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To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65°C). The detection limit fornirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r2 = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution ofnir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.
Microorganisms play an integral and unique role in ecosystem function and sustainability. Understanding the structure and composition of microbial communities and their responses and adaptations to environmental perturbations such as toxic contaminants, climate change, and agricultural and industrial practices is critical in maintaining or restoring desirable ecosystem functions (5, 10, 16). However, because less than 1% of microorganisms have been cultivated (1), characterization and detection of microbial populations in natural environments present a great challenge to microbial ecologists. Current methods for analyzing microbial communities, especially their key functions, are too cumbersome. Rapid, simple, reliable, quantitative, and cost-effective tools that can be operated in real-time and in heterogeneous field-scale environments are needed.
The DNA microarray (or microchip) technology is a powerful tool for studying gene expression and regulation on a genomic scale (7, 9, 13, 14, 24, 27, 32, 34) and detecting genetic polymorphisms (4, 12, 31) in both eukaryotes and prokaryotes. Compared to conventional membrane-based hybridization, glass slide-based microarrays offer the additional advantages of rapid detection, lower cost, automation, and low background levels (25). Although microarray-based genomic technology is potentially an extremely powerful tool for characterizing microbial communities and their biological functions (11), the concept and performance of microarray hybridization have not been tested for environmental applications.
In contrast to studies using pure cultures, microarray-based analysis of environmental nucleic acids presents a number of technical challenges. In environmental studies, the target and probe sequences can be very diverse (29), and it is not clear whether the performance of microarrays with diverse environmental samples is similar to that with pure culture samples and how sequence divergence affects microarray hybridization. Also, environmental samples generally contain humic acid and other organic materials, which may inhibit DNA hybridization on microarrays (19, 21). In contrast to pure cultures, the biomass that can be retrieved from environmental samples is generally low (18). Not surprisingly, information on the performance of microarrays with complex environmental samples is lacking. It is not clear whether microarray hybridization is sensitive enough for detecting microorganisms in environmental samples and whether microarray-based detection can be quantitative. Environmental studies require experimental tools that not only detect the presence or absence of particular groups of microorganisms but also provide quantitative data to help evaluate their biological activities.
To examine the potential of microarray-based genomic technology for environmental studies, a prototype microarray was constructed for understanding the specificity, sensitivity, and quantification of microarray hybridization within the context of complex environmental samples. The prototype microarrays contained approximately 100 functional genes encoding dissimilatory heme- and copper-containing nitrite reductases (NirS and NirK, respectively), ammonia mono-oxygenase (AmoA), and the evolutionarily related methane mono-oxygenase (PmoA). These genes code for key enzymes in the ecosystem processes of denitrification, nitrification, and methane oxidation, respectively. Glass slide microarrays containing DNA fragments from different functional genes for monitoring various environmental processes are referred to in this paper as functional gene arrays (FGAs). Our results evaluate the conditions and performance of glass slide-based microarrays as tools for assessing functional gene distribution in natural samples.
Bacterial strains, environmental clones, and samples.The majority of the denitrifying bacteria used in this study were isolated from continental margin sediments off the coast of the state of Washington (2). The other denitrifying and nitrifying bacteria were obtained from our culture collection at Michigan State University (8, 26). The amoA genes from pure cultures of four nitrifying bacteria were also cloned into plasmids as described previously (37). Plasmid clones containingnirS, nirK, amoA, and pmoA genes from marine sediments were kindly provided by Gesche Braker (3) and Stephen Nold (17). For simplicity, the probe group consisting of amoA and pmoA genes is collectively referred to as amoA unless otherwise noted.
To evaluate the performance of microarray hybridization, marine sediment samples from the Washington margin and soil samples from Michigan State University were used. Marine sediment samples (W303 and W307) (3) were provided by Allan Devol at The University of Washington. Most of the denitrifiers and environmental clones used in this study were isolated from these marine sediment samples (3). The surface soil samples O22, J19, H17, and M24 were from the Cannelton site (J.-Z. Zhou, B. C. Xia, D. S. Treves, T. L. Marsh, R. V. O'Neill, L.-Y. Wu, A. V. Palumbo, and J. M. Tiedje, submitted for publication), a wetland located on the shore of St. Mary's River in northern Michigan. These samples, which contain different levels of organic matter and chromium, were supplied by Terry Marsh at Michigan State University. Yeast genomic DNA was prepared from Saccharomyces cerevisiae ATCC 18824.
DNA purification, quantitation, and PCR amplification.Genomic DNA was extracted from isolates as previously described (35). Community DNA from marine sediments and soils was isolated according to the method described by Zhou et al. (36). DNA concentration was determined in the presence of ethidium bromide by fluorometric measurement of the excitation at 360 nm and emission at 595 nm using an HTS700 BioAssay Reader (Perkin-Elmer, Norwalk, Conn.).
To construct functional gene arrays, nirS, nirK, andamoA genes were amplified from the genomic DNA of pure cultures with primers described previously (3, 17). The desired inserts of the environmental clones were amplified with vector-specific primers (37). 16S rRNA genes were amplified from genomic DNA from pure cultures of denitrifying bacteria using PCR primers (TA-F and TA-R) described elsewhere (22). To determine the effects of probe size on microarray hybridization signal intensity, DNA fragments of varying sizes from different gene regions in Shewanella oneidensis MR-1 were selected. The primers for these DNA fragments were designed based on unpublished genome sequences provided as a courtesy from The Institute for Genomic Research (Rockville, Md.). The size and annotation of the amplified DNA products were 2.3 kb from a putative sigma 54 factor, 1.4 kb from putative RNA polymerase sigma factor N, 0.8 kb from a putative two-component sensor kinase, 0.65 kb from a putative alcohol dehydrogenase homolog, and 0.33 kb from putative heat shock protein A.
Hot-start PCR amplification (6) was carried out using 1 × Taq polymerase buffer (10 mM Tris-Cl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, and 0.1% Triton X-100), 200 μM deoxynucleoside triphosphates, 100 pmol of each primer, 1 ng of plasmid DNA or 100 ng of genomic DNA, and 0.5 U of Taq DNA polymerase in a total reaction volume of 100 μl. Samples were initially denatured at 94°C for 2 min, followed by 30 sequential cycles at 94°C (30 s), 60°C (1 min) for nir andamoA primer sets or 58°C (1 min) for 16S ribosomal DNA and yeast gene primers, 72°C (1 min), and a final extension at 72°C for 7 min. PCR products were analyzed for correct size and the presence of a single product by agarose gel electrophoresis and ethidium bromide staining. The PCR products were then purified using the QIAquick PCR Purification kit (Qiagen, Chatsworth, Calif.) according to the manufacturer's instructions.
Microarray construction and postprocessing.The prototype DNA microarrays for monitoring bacteria involved in nitrogen cycling contained the following PCR-amplified gene products (for details, see our website:http://www.esd.ornl.gov/facilities/genomics/index.html ): (i) 22nirS, 9 nirK, and 4 amoA genes from pure bacterial cultures; (ii) 27 nirS, 9 nirK, 11pmoA, and 7 amoA genes cloned from marine sediment samples; (iii) 16S rRNA genes from pure cultures of 10 denitrifying bacteria as positive controls; and (iv) five yeast genes encoding mating pheromone α-factors (mfα1, mfα2), mating-type α-factor pheromone receptor (ste3), actin (act1), and GTP-binding protein involved in the regulation of the cyclic AMP pathway (ras1) as negative controls. A group of seven nirS genes from pure cultures were represented on the microarray as 1.4-kb fragments. All other nirS genes were 0.76 kb in size. To avoid confusion, the DNA deposited on the array is referred to as the probe, whereas the fluorescently labeled DNA is designated as the target. The size, G+C content, and GenBank accession number for each gene probe are listed in a table on our website.
Purified PCR products were diluted to a final concentration of 200 ng μl−1 in 50% dimethyl sulfoxide (DMSO; Sigma, St. Louis, Mo.). Ten microliters of each sample was then transferred to a 384-well microplate for printing. DNA samples were arrayed with a single pin (ChipMaker 3; TeleChem International, Sunnyvale, Calif.) at a spacing distance of 250 μm on silane-coated 25- by 75-mm glass slides using a PixSys 5500 robotic printer (Cartesian Technologies, Inc., Irvine, Calif.) under conditions of 62% relative humidity. Based on performance in preliminary studies and cost, the silane-modified slides from Cel Associates (Houston, Tex.) were selected for microarray fabrication in this study. All of the 104 probes were arranged as a matrix of 16 rows × 7 columns. The exact location of each gene in the matrix is listed in the table on our website. Each glass slide contained three replicates of the gene probe array. For optimizing hybridization conditions, smaller arrays consisting of nirSgenes were also constructed.
DNA microarrays were rehydrated over a 60°C water bath for 20 s and dried on a heating block at 80°C for 5 s. The DNA on the microarrays was fixed by UV cross-linking at 65 mJ in a UV Stratalinker 1800 (Stratagene, La Jolla, Calif.). The glass slides were then treated with 0.17 M succinic anhydride (Sigma) dissolved in 240 ml of 1-methyl-2-pyrrolidinone (EM Science, Gibbstown, N.J.) and 10.7 ml of 1 M boric acid (pH 8.0) (J. T. Baker, Phillipsburg, N.J.). Immediately following blocking, the DNA was denatured by immersing the slides in deionized water (dH2O) at 95°C for 2 min. The microarrays were then rinsed briefly in 95% ethanol, air dried at room temperature, and stored dry in the dark at room temperature.
To evaluate the quality of printing and the retention of arrayed DNA elements, a single slide from the same printed set of slides was stained for 30 min in a solution of PicoGreen (Molecular Probes, Eugene, Oreg.), diluted 1:200 in 1× Tris-EDTA (TE) buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA). The slides were then washed sequentially in 1× TE, 0.5× TE, and sterile dH2O for 1 min each prior to being scanned with the ScanArray 5000 Microarray Analysis System (GSI Lumonics, Watertown, Mass.).
