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A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.
Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), is a highly transmissible bacterial pathogen with significant morbidity and mortality, particularly in HIV-infected patients. Since 1997, tuberculosis has remained the leading cause of death in South Africa (19, 29), a statistic linked to this country's growing HIV epidemic (25). Moreover, effective treatment measures in patients with active M. tuberculosis have been hindered by increasing cases of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates.
MDR M. tuberculosis strains are resistant to first-line antibiotics rifampin (RIF) and isoniazid (INH), while XDR M. tuberculosis strains are resistant to both RIF and INH as well as fluoroquinolones (FQs) and second-line injectable antibiotic drugs, i.e., amikacin (AMK), kanamycin (KAN), and capreomycin (CAP). Approximately 9.6% of all TB cases are caused by MDR strains, and South Africa continues to report higher percentages of XDR cases each year (20, 28). There are approximately 10,000 newly reported cases of MDR TB each year in South Africa (30), with a mortality rate of more than 10% in patients infected with MDR M. tuberculosis strains (28). The emergence of antibiotic-resistant M. tuberculosis strains underscores the immediate need for rapid and highly accurate diagnosis, particularly in the developing countries of Africa.
Currently, the "gold standard" for identification of MDR strains is culture-based drug susceptibility testing (DST). However, it is time-consuming (weeks to months), technically challenging, and cost prohibitive, especially in resource-limited countries. For example, the Bactec MGIT 960 system (Becton Dickinson, Franklin Lakes, NJ) is an automated, continuously culture-based monitoring platform that measures bacterial oxygen consumption and is used for phenotypic DST. Drug susceptibility tests obtained using the Bactec MGIT 960 system are reliable and reproducible (1, 14); however, this methodology requires handling of viable and potentially infectious cultures, days to weeks until results are obtained, specialized laboratory facilities, expensive consumables, and instrument maintenance.
In recent years, several commercially available nucleic acid-based assays have been developed for determining M. tuberculosis drug resistance and have been approved for use by the World Health Organization (WHO). The Hain LifeScience Genotype MTBDRplus line probe assay (LPA) is one of the most widely used. This test entails nucleic acid extraction, PCR amplification, probe hybridization, and colorimetric visualization on lateral flow strips via an alkaline phosphatase-linked reaction. The GenoType MTBDRplus method cannot detect XDR M. tuberculosis strains and is limited to MDR strains via characterization of mutations in (i) rpoB gene (D516V, H526Y/D, and S531L), (ii) katG gene (S315T), and (iii) inhA promoters. These well-characterized mutations confer resistance to the antibiotics rifampin and isoniazid, respectively. The LPA has been shown to be both sensitive and specific using clinical isolates and/or smear-positive samples (6), but not without several drawbacks. The sensitivity of LPA for all resistance-associated mutations will realistically never reach 100%, since many mutations that confer resistance have yet to be discovered. Additionally, the LPA will not directly detect an amino acid substitution mutation outside a common and characterized mutation site or within a known resistance site but where the amino acid substitution represents an uncommon residue. Furthermore, an undefined mutation indicated by the absence of a wild-type band in LPA may not be phenotypically resistant.
The Ion Torrent is a next-generation semiconductor sequencing platform that utilizes a small chip for detection of released hydrogen ions emitted during DNA polymerization. The Ion Torrent instrument was first marketed in February 2010 by Life Technologies Incorporated (Foster City, CA) and is capable of sequencing megabases of raw sequence reads on a single chip.
In this report, we describe an Ion Torrent Personal Genome Machine (PGM) protocol for rapid (2 days), standardized, and cost-effective full-length gene analysis of M. tuberculosis genes. The developed protocol was used to assess first- and second-line M. tuberculosis drug resistance in 26 geographically diverse MDR and XDR clinical isolates collected between July and November 2011 from South Africa. Ion Torrent sequencing data were compared to data obtained by the Hain LPA and/or phenotypic DST using the Bactec MGIT 960.
