislaw Gertschew gewann Hüseynov eine Bronzemedaille. Anderthalb Monate später erreichte er auch bei den Weltmeisterschaften in Budapest das Viertelfinale und verlor dann gegen den Franzosen Walide Khyar. Mit Siegen über den Mongolen Lkhagvajamts Unubold und den Kasachen Magzhan Shamshadin erkämpfte Hüseynov auch in Budapest eine Bronzemedaille. Weitere anderthalb Monate später fand in Tokio das olympische Judoturnier statt. Hüseynov besiegte zum Auftakt den Briten Ashley McKenzie und schied dann im Achtelfinale gegen den Georgier Luchum Tschchwimiani aus.

Weblinks
 Turnierergebnisse bei judoinside.com

Fußnoten

Judoka (Aserbaidschan)
Olympiateilnehmer (Aserbaidschan)
Teilnehmer der Olympischen Sommerspiele 2020
Teilnehmer an den Europaspielen (Aserbaidschan)
Teilnehmer der Europaspiele 2019
Aserbaidschaner
Geboren 1998
Mann


text:
Puya antioquensis är en gräsväxtart som beskrevs av Lyman Bradford Smith och Robert William Read. Puya antioquensis ingår i släktet Puya och familjen Bromeliaceae.

Artens utbredningsområde är Colombia. Inga underarter finns listade i Catalogue of Life.

Källor

Gräsordningen
antioquensis


Controversy with Dr. Morse (1814) This refers to a legal dispute she had with Morse in 1801.
↑ Adams, Hannah, and Hannah Farnham Sawyer Lee. A Memoir of Miss Hannah Adams. Boston: Gray and Bowen, 1832.
Gary D. Schmidt, A Passionate Usefulness: The Life and Literary Labors of Hannah Adams. — University of Virginia Press, 2004. — ISBN 0-8139-2272-0.


text:
Q: How to delete a specific exe file via batch which can be stored anywhere on the computer? So short from writing out every possible file path. Is there a way to delete a specific exe file that could be anywhere on the computer?
C:
del C:\Users\Student\Downloads\blah.exe
del C:\Users\Student\Documents\blah.exe
del C:\Users\Student\music\blah.exe

A: Open a command prompt window and execute there del /?
You get displayed the help for command del listing also parameter /S for deleting all files matching the file name or file name pattern in all subdirectories.
For example blah.exe is deleted in all subdirectories on drive C: with following command:
del /F /Q /S C:\blah.exe

Excepted are all directories with no permissions for deletion of a file for the current user executing this command.
Perhaps better for your example would be
del /F /Q /S "%USERPROFILE%\blah.exe"


The Money Tree Financial Freedom System full download (PDF files & Videos). Feel free to share this program with your friends on Facebook! Are you tired of living paycheck to paycheck? Sick of owing friends and family money? Do you feel like there's no way out? Rest assured, there is! If you've been searching for a solution that will bring you REAL financial freedom and flexibility, the wait is over. This is an interdisciplinary approach to money mastery that walks you through a proven, 4-Step plan of ACTION for financial success. This is a goal oriented blueprint that will give the secrets to becoming a millionaire, achieving financial independence, creating a literal money tree of diverse income streams, and the courage to carry the great responsibility of wealth. Yes, it will be amazing once you change your life, but then people will start to look up to you!


text:
Back to School News
Sending a Message to Bullies
This week is Anti-bullying Week, which places a really important focus on the prevalence of bullying and the damaging effects it has on children. At Southwold Primary & Nursery School, we dedicate a wealth of time and resources every year to preventing and eradicating bullying.
Anti-bullying Week is organised annually by the Anti-Bullying Alliance (ABA), a unique coalition of organisations and individuals united against bullying. Their vision is to "stop bullying and create safer environments in which children and young people can live, grow, play, and learn." This is a vision that is shared by our school and The White Horse Federation.
Currently, our school has a comprehensive framework in place to prevent, counteract, and punish bullying. Throughout this week, children across the country will be taking part in lessons and activities that raise awareness of bullying, discuss how to identify it, and advise how to deal with it.
Last week, our school hosted anti-bullying ambassador training, which was attended by children and their supervising adults from four other Oxfordshire schools. Those in attendance had been nominated and selected to take a pupil leadership role in promoting a culture of kindness throughout their settings, with an emphasis on filling the role of anti-bullying ambassador.
The training was led by Claire Pike and covered how bullying is characterised (STOP: several times on purpose), what the law says about bullying, and our right to be safe at school. The training also resulted in each school having a child-friendly policy and action plan.
At Southwold, our children planned a variety of activities taking place this week as part of their campaign against bullying. Our slogan for this week is 'Man in the Mirror', as we are focusing on this year's Anti-bullying Week theme 'Change Starts with Us'. The first activities began last week, as children made a 'positive reflections' display which featured a mirror at its centre. The display is placed in the school entrance hall with the intention of encouraging children and parents to say something positive to themselves to boost their self-esteem and then pass on compliments to others.
This week, our school's nominated anti-bullying ambassadors will be known as 'kindness catchers', and they will be on the look out for natural displays of kindness happening among children and staff at the school. When a display of kindness is spotted, the child involved will be awarded a kindness sticker and their act of kindness will be posted on a 'good deed news feed' board for others to read. We are also asking children to sign a pledge this week, showing their commitment to being an active bystander - or, more appropriately, an 'upstander' - in school and online to stamp out the 'no snitching' culture that often prevents people from speaking out against bullying.
Our other activities include kindness lucky dips, taking part in Odd Socks Day yesterday to celebrate diversity, and giving the children access to a suggestions jar, called 'the jar of hearts', so they can make suggestions on how to promote an atmosphere of kindness in our school.
Mrs Izzo, SEND learning mentor, said: "We are so proud of our young people taking this courageous stand against such an important issue. We feel it is incredibly important to set our focus on prevention rather than fix in regards to bullying. From the youngest to the eldest pupil, all are encouraged to show kindness and other value-based themes in all we do at school, at home, and in the wider community. We want to create an atmosphere of kindness, and we recognise that change begins with each and every one of us."
Although this week is an opportunity for us to focus and address the issue of bullying, we would like to reassure parents, pupils, and staff that every effort is being made throughout the year to make our school a safe, enjoyable, and bullying-free environment.
Go to ParentPay
Next in School News
We've Got a New Set of Wheels! Fundraising Fun in Festive Knits Stay and Play Sessions Starting in January 2020