Preparation of fluorescently labeled DNA.Two methods were employed to fluorescently label DNA. In a direct labeling procedure, 1 pg to 2.5 μg of genomic DNA was denatured by boiling for 2 min and immediately chilled on ice for labeling. Each 40-μl labeling reaction mixture contained denatured genomic DNA; 1.5 μg of random hexamers (Gibco BRL, Gaithersburg, Md.); 1× EcoPol buffer (New England Biolabs, Beverly, Mass.); 50 μM dATP, dTTP, and dGTP; 20 μM dCTP; 10 μM Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.); 2.5 mM dithiothreitol; and 10 U of the large Klenow fragment of DNA polymerase I (New England Biolabs). The reaction mixture was incubated at 37°C for 2 h, heat treated in a 100°C heating block for 3 min, and chilled on ice. Labeled target DNA was purified with a QIAquick PCR purification column according to the manufacturer's instructions, concentrated in a Savant SC110 Speedvac (Savant Instruments, Inc., Holbrook, N.Y.) at 40°C for 1.5 h, and resuspended in 10 μl of dH2O for hybridization, except for sensitivity experiments, in which the labeled target DNA was resuspended in 2 μl of dH2O.
For labeling by PCR amplification, each reaction mixture contained 1×Taq polymerase buffer; 20 ng of genomic DNA template or 10 pg of plasmid containing the desired target gene; 100 pmol each ofnirS, nirK, amoA, 16S rDNA, or vector-specific primers; 200 μM dATP, dTTP, and dGTP; 80 μM dCTP; 50 μM Cy3- or Cy5-dCTP (Amersham Pharmacia Biotech); and 0.5 U of Taq DNA polymerase in a 100-μl reaction volume. PCRs were programmed for 30 sequential cycles, and the labeled PCR products were purified as described above.
Microarray hybridization.Microarray experiments for testing specificity were carried out in duplicate (a total of six replicates per gene probe), while all other microarray hybridizations were performed in triplicate (a total of nine replicates per gene probe). Hybridization solutions contained 3× SSC (1× SSC contained 150 mM NaCl and 15 mM trisodium citrate), 1 μg of unlabeled herring sperm DNA (Promega, Madison, Wis.), and 0.3% sodium dodecyl sulfate in a total standard volume of 15 μl. A reduced hybridization solution volume of 2 μl was used for testing detection sensitivity and monitoring target genes in environmental samples. In this case, the hybridization solution was deposited directly onto the immobilized DNA prior to placing a coverslip (6.25 by 8 mm) over the array. Fluorescently labeled DNA was denatured in the hybridization solution at 100°C for 2 min, cooled to ambient temperature, and deposited onto glass coverslips. The microarray (array side down) was placed on the coverslip and then into a waterproof slide chamber (TeleChem International). Fifteen microliters of 3× SSC was dispensed into the hydration wells on either side of the microarray slide, and hybridization was carried out for 12 to 15 h at 45 or 65°C. For experiments determining the effect of temperature on signal intensity, hybridization was carried out at 40, 45, 50, 55, 60, 65, or 75°C. Following hybridization, the arrays were washed with 1× SSC, 0.2% SDS and 0.1× SSC, and 0.2% SDS for 5 min each at ambient temperature and then with 0.1× SSC for 30 s (ambient temperature) prior to being air dried in the dark. For experiments testing the effect of washing conditions on microarray hybridization signals, slides were washed in four separate wash treatments: (i) 1× SSC + 0.2% SDS (twice, 5 min) and dH2O (30 s); (ii) 1× SSC + 0.2% SDS (once, 5 min), 0.5× SSC + 0.2% SDS (once, 5 min), and dH2O (30 s); (iii) 1× SSC + 0.2% SDS (once, 5 min), 0.1× SSC + 0.2% SDS (once, 5 min), and dH2O (30 s); and (iv) 1× SSC + 0.2% SDS (once, 5 min), 0.01× SSC + 0.2% SDS (once, 5 min), and dH2O (30 s).
Array scanning and quantitative analysis of hybridization signals.Microarrays were scanned initially at a resolution of 50 μm to obtain a quick display image and then at 5 μm with the scanning laser confocal fluorescence microscope of the ScanArray 5000 System. The emitted fluorescent signal was detected by a photomultiplier tube (PMT) at 570 nm (Cy3) or 670 nm (Cy5). For sensitivity experiments and analysis of environmental samples, the laser power and PMT gain were both 100%. For all other microarray experiments, the laser power was 95% and the PMT gain was 90%.
The scanned image displays were saved as 16-bit TIFF files and analyzed by quantifying the pixel density (intensity) of each hybridization spot using the software of ImaGene version 3.0 (Biodiscovery, Inc., Los Angeles, Calif.). A grid of individual circles defining the location of each DNA spot on the array was superimposed on the image to designate each fluorescent spot to be quantified. Mean signal intensity was determined for each spot. The data sheet from ImaGene was then exported to Excel for further processing. The local background signal was subtracted automatically from the hybridization signal of each separate spot. Fluorescence intensity values for the five yeast genes (negative controls) were averaged and then subtracted from the final quantitation values for each hybridization signal. Statistical analysis was performed using SigmaPlot 5.0 (Jandel Scientific, San Rafael, Calif.).
Optimization of parameters for microarray fabrication.The following buffers for DNA deposition were also evaluated: 3× SSC, 1.5× SSC + 50% DMSO, 1× TE, 0.5× TE + 50% DMSO, 10 to 50% DMSO (at increments of 10%), and dH2O. DMSO at a concentration of 50% was the most appropriate buffer for microarray fabrication in terms of hybridization intensity, spot homogeneity, and evaporation loss during printing (data not shown). Arrayed DNA that was rehydrated at 60°C for 20 s, in contrast to 0 and 10 s, gave the highest and most reproducible signal intensities following microarray hybridization (data not shown). When 50% DMSO and a 20-s rehydration time were used, differences in the denaturation time (ranging from 2 to 4 min at 1-min increments) for arrayed genes did not have a significant effect on the fluorescence intensity (data not shown). Hence, a denaturation time of 2 min was selected as part of the standard protocol.
The effect of DNA probe concentration on hybridization intensity was also examined. PCR-generated nirS DNA was prepared for array deposition in 50% DMSO at various concentrations and rehydrated for 20 s at 60°C prior to hybridization. No substantial difference in hybridization signal intensities for nirS was observed when the DNA concentration was greater than 100 ng μl−1(data not shown). A DNA probe concentration of 200 ng μl−1 was therefore used for microarray construction in later experiments.
Specificity of DNA microarray hybridization.To determine the specificity of microarray hybridization, functional gene arrays consisting of nitrite reductase genes (nirS andnirK), ammonia mono-oxygenase genes (amoA), and methane mono-oxygenase genes (pmoA) were used. PicoGreen staining indicated that the shapes (diameters) of the DNA spots on the glass slides were uniform (data not shown).
Microarray hybridization was performed in duplicate at 45°C (low stringency) and at 65°C (high stringency) for 12 h with genomic DNA from pure cultures, and the arrays were washed and scanned as described above (see Materials and Methods). At low stringency, all of the labeled target DNAs hybridized to their cognate genes from both pure cultures and environmental samples (data not shown). By contrast, probes hybridized strongly to their complementary sequences from pure cultures at 65°C, with no or only weak cross-hybridization with genes cloned from environmental samples (Fig. 1A to D). At high stringency, Cy3-labelednirS from the γ-proteobacterium Pseudomonas stutzeri E4-2 hybridized strongly with nirS genes from other P. stutzeri strains that were 92 to 100% identical (Fig. 1A, 5c, 6c, 10c, and 10d). All of the other nirS gene sequences that were less than 75% identical (Fig. 1A, 1a to 15a, and1b to 4c) did not hybridize, except for three nirS genes (7c to 9c), which showed weak hybridization. The nirK genes showing more than 88% identity hybridized strongly with the labelednirK gene C3-2 (Fig. 1B, 11d to 16d), while thenirK genes displaying less than 79% identity showed no visible hybridization with the labeled target DNA (Fig. 1B, 16a and 7b to 14b). The Cy5-labeled amoA target gene fromNitrosospira sp. NP39-19 showed hybridization to the sequences from pure cultures of Nitrosolobus andNitrosospira species with 87 to 100% sequence identity (Fig. 1C, 6f to 9f) but not to the genes from environmental clones with sequence identities of less than 80% (Fig. 1C, 4e to 5f). The 16S genes from different bacteria hybridized very well with the labeled target 16S gene (Fig. 1D, 10f to 3g). Cross-hybridization among different gene groups was not observed at either temperature, regardless of whether the probe was prepared by random primer labeling or PCR labeling with gene-specific primers (Fig. 1). In addition, no hybridization was observed with any of the five yeast genes on the array. These results indicate that different levels of hybridization specificity can be achieved using glass slide microarrays.
Detection sensitivity of FGA hybridization.The detection sensitivity of hybridization with the gene array (104 genes) was determined using genomic DNA from both pure cultures and soil community samples (Fig. 2). Genomic DNA (at concentrations of 0.5, 1, and 5 ng) from a nirS-containing denitrifier, P. stutzeri E4-2, was randomly labeled with Cy5 as described. At high stringency, strong hybridization signals were observed with 5 ng of DNA for both nirS and 16S rRNA genes, whereas hybridization signals were weaker but detectable with 1 ng of DNA (Fig. 2A). The hybridization signal at low DNA concentrations was stronger for 16S rRNA genes than for nirS genes. Hybridization signals were measurable with 0.5 ng of genomic DNA, but the fluorescence intensity was poor (Fig. 2A). Therefore, the detection limit with randomly labeled pure genomic DNA under these hybridization conditions was estimated to be approximately 1 ng.
The detection sensitivity of microarray hybridization was also evaluated with community genomic DNA isolated from soil sample O22, which contained a large amount of organic matter. Community DNA was randomly labeled with Cy5 as described. All of the arrayed genes, with the exception of the five yeast genes, showed hybridization with 50 and 25 ng of labeled community DNA (Fig. 2B). Only the 16S rRNA genes could be detected when as little as 10 ng of soil community DNA was used in the hybridization reaction (Fig. 2B). Thus, with this microarray hybridization system, the detection sensitivities of nirSand 16S genes in this soil sample were considered to be approximately 25 and 10 ng of total environmental DNA, respectively.
Microarray hybridization-based quantitation.To evaluate whether microarray hybridization can be used as a quantitative tool, the relationship between target DNA concentration and hybridization signal was examined. Genomic DNA from a pure culture of thenirS-containing denitrifier P. stutzeri E4-2 was fluorescently labeled with Cy5 as described and hybridized in triplicate with the microarray at total concentrations ranging from 0.5 to 100 ng. The fluorescence intensities obtained at each DNA concentration for nine data points (three independent microarrays with three replicates on each slide) were averaged, and the log of the concentration was compared to the corresponding log value of the mean fluorescence intensity (Fig. 3A). Within a DNA range of 0.5 to 100 ng, a linear relationship (r2 = 0.89) was observed between signal intensity and target DNA concentration; however, this relationship was stronger (r2 = 0.96) within a range of 1 to 100 ng, suggesting that microarray hybridization is quantitative for pure bacterial cultures within a limited range of DNA concentration.
Since environmental samples contain a mixture of target and nontarget templates, the presence of other nontarget templates could affect microarray-based quantification. To determine whether microarray hybridization is quantitative for targeted templates within the context of environmental samples, 11 nirS, nirK, andamoA genes (plasmid DNA) were mixed together in a series of twofold differences in concentrations ranging from 1 pg to 1 ng. The pairwise similarities of these genes are less than 80%. The mixed plasmid DNA was randomly labeled with Cy5 in triplicate and hybridized with the microarrays. Similarly, a linear relationship (r2 = 0.94) was observed between signal intensity and target DNA concentration (Fig. 3B), suggesting that microarray hybridization is also quantitative for mixed DNA templates.
The accuracy of quantitative measurements obtained from hybridization-based methods relies on hybridization signal intensity, which in turn depends on the fragment size and G+C content of the probes on the glass slides. The effects of size and G+C content of arrayed DNA elements on hybridization were therefore investigated. DNA fragments of different sizes, ranging from 0.33 to 2.3 kb, were amplified by PCR from different open reading frames distributed throughout the sequenced S. oneidensis MR-1 genome. The G+C content of the DNA fragments varied from 41.8 to 49.6%. Within this narrow percentage range, no substantial difference in hybridization intensity was observed due to G+C bias; however, a pronounced impact on signal intensity may be evident across a broader range of percentage of G+C values. The signal intensity increased linearly as the size of the probe increased up to about 1 kb (Fig. 3C), whereas no substantial difference in signal intensity was observed when the probe size was greater than 1.4 kb. This result suggests that the effect of probe size on signal intensity is negligible when the probe is larger than about 1.5 kb.
Effect of sequence divergence and hybridization stringency on signal intensity.The target functional genes in environmental samples may be highly divergent. Therefore, it is important to understand how sequence divergence affects microarray hybridization. Since, unlike amoA and pmoA genes, the sequence identity of the partial nirS and nirK genes on the arrays is not well distributed within the sequence range of 60 to 100%, only amoA genes were selected for examining the effect of sequence divergence and hybridization stringency on signal intensity. The relationship of sequence homology among amoAgenes to hybridization signal intensity at low stringency (45°C) was investigated. A nonlinear S-shaped curve (r2 = 0.83) between the hybridization intensity and gene identity for the amoA genes was observed (Fig.4A). At 45°C, little hybridization was observed for probes showing 60 to 70% sequence identity to the labeled target DNA, whereas the hybridization signals increased exponentially within the range of 70 to 75% identity. No substantial difference in fluorescence intensities was detected foramoA and pmoA probe genes exhibiting 80 to 100% identity to the labeled target DNA (Fig. 4A). Microarray hybridization was also carried out at 40, 50, 55, 60, 65, 70, or 75°C to test the effect of temperature on signal intensity. Nonlinear S-shaped curves ( r2 = 0.84 to 0.96) showing saturation at sequence identities of about 80% were observed when hybridization temperatures were below 65°C (data not shown), whereas nonlinear hyperbolic curves with no saturation ( r2 = 0.83 to 0.84) were obtained at hybridization temperatures of 70 and 75°C (data not shown). This suggests that the effect of sequence divergence on signal intensity may be negligible at certain temperatures for genes displaying sequence identities above a certain limit.
In order to understand the effect of hybridization temperature on signal intensity, eight amoA genes exhibiting a wide range of sequence identities to the labeled target gene were examined. TheamoA gene on plasmid NP39-19 was labeled with Cy5 with a vector-specific primer. The relationship of signal intensity to temperature was fitted with nonlinear statistical models, and the calculated hybridization intensities based on the fitted nonlinear models were then plotted against hybridization temperatures (Fig. 4B). All of the experimental data fit the nonlinear models, as evident from the determination coefficient values ( r2 = 0.78 to 0.96) (Fig. 4B). As expected, signal intensity decreased as the hybridization temperature increased and gene sequence identity decreased (Fig. 4B). Temperature melting profiles were found to be sequence-dependent. For example, at temperatures below 50°C, signal intensity decreased very slowly for genes with sequence identities ≥62%, and genes with >80% identity showed only a small decrease in signal intensity at temperatures below 60 or 70°C (Fig. 4B). The signal intensity for most of the genes decreased dramatically when the hybridization temperature was above 60°C. In addition, at temperatures below 50 or 60°C, no obvious differences in signal intensity were observed among genes with identities greater than 80%. When the hybridization temperature was above 70°C, little or no hybridization was evident even for genes showing about 90% identity to the labeled target DNA. These results suggest that hybridization specificity can be achieved by adjusting the hybridization temperature.
The effect of washing conditions on microarray hybridization at 45°C was also examined by varying the concentration of SSC from 0.01× to 1×. The hybridization data under different washing conditions were fitted with nonlinear models ( r2 = 0.84 to 0.96), and the calculated hybridization intensity was plotted against salt concentration (Fig. 4C). As expected, signal intensity generally increased as the salt concentration increased, and the effect of washing stringency on signal intensity was found to be sequence dependent. Significant differences were observed under different salt concentrations for gene sequences that were about 70% identical to the labeled target DNA, whereas little or no difference was obtained for genes with identities greater than 80%.
Microarray-based detection of target genes in marine sediment and soil samples.To evaluate the potential applicability of DNA microarrays for microbial community analysis, community DNA from two marine sediment samples and four surface soil samples was directly labeled with Cy5 using the random primer labeling method and hybridized with the FGAs in triplicate. Strong hybridization signals above the background to the yeast genes were obtained with both marine sediment and soil samples (only the results for the marine sediment sample W303 and the soil sample O22 are shown in Fig.5). No hybridization with the five yeast control genes was observed. The average variation in signal intensity for all of the gene replicates in the samples was 12.8% with a standard deviation of 7.7%. The signal variation was lower for the soil sample (10.7% ± 6.9%) than for the marine sediment sample (14.9% ± 8.0%).
As shown in Fig. 5, microarray analysis of the composition of marine sediment sample W303 indicated that the most abundant genes appeared to be those genes showing sequence similarity to nirS from cultured denitrifiers (bars 28 to 42). P. stutzeri (bar 30) and A. tolulyticus (bar 40) nirS genes showed the highest hybridization signal intensity with labeled DNA from the marine sediment sample. Genes similar to nirS from environmental clone pB20 (bar 4) displayed high abundance relative to the other environmental clones (Fig. 5). Similar to what is observed in the distribution of nirS, gene sequences hybridizing to thenirK genes of cultured denitrifiers (Pseudomonassp. G179 [bar 15], Bacillus azotoformans [bar 16], andCorynebacterium nephridii [bars 17 and 18]) were generally more abundant than genes hybridizing to the nirK genes from environmental clones. By contrast, genes similar to amoAand pmoA genes appeared to be evenly distributed among environmental clones and cultured nitrifiers.
Although most of the functional gene probes were derived from marine sediment environments, these probes also hybridized well with community DNA from soil samples, suggesting that these types of nitrifier and denitrifier genes are present in both environments. The distribution patterns for nirS- and nirK-like genes in the surface soil sample O22 generally resembled those patterns obtained for the marine sediment sample (Fig. 5). Genes showing the highest abundance based on hybridization signal intensity more closely resembled nirS and nirK genes from cultured denitrifiers than the environmental clones. For example,nirS genes similar to those from P. stutzeri (bar 30) and A. tolulyticus (bar 40) appear to be abundant in both the marine sediment and surface soil samples as indicated by hybridization with nirS probes (Fig. 5). However, genes similar to amoA and pmoA sequences from both environmental clones and cultured nitrifiers were less evenly distributed in the surface soil sample.
Specificity is one of the most critical parameters for any technique used to detect and monitor microorganisms in natural environments. The specificity of probe-target association depends on the degree of sequence divergence, which can be very high among target genes in natural microbial communities. However, different levels of specificity can be achieved by adjusting microarray hybridization conditions, depending on the experimental objectives. While microarray hybridization under conditions of low stringency allows the detection of microbial populations with a broad range of sequence divergence, high-stringency conditions permit the detection of more specific microbial populations. For example, at low stringency (45°C), all of the amoA and pmoA genes on the array hybridized with Cy5-labeled amoA target DNA fromNitrosospira sp. NP39-19 (Fig. 4A), and no cross-hybridization was observed for nontarget genes. By contrast, at high stringency (65°C) only the sequences from pure cultures ofNitrosolobus and Nitrosospira species with 87 to 100% identity showed strong hybridization, while genes from environmental clones with sequence identities of less than 80% did not show any detectable hybridization (Fig. 1C). At extremely high stringency (75°C), only the sequence displaying 100% identity showed strong hybridization, while the gene from the environmental clone with 90% identity showed very little hybridization (Fig. 4B). Since there were no amoA gene sequences with identities between the ranges of 80 to 90% and 90 to 100%, it is uncertain what minimum sequence divergence is required for differentiation at high or extremely high stringency. We estimate that our microarray hybridization conditions can differentiate sequences exhibiting a dissimilarity of approximately 15 to 20% at 65°C and 10% at 75°C. The level of specificity could be further improved by introducing formamide into the hybridization buffer.