What skills do teachers need to teach 21st century graduates? Join in a discussion with education leaders about changes to teacher evaluation and support as we look at teacher training and retention.
Carolina Classrooms: College and Career Readiness - Watch Now!
Carolina Classrooms: Making College Affordable - Available Online!
Carolina Classrooms: Technology in SC Schools - View Online!
Mark your calendar to tune in November 23 at 7:00 p.m. on ETV when the topic on Carolina Classrooms will be technology in our schools. Carolina Classrooms is ETV's monthly educational series for teachers, parents and all citizens invested in the education of our children.
Carolina Classrooms, ETV's monthly educational series, focused on education options for SC students in the October 26, 2016 program. View online at Carolina Careers!
Bullying and Suicide Prevention - View Online!
Carolina Classrooms: Education Options for SC Students Oct. 26 at 7 p.m.
Carolina Classrooms: Celebrating Teachers - View Online Now!


Diesel usage is an area where most Transport companies constantly strive to cut costs on. It is the biggest single cost to Haulage Firms, with an average of over 30% of total costs being fuel expenditure. Every Haulage firm targets this area in an effort to reduce costs, with the vast majority of firms daily exploring solutions to the ever-increasing price of diesel.
The Driver is the person who can most affect diesel usage on a day-to-day basis. Driving habits are imperative in the saving of fuel costs. Driving aggression, braking, gear changes, acceleration, idling and over-speeding are just a few of the things which impact fuel consumption in the truck. The driver is in complete control of these things, and if sufficiently motivated and trained, should be in a position to save fuel by improving their driving performance. Improved incentives and proper driver training are the two obvious methods that companies can implement in order to improve driver performance and subsequently reduce fuel consumption. Haulage Firms have monitoring systems in place in order to survey the drivers performance. This enables them to offer incentives to drivers in terms of their performance and driving standards, and keep an eye on their driving habits and fuel consumption. Accurate monitoring systems will lead to fair Driver evaluations, culminating in improvements in fuel consumption and increased driving performance.
Reducing the Speed Limiter Calibration on the trucks by just 5kmph can reduce fuel costs significantly. Although it will increase the target lead time on deliveries, if managed and planned properly this will not have a great impact. This will not only increase savings on diesel costs, but reduce potential mechanical issues with the vehicle, culminating in a safer truck with a higher performance.
Regular maintenance checks on trucks and trailer units will aid the reduction in fuel consumption. Regular checks of tyres, clutch, engine, spark plugs, air filter, and oil, and a regular monitoring of the vehicles condition and roadworthiness can go a long way to reducing fuel consumption. Trucks operating in their optimum condition will burn less fuel than trucks that aren't, and maintaining these vehicles and keeping them at a high performance level can ensure that diesel costs are reduced.
Another fuel reduction measure would be the Specification and type of the tractor units. The age of the truck is a big factor, with newer models using less fuel than older models, due to improved engines etc. The weight, condition, load, aerodynamics, intake, exhaust, rolling restrictions and mechanics of the vehicle are all factors that can aid in the reduction of fuel consumption.
Weather & Road Conditions, Traffic Congestion etc.
Weather has a major impact on fuel consumption, as it can lead to both poor road conditions and potential traffic congestion issues. These can all affect fuel consumption levels, and although they are mostly unavoidable problems, good planning can mean that trucks take a different route, with possible smoother surfaces and less traffic. This can also help reduce wear and tear. In hot conditions, air conditioning can cause fuel consumption to rise, however, there is a solution to this. Leaving the windows open and air conditioning off a lower speed, and then switching when at a higher speed can actually reduce diesel consumption levels.