At high stringency (65°C), all of the sequences with >80 to 85% identity hybridized well with their labeled cognate target gene when a single target template was present (Fig. 1). However, in the case of mixed target templates, it is expected that the labeled target templates will preferentially hybridize with their perfect-match probes rather than with their mismatch probes, because the rate of hybridization will occur more rapidly and the probe-target association will be more stable between the perfectly matched sequences than between the mismatched sequences. As a result, the mismatch sequences will have less effect on the hybridization signal intensity of the target genes when the perfect-match sequences are present. To illustrate this, the nirS gene from P. stutzeriE4-2 was labeled with Cy3 in triplicate using PCR amplification andnirS-specific primers. The Cy3-labeled E4-2 DNA was divided into two aliquots, and one aliquot (∼100 ng) was mixed with an equal amount of PCR-amplified D8-12 products. The nirS gene sequences of these species are 92% identical. The labeled target DNA was hybridized with the microarrays at 65°C. Equivalent hybridization signals were obtained for E4-2 in both treatments (Fig.6). However, the hybridization signal for D8-12 decreased by ∼75% when the perfect-match sequences from D8-12 were present (Fig. 6). These results support the expectation that mismatch sequences will have less effect on the hybridization signal intensity of target genes when perfect-match sequences are present.
The amoA and pmoA sequences having less than 80% identity showed some hybridization even at 65°C (Fig. 4B). This weak hybridization could be due to the amoA sequences that are less than 80% similar or to the primer sequences used for cloning and PCR amplification. Since all of the amoA and pmoAgene fragments on the arrays were first cloned with theamoA-specific primers, followed by amplification from plasmid clones with vector-specific primers, they have 80 bp of identical sequence at each end. Weak signals were also observed in some other experiments for the nirS gene fragments cloned from environmental samples but not for the nirS gene fragments from pure cultures when the target genes were labeled with vector-specific primers (data not shown). The arrayed nirSfragments cloned from environmental samples have ∼40 bp of identical sequence at each end, while those amplified from pure genomic DNA had 18 to 22 bp of identical sequence at each end. The presence of these vector-specific sequences in the nirS gene fragments could contribute to the weak hybridization signals observed in the microarray analysis. In addition, a good linear relationship was obtained for the mixed nine sequences that are all less than 80% similar (Fig. 3B). This also implies that the microarray hybridization is not influenced by sequences with less than 80% identity. Otherwise, no linear relationship between signal intensity and DNA concentration could be obtained.
To investigate the potential contribution of vector-specific sequences to hybridization intensity, the hybridization signals observed for the other target and nontarget probes in the above experiments were also analyzed (Fig. 6). Since genomic DNAs were used as templates, the vector primer sequence portion of the arrayed probes should not affect microarray hybridization. No substantial differences in hybridization signal intensity were observed among nirS genes with less than 80% sequence identity, or between the nontarget nirSgenes and nirK, amoA, 16S, or yeast genes (Fig. 6). The signal intensities of the nontarget nirS genes and blank controls were extremely low and comparable, suggesting that no hybridization occurred among the nirS sequences that were less than 80% identical. These results implied that the weak hybridization signals observed in Fig. 4B for the amoA genes of less than 80% sequence identity are most likely due to the continuous stretch of primer sequences used for cloning and amplification.
The potential applicability and usefulness of DNA microarray hybridization to environmental samples will depend not only on the arrayed probe number, specificity, and representation, but also on the sequence diversity of the target genes in natural environments. Analysis based on published sequences and our own unpublished sequences indicates that the genes involved in many important biogeochemical processes such as nitrogen fixation (e.g., nifH), denitrification (e.g., nirS, nirK), sulfite reduction (e.g., dsrA/B) are diverse in natural environments. Many cognate sequences are less than 80 to 85% similar and hence should be resolvable. Collaborative efforts are needed to recover more specific, representative probe sequences from a variety of environments to build a more generally useful array.
If it is useful to differentiate sequences with more than 80 to 85% identity, oligonucleotide microarrays containing short oligonucleotide sequences could be used. Oligonucleotide-based microarrays have the advantage of minimizing the potentially confounding effects of cross-hybridization (33) and are uniquely suited for detecting genetic mutations and polymorphisms. Previous studies have indicated that single-nucleotide differences can be distinguished by oligonucleotide microarray hybridization (reviewed in reference23). However, since only a very small portion of a gene is used for detection in oligonucleotide microarray hybridization, the signal intensity is generally low and thus detection sensitivity will be lower. In order to fully characterize microbial communities, both types of microarrays may be needed.
Sensitivity is another critical parameter for environmental application and may be the most difficult challenge. Using our optimized hybridization protocol with the reduced hybridization solution volume (2 μl), nirS genes were detected in only 1 ng of labeled pure genomic DNA and 25 ng of bulk community DNA from the surface soil samples (Fig. 2). We found that the detection sensitivity was approximately 500-fold lower when 15 μl of hybridization solution was used than when the reduced volume was used (data not shown). Also, we found that fresh reagents were very important in achieving highly sensitive detection and the detection sensitivity often varied with the batch of fluorescent dyes used. The level of detection sensitivity obtained with the reduced volume should be sufficient for many studies in microbial ecology. However, the sensitivity obtained with the glass-based microarray hybridization may still be 1,000 to 10,000-fold less than with PCR amplification (37), and probably 1,000-fold less than the one obtained with membrane-based hybridization (30). One of the main reasons that the sensitivity of glass-based microarray hybridization is lower than that of the membrane-based hybridization is probably that the probe-binding capacity on glass surfaces is much lower than that on porous membranes. We are currently exploring ways to enhance the level of detection sensitivity.
The quantitative aspects of microarray hybridization have not been well established. The accuracy of microarray-based quantitative assessments is uncertain due to inherent variations associated with array fabrication, labeling, target concentration, and scanning. Comparison of microarray hybridization results with previously known results suggested that microarray hybridization appears to be quantitative enough for detecting differences in gene expression patterns (7, 15, 28). DNA microarrays were also used to measure differences in DNA copy number in breast tumors (20). Single-copy deletions or additions can be detected, suggesting that microarray-based detection can be potentially quantitative. In this study, a linear relationship was observed between hybridization signal intensity and target DNA concentration for a pure culture and a population of mixed DNAs (Fig. 3A and B), suggesting that DNA microarrays may potentially be used for quantitative analysis of environmental samples. With our optimized protocol, experimental variation between array slides was reduced to below 15% (data not shown). This is consistent with the findings of microarray studies on gene expression (2).
The challenge in quantifying the abundance of microbial populations in natural environments based on hybridization signal intensity is how to distinguish differences in hybridization intensity due to population abundance from those due to sequence divergence. One possible solution is to carry out microarray hybridization under conditions of various levels of stringency. Based on the relationships among signal intensity, sequence divergence, hybridization temperature, and washing conditions determined in this study, it should be possible to distinguish, to some extent, the contributions of population abundance and sequence divergence to hybridization intensity. For instance, our results showed that at about 55 to 60°C, sequence divergence had little or no effect on signal intensity for amoA genes with greater than 80% identity to the labeled target DNA (Fig. 4B). This suggests that under such hybridization conditions the effect of sequence divergence on signal intensity is negligible for genes with >80% sequence identity; therefore, any significant differences in signal intensity are most likely due to differences in population abundance. Another possible solution to this problem is to use microarrays containing probes extremely specific to the target populations, such as those used in oligonucleotide microarrays.
This work evaluates specificity, sensitivity, sequence divergence and quantitation of DNA microarrays for environmental application. While this tool holds much potential for environmental studies, more development is needed, especially for improved sensitivity, quantitation, and the biological meaning of a detectable specificity before it can be used broadly and interpreted meaningfully.
This research was supported by the United States Department of Energy under the Natural and Accelerated Bioremediation Research, the Biotechnology Investigations—Ocean Margins, and Microbial Genome Programs of the Office of Biological and Environmental Research. Oak Ridge National Laboratory is managed by University of Tennessee—Battelle LLC for the Department of Energy under contract DE-AC05-00OR22725.
- Received 13 March 2001.
- Accepted 21 September 2001.
- Copyright © 2001 American Society for Microbiology


text:
some longish essays by some computer programmer dude, alot about how we as a society educate our children
Archive for May, 2006
some long essays26 May, 2006
great weekend, but15 May, 2006
started with a not-boss giving me a bottle of wine.
Played a silly game with friends on Friday night.
Saturday, relaxed, played in my garden, studied, then went to fancy yummy dinner and then pool for post-bachelor party. We were great at avoiding those holes on the table.
Sunday, long run, nice walk through park, then post-b-day-BBQ.
it's been awhile13 May, 2006
First, I learned last night that tarantulas can climb up a glass window. I tried not to watch. But I couldn't turn away. I wonder if tarantulas can crawl across the ceiling. Crap! I am never gonna be able to get to sleep now.
I saw "Thank you for smoking".
I was entertained.
I give it a B+
I am slowly getting back into shape. Been on a couple of 6 mile jogs w/ SnarkyKat, and I lived.
I read "the Shattered Land" book 2 in I suppose a trilogy. Easy read. But it bored me. I don't care about the characters. And when a diety has to rescue them twice, I'm thinking poor writing/they don't deserve to be heroes.
I give it a C-

subset: commoncrawl


text: Zans Posted July 18, 2019 Hey, not sure if it's correct subforum to post, apologies if it's not. A lot of people here seem to aquired ticket to hell for a reasons other than mental illness. Like insomnia, some medical issue or situational anxiety. I wonder about those who been hopelessly depressed prior getting on meds and what happened after tapper? Did your condition changed? I rotated many pills over course of half a year and none did help. Curently the one I am on helps SLIGTHLY but as we all now it's a dead end. So, please share what changed for you after you taper was over ☺ Thank you all 0 Share this post Link to post


text:
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In late December 2016, the Washington Post published an explosive story: the wily Russians had successfully hacked into the US electric grid, threatening us all. "Breaking," the Post wrote on Twitter: "Russian hackers penetrated US electricity grid through a utility in Vermont." The reaction from politicians and so-called journalists was duly histrionic. "Vermonters and all Americans should be both alarmed and outraged that one of the world's leading thugs, Vladimir Putin, has been attempting to hack our electric grid, which we rely upon to support our quality-of-life, economy, health, and safety," Vermont Governor Peter Shumlin said in a statement, forgetting to add "precious bodily fluids" to the list. "This episode," he continued, "should highlight the urgent need for our federal government to vigorously pursue and put an end to this sort of Russian meddling." Lawmakers from Vermont issued equally alarmist statements, adding fuel to the anti-Russian hysteria that continues to dominate our national discourse, Russiagate's spectacular faceplant notwithstanding. (Anyone who thinks that the conclusion of the Mueller probe, that there was no Trump-Russia election conspiracy, will give the media a moment of pause is deluding himself: the New Cold War is here to stay.)
Two days after the Post's sensational hacking story, the paper published a related article, this one a little less exciting. The headline read: "Russian government hackers do not appear to have targeted Vermont utility, say people close to investigation." The original news story, like dozens of other Russia-related "bombshells," was a fabrication. In reality, some malware was detected on a single laptop used by an employee at the Burlington Electric Department. "The company said in its statement that the laptop was not connected to its grid systems." And while the malware was allegedly linked to a "Russian hacking operation," the Post was forced to report that "officials say it is possible that the traffic is benign, since this particular [Russian] IP address is not always connected to malicious activity."
How disappointing. Still, the story wasn't a total waste. In its original, amended article, the Post made sure that its readers understood that "Russia has been accused of in the past of launching a cyberattack on Ukraine's electrical grid," citing allegations made by Petro Poroshenko, who is losing to a comedian in the current presidential elections. So while Putin may not have hacked our power grid, it's possible that he did hack Ukraine's. Moreover, in 2014, "federal authorities reported that a Russian malware known as BlackEnergy had been detected in the software controlling electric turbines in the United States." Don't despair too much—Putin is still coming for us.
It appears that the Post is on board with the idea that electric grids are logical targets for states engaging in cyberwarfare attacks—so long as the victim isn't an adversary of the United States. On March 25, the Post reported that Venezuela was dealing with its second major blackout in two weeks, this one affecting "as many as 16 of Venezuela's 23 states." Venezuelan President Nicolas Maduro asserted on both occasions that his government found evidence that the outages were caused by a cyberattack carried out by individuals associated with Washington's hand-picked opposition leader and "interim president" (more on which later), Juan Guaido. The Post notes Maduro's charge in passing before writing that Trump "has denied any role in the outages," and furthermore that "electricity experts" have blamed "years of government graft and incompetence," ruling out a cyberattack.
Reporting on the first blackout, the Post quotes Guaido as stating that "at least 17 deaths had occurred at hospitals as a result of the outages," and that "the power outage resulted from poor maintenance—specifically, the failure to trim vegetation near major power lines, which caught fire." While the Post was unable to "independently confirm" the deaths, it's not unreasonable to assume that they happened. It would be surprising if people didn't die as a result of the blackouts, given the dire shape of Venezuela's economy.
So who or what is causing them? Maduro says we're responsible. The Post isn't buying it, maintaining a cool agnosticism that was conspicuously missing from its coverage of the Vermont hacking hoax. Can we take Maduro's word for it? No, just as we can't take any politician's word for anything. Politicians lie; that is their job. Let's ignore Maduro's comments, then, and look at the matter objectively. First, cui bono? Obviously Guaido and his gringo ventriloquists. The worse things get for the Venezuelan people, the better things look from Washington. As Guaido himself said, "The time has come to agitate in every state, in every community, to get water back, get electricity back, get gas back."
The opportunities afforded by massive power outages in Venezuela were understood, and articulated, back in 2010 by the Center for Applied Nonviolent Action and Strategies (CANVAS), a shifty non-profit organization that specializes in "strategic nonviolent conflict," which doesn't sound at all Orwellian. CANVAS was founded by anti-Milosevic activists in Serbia and, for the better part of this century, has conspired with like-minded entities to bring about a coup in Venezuela. In documents published by WikiLeaks, CANVAS claims that "we are definitely ramping up activity in Venezuela." That was in 2010. The same batch of documents has the group stating that "US networks are definitely involved" in Venezuela operations, referring to such networks as "the usual establishments." Intriguingly, part of CANVAS' "activity" in Venezuela involved fantasies about a catastrophic failure of the country's electricity system. In a lengthy investigative report, Dan Cohen and Max Blumenthal quote the following excerpt from an internal CANVAS memo:
[The collapse of Venezuela's power grid] could be the watershed event, as there is little that Chavez can do to protect the poor from the failure of that system. This would likely have the impact of galvanizing public unrest in a way that no opposition group could ever hope to generate. At that point in time, an opposition group would be best served to take advantage of the situation and spin it against Chavez and towards their needs.
[The collapse of Venezuela's power grid] could be the watershed event, as there is little that Chavez can do to protect the poor from the failure of that system. This would likely have the impact of galvanizing public unrest in a way that no opposition group could ever hope to generate. At that point in time, an opposition group would be best served to take advantage of the situation and spin it against Chavez and towards their needs.
Guaido has ties to CANVAS. A mere coincidence, no doubt.
In a follow-up article, Blumenthal notes the interesting reaction of US officials to the Venezuelan blackouts. Mike Pompeo: "No food. No medicine. Now, no power. Next, no Maduro." Meanwhile, Little Marco Rubio informed his Twitter followers that Venezuela's "backup generators have failed." Afterwards Jorge Rodriguez, Vice President of Communication, Tourism and Culture, stated during a press conference that Rubio's tweet was posted before Venezuelan officials knew anything about the backup generators. "How did Marco Rubio know that backup generators had failed?" Rodriguez asked. "At that time, no one knew that." A mere case of clairvoyance, no doubt.
A coup in broad daylight
Even without any of the forgoing oddities, it's impossible to dismiss Maduro's explanation for the blackouts when they're occurring in the context of a US-led regime change operation, about which Trump and his henchmen have been perfectly frank. It's pretty remarkable how transparent and shameless they've been about their predatory, imperialist motives. It's a rum coup that doesn't have some mitigating cover story, however unconvincing. At least George and Dick pretended to believe that Saddam had WMD and friendly relations with Osama. At least we were told that Assad, Gaddafi and Milosevic were genocidal butchers. From Trump we get the ugly, unvarnished truth, which makes it tricky for the media to accomplish their usual task of selling an imperialist crime to the American people.
Shortly after Guaido announced that he was electing himself president in January, Rubio wrote on Twitter: "Biggest buyers of Venezuelan oil are Valero Energy and Chevron. For the sake of these US workers I hope they will begin working with [the] administration of President Guaido and cut off [the] illegitimate Maduro regime."
Still more explicit was John Bolton, who appeared on Fox News (sic) and said: "We're looking at the oil assets. That's the single most important income stream to the government of Venezuela. We're looking at what to do to that." He continued: "We're in conversation with major American companies now that are either in Venezuela, or in the case of Citgo here in the United States … It will make a big difference to the United States economically if we could have American oil companies really invest in and produce the oil capabilities in Venezuela."
In his recent book, former Deputy Director of the FBI Andrew McCabe wrote that, during a national security meeting, Trump went off on a tangent about Venezuela. "That's the country we should be going to war with," the president said. "They have all that oil and they're right on our back door." He made similar comments to advisers in August 2017, asking them how they felt about the prospect of invasion. This is consistent with what appears to be Trump's long-held approach to foreign policy, to wit, that the US should only go to war with countries in close proximity. For example, asked in 1999 about the Vietnam War, Trump had this to say: "I think that Vietnam was a disaster. It was too far away. It didn't affect us. I guess if you do it, you have to go to win. We didn't go to win. But I think Vietnam would have been a good place for us to stay out of." His backdated opposition to the invasion of Iraq, and our Middle East wars generally, follows the same line of thought.
Understanding that all our "great" presidents have had their own wars to define their legacies, Trump evidently chose Venezuela as the arena for his. That was logical enough: Washington has been aggressively meddling in Venezuela's domestic affairs since Chavez was elected president in 1998, supporting the abortive right-wing coup against him in 2002. In 2015, Barack Obama imposed unilateral sanctions on Venezuela after designating the small Latin American country a "national security threat," helping to lay the groundwork for Trump's coup, which, again, the media heartily support. As I and many others have noted, The Resistance abruptly falls silent, or rather falls into line, whenever Trump sets off down the war path. See their reaction to his illegal cruise missile strikes against Syria. When he does the reverse—e.g. pretending that he's going to withdraw from Syria—they have a fit and make lots of noise about how dangerous and irresponsible he is. War is Peace, after all.
Making the people of Venezuela scream
Economic sanctions have been the foundation of numerous US-orchestrated coups and/or invasions since the end of World War Two. Venezuela is no exception. In addition to targeting the oil industry, Washington has gone after the banking sector and blocked Caracas from recovering more than $1 billion worth of gold it had previously deposited in foreign banks. The US is also threatening to sanction countries that defy its sanctions, such is the extraordinary degree of our hubris. Per Reuters, "the State Department made clear that any kind of oil trade, whether it be direct, indirect or barter, would be considered a breach" of the rules it recently made up. Here's how one source summarized things: "This is how the United States operates these days. They have written rules and then they call you to explain that there are also unwritten rules that they want you to follow." Obey our arbitrary and illegitimate rules, even the ones you don't know about, or else!
The idea is to make Venezuela's "economy scream," to borrow Nixon's phrase. Of course, it would be more accurate to say that Trump intends to make the Venezuelan people scream—to manufacture a humanitarian crisis that might open the door for the use of military force. Denouncing Washington's economic warfare, Special Rapporteur to the UN Idriss Jazairy said that "sanctions which can lead to starvation and medical shortages are not the answer to the crisis in Venezuela," adding that external "coercion … is in violation of all norms of international law." In an interview with The Independent, former UN official Alfred de Zayas stated bluntly that, in addition to being illegal, "sanctions kill." He likened them to "medieval sieges," explaining that "twenty-first century sanctions attempt to bring not just a town, but sovereign countries to their knees." Zayas added that he has been shunned not only by the media but also by the UN, the leaders of which are apparently averse to honest judgments of the situation in Venezuela.
The Trump regime has all but echoed Zayas' assessment, albeit from a different point of view. "The effect of the sanctions is continuing and cumulative," one official told reporters. "It's sort of like in Star Wars when Darth Vader constricts somebody's throat, that's what we are doing to the regime economically." That's mostly accurate—we just need to substitute "population" for "regime."
Anyone who wants to estimate the general outcome of the "continuing and cumulative" sanctions on Venezuela doesn't have to look back very far into the past. The obvious and most obscene example is Iraq. Throughout the '90s, an untold number of civilians were killed by UN-approved sanctions that paralyzed the entire economy. Denis Halliday, one of two UN humanitarian aid coordinators who resigned in protest, wrote in 2003 that "we, the United Nations, killed more Iraqis through UN sanctions—probably a million people, particularly children—in those 13 years than Mr. Bush the First, Mr. Clinton and then Mr. Bush the Second did with bombs." Jutta Burghardt was appointed Representative and Country Director of the World Food Program in Iraq in 1999, and resigned in 2000. In her words, "There can be no doubt whatsoever that the imposition of the sanctions on Iraq by the Security Council is leading to partially or totally destroying a nation, and that this concept was and continues to be deliberately and intentionally applied."
Media manipulation
Partial or total destruction: that's what the Trump regime has in mind for Venezuela, and what Guaido is going along with. For the media to pretend otherwise is a disgrace. But pretend they do, in service, as always, to their masters in Washington. On February 5, the New York Times editorial board wrote that "There is no question that President Maduro must go," having "led his oil-rich country into utter ruin." No mention of US sanctions and their impact on Maduro's ability to govern.
Before continuing I'd better clarify something: Maduro's flaws and corruption are none of my concern. Not being Venezuelan, I'm in no position, nor do I have any right, to do anything about them. Venezuela's domestic affairs are the business of the Venezuelan people, the overwhelming majority of whom oppose American intervention of any kind. The degree to which Maduro is responsible for the crisis is debatable, and I have no interest in engaging in that debate. What matters, as far as I'm concerned, is that there is a crisis, and that the US is deliberately and criminally exacerbating it. Every American citizen has not only a right but an obligation to speak out against the crimes committed by their government in their name, and moreover against the distortions and outright lies promulgated by the corporate media.
Returning to the Times, the news media's chief distorter, its editors argue that "The only solution is an interim government under Mr. Guaido, who as the head of the National Assembly has a legitimate claim to the presidency under the Venezuelan Constitution." Nonsense. The "legitimate claim" the Times writes of is not legitimate at all. Guaido has invoked Article 233 of the Venezuelan constitution, which states that, should the office of the presidency become vacant, the leader of the National Assembly shall serve as interim president until an election is held. The problem is that the office of the presidency is not vacant: it's held by Nicolas Maduro. Trump and his sock puppet Guaido claim that Maduro is not really president because the last election was, according to them, illegitimate. But as Roger Harris writes in CounterPunch, Article 233 says nothing about illegitimate elections. And anyway,
The Supreme Tribunal of Justice (TSJ), which is the separate supreme court branch of the Venezuelan government has certified Maduro's reelection, as have independent international observers. Further, no appeal was filed by any of the boycotting parties, while all participating parties—including opposition ones—signed off on the validity of the election after the polls closed.
The Supreme Tribunal of Justice (TSJ), which is the separate supreme court branch of the Venezuelan government has certified Maduro's reelection, as have independent international observers. Further, no appeal was filed by any of the boycotting parties, while all participating parties—including opposition ones—signed off on the validity of the election after the polls closed.
It was only after Maduro was sworn into office for his new term that Washington decided to hand-pick another president. Article 233 is irrelevant. Guaido has no legal claim to the presidency. Put simply (by Alfred de Zayas on Democracy Now!), "We have here an unconstitutional situation in which the legislature is usurping competences that belongs to the executive and to the judiciary. The judiciary has already declared all of these actions and declarations of the National Assembly to be unconstitutional." Maduro is president; Guaido is not. Despite what the almighty New York Times says.
Burning aid and military pretexts
To reiterate: the vast majority of Venezuelans do not support any US intervention in their country, especially military intervention. That hasn't stopped "President" Guaido, the man who claims to represent the Venezuelan people, from inviting the US to use military force against Maduro. After the government refused to allow "humanitarian aid" from the US into Veneuzuela (probably the most farcical scene in this unfolding tragedy) Guaido took to Twitter, where he implied that the US should perhaps attack his country. "Today's events force me to make a decision," he wrote: "to formally propose to the international community [aka the United States] that we must have all options open to secure the freedom of our country." We all know what "all options" means. Trump has been saying it over and over for a year and a half. Now Guaido is saying it, as though this were a literal puppet show.
February's "humanitarian aid" stunt was embarrassing to witness. Erroneously claiming that Maduro was not letting in any aid, the US sent a fleet of trucks to the Venezuelan border via Colombia, knowing that Maduro would put up a blockade. When he did, Washington and its media hacks feigned outrage and, like a broken record, repeated the bald-faced lie that Maduro was blocking all aid shipments. In reality, Maduro has appealed to the UN for assistance and let more than 1,000 tons of food and medicine into the country. He blocked the US aid shipment—labeling it a Trojan Horse—because it was so obviously an act of imperialist theater, which is also why real aid organizations like the Red Cross distanced themselves from it. "We will not be participating in what is, for us, not humanitarian aid," said Christoph Harnisch, spokesman for the International Red Cross in Colombia. Likewise, the UN wanted nothing to do with the ruse, stating that "humanitarian action needs to be independent of political, military or other objectives."
Most of the major media ignored these criticisms, preferring instead to depict Maduro as a monster who deliberately starves his people. He even sets humanitarian aid on fire! That's what we were told by Trump's flunkies when one of the trucks went up in flames along the border. Videos and images of the burning truck spread like wildfire (no pun) across social media. The settled-upon narrative was that Maduro had ordered his security forces to firebomb the truck. "What kind of a sick tyrant stops food from getting to hungry people?" Mike Pompeo asked on Twitter. "The images of burning trucks filled with aid are sickening." Similar statements were made by the rest of the goon squad and then amplified by the usual media suspects.
The story, like the Vermont hacking story, was phony. So phony, in fact, that the New York Times corrected the record a few weeks later. Pointing to a video that showed an anti-Maduro protester throwing a Molotov cocktail, part of which went sideways and landed on the truck, the Times wrote that "the opposition itself … appears to have set the cargo alight accidentally." The Times also reported that the trucks contained no medicine, contrary to statements made by various government officials. Before you give the Times too much credit, consider that the video footage cited in the article was available on the internet, and shared by several journalists, almost immediately after the incident occurred. Rather than make the minimal effort required to debunk the fake story concocted by the State Department, the major media chose to run with it, which simply confirms yet again that much of what passes for journalism today is nothing more than mindless propaganda, the purpose being to manipulate the American public into supporting Washington's delinquent behavior.
At this point I wouldn't be surprised if CNN told us that Nicolas Maduro was stealing incubators away from babies, or attacking protesters with sarin gas, or giving his troops Viagra to facilitate mass rape, or enriching uranium at secret nuclear sites. We already know that he has "ties to Hezbollah." That means he's capable of anything. It's only a matter of time before we're fed the next horror story out of Venezuela. With the 2020 elections around the corner, Trump the Conquistador will be looking for any excuse to launch his war, the one that will secure his reelection—and his place in American history.
*(Top image: Guaido and Pence shake hand at a meeting of Lima Group on February 25, 2019 in Bogota, Colombia. Credit: Reuters)
Michael Howard is a writer and teacher living in Vietnam. Contact him at firstname.lastname@example.com.
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A long time ago I visited a place in Calcutta, India, where Mother Teresa used to operate. According to the Catholic Church and Western pr...


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You have found the perfect job? It's time to work on your application. We have some tips for you!
Data Hunter for Data Intelligence (m/f/d) in Group Data Analytics
Group Data Analytics (GDA) is a department in Allianz's global headquarters. We are responsible for shaping Allianz's strategic agenda for Data Analytics and Artificial Intelligence (AI), and executing Data Analytics and AI solutions at scale, across the entire Allianz Group. Our mission is to add tangible value to our business by driving future business growth and productivity, and enable data-driven transformation and decision-making. ~crlf~ ~crlf~We do this by developing and deploying globally scalable Data Analytics and AI solutions. We work closely with our colleagues driving the new global business model (Allianz Customer Model), to scale the impact of analytics and AI in Claims, Sales, and Operations. We co-develop reusable assets with OEs across the world and work on strategic business intelligence projects to improve data-driven decision-making. ~crlf~ ~crlf~We believe that our long-term success also rests on enabling the business and empowering employees to adopt Data Analytics and AI. We do this by creating transparency on AI capabilities and assets, providing expert knowledge to local data science teams, working towards common architecture and coding principles, and running communities of practice.~crlf~ ~crlf~We are looking for data hunters for our new Data Intelligence team. S/he will identify and implement solutions to improve and exploit the data in insurance processes. S/he will work in interdisciplinary team setups alongside business owners, business domain experts and technical experts. The data hunter will define how to assess and implement identified solutions and innovations to make better use of our internal and external data. ~crlf~ ~crlf~Besides the work on specific high-impact projects, the data hunter will be responsible for making internal and external data discoverable for data consumers. Also, s/he will identify opportunities where internal data can be complemented by external data to build superior solutions and improve processes. ~crlf~ ~crlf~The data hunter will work in an interdisciplinary team reporting directly to the Head of Business Data Improvement.
##Performs detailed business analysis to identify the potential for better use of internal data and synergies with external data ~crlf~##Coordinates the planning and execution of data intelligence projects with the following core activities: ~crlf~##Analyzes data situations based on facts (e.g. data-driven process analysis) and defines areas of improvement, transformation and innovation~crlf~##Runs impact analysis of missing data and poor data quality on business core processes and feasibility of potential analytics solutions~crlf~##Defines opportunities and potential solutions (e.g. business intelligence, data analytics solutions) to unlock the value of data ~crlf~##Defines the conceptual/ content data requirements for data analytics and business intelligence solutions ~crlf~##Quantifies the potential of individual data intelligence measures and suggests an adequate prioritization of activities~crlf~##Develops comprehensive and scalable blueprints how to unlock the potential of data across the Group and drives the implementation of such blueprints~crlf~
Qualification:~crlf~##Outstanding PhD/Master's degree in Science / Engineering / Economics / Business Administration~crlf~##Practical experience in Data Management, Data Analytics, Artificial Intelligence, Business Intelligence~crlf~##Broad insurance domain knowledge (e.g. data in use, objectives, metrics, best-practices)~crlf~##Program/project management experience in data and transformation projects~crlf~##Knowledge and experience in agile methodologies ~crlf~##Basic understanding and/or strong interest in data analytics/AI ~crlf~##Fluency in English is required, additional languages are a plus~crlf~ ~crlf~Experience & Key Skills:~crlf~##+ 5 years of professional experience in the insurance, consulting or technology sector~crlf~##A passion for identifying and solving analytical problems and for translating your insights into data-driven and actionable recommendations~crlf~##Strong communication and storytelling abilities to mobilize others without authority~crlf~##Strong analytical, conceptual and problem solving skills~crlf~##Strong project management capabilities~crlf~##Ability to get up to speed on complex strategic and operational topics and to consolidate and evaluate large amounts of information~crlf~##Strong communication, negotiation, mediation and diplomatic skills, ability to work with different cultures and manage numerous interfaces~crlf~##Entrepreneurial can-do attitude and drive with out-of-the-box-thinking focus on quality execution and delivery of task, result-oriented, customer-focused, pro-active, flexible and energetic team player with ability to work under pressure~crlf~##Being willing to 'go the extra' mile for our customers, partners and other business units~crlf~
Senior Recruiter: Ms. Zhiva Hristova Phone: +49 89 3800 14379~crlf~Please submit your applications only via the tool (blue button above).
Allianz is the home for those who dare – a supportive place where you can take the initiative to grow and to actively strengthen our global leadership position. By truly caring about people – both its 85 million private and corporate customers and more than 142,000 employees – Allianz fosters a culture where its employees are empowered to collaborate, perform, embrace trends and challenge the industry. Our main ambition is to be our customers' trusted partner, instilling them with the confidence to grow. If you dare, join us at Allianz Group.~crlf~~crlf~Allianz is an equal opportunity employer. Everybody is welcome, regardless of other characteristics such as gender, age, origin, nationality, race or ethnicity, religion, disability, or sexual orientation.
Allianz SE is the global headquarters of the Allianz Group. Our employees reflect the Group's geographic and functional diversity. Located in Munich, Allianz SE provides the perfect opportunity to start or continue with your international career.
Please submit your complete application documents (incl. CV, certificates, references and motivation letter)~crlf~~crlf~We are looking forward to receiving your application on www.allianz.com/careers.~crlf~~crlf~Allianz SE is committed to employment equality and therefore welcomes applications from all people regardless of gender identity and/or expression, sexual orientation, race or ethnicity, age, nationality, religion, disability, or philosophy of life.~crlf~~crlf~
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You're invited!
With the objective to humanize mental healthcare, insyaAllah next year we will be holding a workshop on World Health Organization's (WHO) Quality Rights module. This will be a continuation from our last Quality Rights workshop, which you can read more here. What is Quality Rights, you might ask? The paragraph below from WHO's website explains it in a nutshell:
"WHO QualityRights aims to improve the quality and human rights conditions in inpatient and outpatient mental health and social care facilities and empower organizations to advocate for the rights of people with mental and psychosocial disabilities. The care available in mental health facilities around the world is not only of poor quality but in many instances actually hinders recovery. It is common for people to be locked away in small, prison-like cells with no human contact or to be chained to their beds, unable to move. Violations are not restricted to inpatient and residential facilities however; many people seeking care from outpatient and community care services are disempowered and also experience extensive restrictions to their basic human rights."
Coming back to the workshop, our esteemed panels are:
The topics to be covered are as follows:
- Conducting the visit to current mental health facilities to assess its human rights condition. This will be covered by Dr. Abdul Kadir
- Religion and healing, which will be covered by Ustaz Mohd Noor Deros.
Details of the program:
- Venue: MIASA (click here to locate us on Google Maps)
- Date: Sunday, 7th January 2018
- Time: 10.00am – 3.00pm
You may register by filling in the contact form below:
Or, you can also send us a Whatsapp message at:
- 013-878 1322
- 019-236 2423
If you have any questions or comments, feel free to drop a comment below. Hope to see you at the workshop, insyaAllah!


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Terra Map: GPS Hiking maps 4+
Outdoor trails & topo charts
GEC s.r.l.
- Free
- Offers In-App Purchases
An app to satisfy all your Hiking and Biking's needs, developed with the help of professional hikers and bikers. You will be able to download the best topo maps and enjoy your new outdoor season. Press the RECORD button, put the phone in your pocket and record your day.
►► Features:
• Optimised for both iPhone and iPad use.
• Amazingly clear maps, thanks to the Retina technology usage.
• Easy to use user interface.
• You can download maps (for FREE) on the device, to be used later without any internet connection.
• Topo Maps for US, Canada, Europe, South America, Asia, Africa, Oceania (the whole world !!!).
• Expand your horizons with mapXpan (patent pending).
• Share tracks, tours, markers and photos in various formats (KMZ, KML, GPX).
• Share your track together with all the photo taken along in one single file.
• Add the Satellite images as overlay of your map.
• Optimized battery usage with battery monitoring.
• Store your track without the need of cellular data or phone signal.
• Switch between different map colouring.
• Customize your Marker's icon and Track's color.
• Measure distances on the map.
• Load any track, tour and marker from internet communities (KMZ, KML, GPX even if zipped)
• Graph of the elevation profile and speed for each track.
• Search objects around you and around a point selected on map
• Search world places by name
Terra Map lets you download — for FREE !!! — the best topo maps with elevation contours, trails, huts, parks, etc. and no additional purchase is required. You can download the maps at home within your device and then use them without any Internet connection need. Check our maps at: www.globalterramaps.com/terraviewer.html
Why not sharing your favorite markers on Facebook or sending an email with your last daily track, with all the pictures you captured (Note 1) to show them on Google Earth™ or Maps ? With Terra Map it is just a click.
Search by name of important places worldwide, search of objects around the GPS position or a map position.
Record all important info of your track and plot the graph of elevation and the graph of the speed.
► mapXpan
This new mode will greatly extend your display; you will have the details you need for a safe navigation seamlessly integrated with a large overview area. Using this new mode you will see a map 64 times larger than the standard mode without losing any detail in the center part of the screen.
Unlike many other apps, Terra Map is designed to use at the best the battery power to last for one full day (Note 2). The Battery Monitoring will stop the track recording when the battery is too low, in order to ensure the phone is still usable at the end of your outdoor's day.
► iOS BACKGROUND MODE
You can record GPS track while Terra Map is in background and while the device is locked.
- Unlimited: you can download and update your charts forever.
- Subscription: it is an year auto-renewable subscription, it will automatically renews unless auto-renew is turned off at least 24-hours before the end of the current period. Account will be charged for renewal within 24-hours prior to the end of the current period. Subscriptions may be managed by the user and auto-renewal may be turned off by going to the user's Account Settings after purchase.
Payment will be charged to iTunes Account at confirmation of purchase
Terms of Service: http://www.globalaquamaps.com/Acknowledge.html
Privacy Policy: http://www.gec-it.com/GEC-privacy.pdf
Note 1. Pictures captured by Terra Map while recording the track are automatically linked to the track itself, and sent with the track when you share it.
Note 2. The effective battery duration depends by the battery condition, by the temperature and the device usage.
What's New
Version 20.1
• Solved an issue related to the map shading
• Increased readability of text on the map
Ratings and ReviewsSee All
Gotta love offline maps!
This is a nice program as long as you download the maps you need before you are offline. I would give it 5 stars except for the intermittent problem that I am having with it marking my track. Yesterday I thrashed through brush for three hours uphill to mark a fence line, when I got to the top the track hadn't registered even tho the little icon was still flashing as active. Grrrrr!
Developer Response ,
thanks for your words and we are sorry you had a problem using our app. What we could suggest about tracking is to pay attention to where you place the phone. You need to put it in a position that gives a "clear sky view" and not under some item that hidden the sky. Check also the quality of the GPS signal (box in the bottom side of the screen) because in some condition (big trees) the signal could be lost.
Best Regards
Good app with great support
Love this app for being easy to understand and easy to use, plus the maps are very good.
I had a problem with OS13 update and the app. Emailed support, and had an answer and fix quickly.
Edit: The developer helped me with this, so I'm raising my rating.
Original Rating: "I notice that the maps leave artifacts from the relief when looking at the topo. It's very annoying."
From the developer message:
Hello, we have to keep a limited number of height samples to keep memory occupation acceptable and our library works with a tessellation of images of 256x256 pixels so when the system is zoomed the shading can appear unrealistic.
You can switch off the shading using the menu Settings —> Shading
We already switch off it automatically at the given some level, we can anticipate the switch off in future releases.
Developer Response ,
thanks for your feedback. Could you kindly send an email (firstname.lastname@example.com) with an image explaining what is exactly the problem you are seeing ? We will investigate to improve our app.
Best Regards
Terra Map Support Team
- Seller
- GEC s.r.l.
- Size
- Category
- Navigation
- Compatibility
Requires iOS 10.0 or later. Compatible with iPhone, iPad, and iPod touch.
- Languages
English, Dutch, French, German, Italian, Portuguese, Spanish
- Age Rating
- 4+
- Location
- This app may use your location even when it isn't open, which can decrease battery life.
- Copyright
- © GEC srl 2013 - 2020
- Price
- Free
- In-App Purchases
- Pro Version $8.99
- Pro Version $4.99
Family Sharing
With Family Sharing set up, up to six family members can use this app.


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I am delighted to welcome the Justice Mental Health and Fair Trial Report into mental health and the criminal justice system as a valuable contribution to a continuing debate over the appropriate ways to cater for mental health illness and neurological illness at all stages of investigation, prosecution and disposal of offences.
In 2001, Mind – the mental health charity – highlighted the difficulties which individuals with mental health conditions had in securing effective access to justice. Its views were recently summarised in a searching paper written by Professor Lee-Ann Fenge and others on the relationship between mental health and the criminal justice system. They said this,
'People with mental health conditions and learning disabilities tend to experience greater difficulties in accessing justice than other groups, and are one of the most socially excluded groups within society as they experience greater discrimination, disadvantage and stigma.


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Last year was the first year that non-European/Pākehā students made up more than half the number of students in Catholic schools in New Zealand.
Using Ministry of Education school roll data as of July, the New Zealand Catholic Education Office reported that students of European/Pākehā ethnicity made up 49.9 per cent of those attending Catholic schools in 2018. The next largest group was Pasifika students (15.7 per cent), Asian (15.3 per cent) and Māori (14.5 per cent).
In 2009, the figures were European/Pākehā (58 per cent), Pasifika (16 per cent), Māori (13 per cent) and Asian (11 per cent).
Schools in Auckland diocese and Wellington archdiocese, with their more multi-cultural urban populations, have more ethnically diverse student populations, followed by Hamilton and Palmerston North, the NZCEO report stated.
In Auckland diocese, the ethnicity breakdown of the student population at Catholic schools given in the report is European/Pākehā (39 per cent), Pasifika (28 per cent), Asian (19 per cent) and Māori (9 per cent). There is a further category denoted as "other" that completes the student population.
In Wellington archdiocese, the figures are European/Pākehā (47 per cent), Pasifika (18 per cent), Māori (16 per cent) and Asian (15 per cent).
The percentages in the NZCEO report of European/Pākehā students in other dioceses are: Hamilton (57 per cent), Palmerston North (59 per cent), Christchurch (64 per cent) and Dunedin (66 per cent).
Catholic Education Services manager for Auckland diocese Philip Mahoney told NZ Catholic he is not surprised at the ethnicity figures, based on what he and his staff see at school Masses and in doing school reviews.
"[I]t has been obvious in the last couple of years that the proportion of European students is a lot smaller than in years past," Mr Mahoney said.
He noted, though, that the European/Pākehā proportion of Catholic schools in Auckland has been under 50 per cent for some time.
Growth in the proportion of Asian students, especially Indians and Filipinos, has been noticeable.
Mr Mahoney said that at some Auckland Catholic secondary schools, it can be seen that, of the students who are collecting academic prizes and who are taking roles in school liturgies, such as being ministers of the Eucharist, a minority are "New Zealand European students".
The growth in Indian and Filipino families in parishes in Auckland is being reflected in the schools, he believed.
"In Auckland, our schools have had strong Pasifika representation for quite some time and some of our schools are almost 90 per cent plus Pasifika," he said, "but the change has been in the growth of what is broadly categorised as Asian."
He noted that Indian and Filipino people have their own cultures, which are "quite different", but for the report they "are broadly categorised as Asian".
An increasingly diverse school population is a positive, but it also brings challenges, Mr Mahoney said.
"It is a positive overall because they bring different cultural perspectives around not only the worldview in terms of the countries they have come from, which are quite different both in terms of size and structure and how they operate and degrees of wealth and poverty that we see in New Zealand, so they bring that, but they bring different cultural attitudes towards education, towards achievement, what they value, possibly
sometimes a lot of value on academic achievement, perhaps not so much on sports or if it is sports, it is different sports to what have been customary for the European [population]."
But having families who are relatively new to New Zealand and its education system can pose challenges.
"A lot of our schools are moving to 'innovative learning environments' with rebuilds," Mr Mahoney said. "Although that is also new for European New Zealander families who didn't experience that with the parents' generation, they have seen it happening for a little bit longer than perhaps some of the new, recently-arrived Filipino, Indian families who don't have schools like that. So that is one challenge for them as families and
for their children to adapt to learning in wide open spaces."
Mr Mahoney said there can be challenges concerning "the religious background that they bring, again this possibly also has a lot more traditional devotional practices in it, which they seem to, after a little while, develop of their own accord in parishes, sometimes with the blessing of the parish priest as well, and sometimes alongside. . . other practices, so we see a growth of sodalities or devotions which are not common in New Zealand. . .".
"For those children, who possibly attend some of that with their parents, . . . there is a little bit of an expectation [from] the parents that there will be some kind of devotional practices in the schools, and so the schools need to be aware that there is an expectation that a Catholic school will offer the rosary in the month of the rosary, and things like that, which we normally do, but not to the extent that they might expect."
The changes in the ethnic make-up of the student population in Catholic schools in Auckland is not limited to the students. Mr Mahoney said it is represented among teaching staff too.
He said that, particularly in primary schools, when his staff visit on reviews, "we are seeing a lot more schools employing Filipino and Indian teachers", notably for the tagged positions. (A teacher in a tagged position must be willing and able to take part in religious instruction in the school).
A lot of the names on the forms which come to Mr Mahoney's office for approval for tagged positions in Catholic schools are Filipino or Indian, he said.
The NZCEO report also noted that, as of July, there were 66,888 students in Catholic schools, representing 8.3 per cent of the New Zealand schooling network.
This was an increase of 160 over the previous year. But, proportionately, it represented a decline of less than 0.1 per cent in terms of the overall New Zealand schooling network. In 2011 and 2012, the proportion of students at Catholic schools as against the overall school network was 8.6 per cent.
In 2018, in the Taranaki region, students in Catholic schools made up 15 per cent of the overall school network in the region. In the West Coast and Southland, the figure was 14 per cent. In Marlborough, the figure was 2 per cent and it was 4 per cent in Tasman and Northland.
Of the Catholic school population, 44 per cent were year 1-6 primary students, 39 per cent year 9-13 secondary students and the remainder year 7-8 intermediate level who may have been in primary or secondary schools.
There are 237 Catholic schools in New Zealand, with the average Catholic school size being 280 students. The average Catholic primary school roll is 175 and the average Catholic secondary school roll is about 750.
There were 23,007 students in Catholic schools in the Auckland region, 9644 in the Wellington region and 8110 in the Canterbury region.


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White Heron, a specialty tea purveyor and coffee roastery has a new face in the charming New England city of Portsmouth, N.H., with the opening of the White Heron Coffee & Tea Shop at 601 Islington St.
The coffee and tea company was founded by Jonathan Blakeslee in 2005, and has steadily expanded its retail presence at farmers markets, as well as local grocery stores and restaurants. Now Blakeslee has opened a 38,000-square foot retail space — with a separate production and packaging space that makes room for a U.S. Roaster Corp. drum roaster — including a 39-seat cafe.
For more on White Heron:
Nick Brown
Nick Brown is the editor of Daily Coffee News by Roast Magazine. Feedback and story ideas are welcome at publisher (at) dailycoffeenews.com, or see the "About Us" page for contact information.


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FirstACTS® student behavior tracking system
A web-based tool for easily communicating incidents on the bus
Communicate easily so you can act effectively
Student conduct issues on even a single bus route can throw off the whole school day for students, parents or caregivers, and school officials. FirstACTS (Active Conduct Training System) helps you prevent that by addressing:
- Unsafe behavioral issues
- Time-consuming paper-based reporting
Facilitates timely action to resolve student behavior incidents
We work with districts in 39 US states and 7 Canadian provinces
Partner with First Student and you'll get:
Instant Communication
Cloud-based incident reporting helps communicate directly with school administrators, which gives everyone the confidence that issues will be addressed promptly.
Easy Documentation
This cloud-based tool eliminates tedious and time-consuming tasks of completing, delivering, filing, and tracking paper documents, which helps increase efficiency.
You can adapt the application to your workflow and interfaces, then customize it according to your district's needs.
The application is safe and secure, allowing you to confidentially track student behavior history, identify trends, and send incident reports to the appropriate personnel.
Download 10 Strategies for Safety Success
FirstACTS – School Bus Behavior Active Conduct Tracking System
FirstACTS is web-based application that efficiently reports and tracks student behavior concerns on the school bus. FirstACTS connects drivers, transportation leaders, and district administrators to keep your children safe each and every day.