Clinical isolates.A random sampling of 26 drug-susceptible, MDR, and XDR clinical isolates (collected between July and November 2011) was selected from clinical isolates from the Gauteng and Kwa-Zulu Natal provinces of South Africa. The clinical specimens were initially confirmed by conventional culture methodology. The H37Rv M. tuberculosis lab strain was included as a sequencing control throughout the protocol. All M. tuberculosis isolates used were archived strains from pure-culture MGIT 960 system tubes (Becton Dickinson, Franklin Lakes, NJ), with species identification and genotypic resistance to rifampin and isoniazid determined using the Genotype MTBDRplus assay (Hain Lifescience, Germany) according to the manufacturer's instructions. Phenotypic resistance for first- and second-line drugs was determined using the MGIT 960 system as previously described (17). Critical concentrations for ofloxacin and kanamycin (second-line drugs) were 2.0 μg/ml and 5.0 μg/ml, respectively. Resistance to first- and second-line drugs was determined using standard diagnostics algorithms as previously described (17, 27).
DNA preparation.M. tuberculosis isolates were handled in blinded fashion throughout. M. tuberculosis samples (0.5 ml) were pipetted into cryovial tubes containing 1.5 ml PrimeStore molecular transport medium (MTM; Longhorn Vaccines & Diagnostics, San Antonio, TX). PrimeStore MTM is a clinical transport solution that inactivates a broad range of microbes (5), including M. tuberculosis (8; A. W. Dreyer, N. A. Ismail, S. V. Omar, P. B. Fourie, K. Baba, and A. A. Hoosen, unpublished data), and preserves and stabilizes released RNA and DNA for safe ambient-temperature shipment (4, 5). Inactivated samples were transported from South Africa to San Antonio, TX, at ambient temperature (3 to 4 days) and stored at 5°C until used. Total DNA (50 μl) was purified from a 200-μl aliquot of PrimeStore MTM containing inactivated culture using a Qiagen EZ1 advanced robot and EZ1 DNA tissue kit (catalog no. 953034) according to the manufacturer's recommendations (Qiagen Inc., Germantown, MD).
Primer design.Novel PCR primers were designed for amplification of full-length coding regions for each M. tuberculosis gene of interest (Table 1). Primer pairs for rpoB (2 sets of primers), katG, pncA, gyrA, and rrs (16S) gene amplification were designed using the genome sequence of M. tuberculosis strain H37Rv (GenBank accession no. NC_000962) as a reference. Primer secondary structure, melting temperature, and potential primer-dimer formation were determined using LaserGene 9.1 (DNAStar, Madison, WI) and PrimerExpress 3.0 (Life Technologies, Foster City, CA). All oligonucleotides were synthesized using standard, desalted primers (Integrated DNA Technologies [IDT], San Diego, CA).
PCR amplification.Amplification reactions for all M. tuberculosis gene targets were designed and optimized to be used under one standardized set of thermocycling parameters. All PCR master mixes were prepared using Platinum Taq DNA polymerase, 10× buffer, and 50 mM MgCl2 (part no. [PN] 10966-034; Life Technologies). Amplification was carried out in a 50-μl final volume reaction mixture containing 24.1 μl Ambion nuclease-free water (catalog no. AM 9932; Life Technologies), 5 μl 10× PCR buffer, 2 μl 50 mM MgCl2 (2 mM final concentration), 0.4 μl PCR nucleotide mix ultrapure deoxynucleoside triphosphates (dNTPs) (200 μM final concentration for each dNTP, PN 77119; USB, Santa Clara, CA), 0.5 μl Platinum Taq DNA polymerase (2.5-U final concentration), and 2 μl primer blend (rpoB, katG, pncA, gyrA, or rrs gene; 0.4 μM final concentration for each primer). To each 34-μl master mix reaction mixture, 16 μl extracted DNA was added to bring the total volume to 50 μl. Reactions were carried out in MicroAmp optical 96-well reaction plates (PN N801-0560; Life Technologies) and capped using MicroAmp 8-cap strips (PN 4323032; Life Technologies). Amplification was performed using an ABI 2720 thermocycler (Life Technologies). Thermocycling parameters were 95°C for 2 min, followed by 40 cycles at 95°C for 30 s, 55°C for 15 s, and 72°C for 2 min, with final extension at 72°C for 5 min. Resulting amplicons were confirmed by addition of 5 μl PCR product with 1 μl GelPilot loading dye, 5× (PN 1037649; Qiagen), on 1% (wt/vol) molecular-biology-grade agarose (catalog no. BP1356; Fisher Scientific, Pittsburgh, PA) with ethidium bromide (0.1-μg/ml final concentration, catalog no. 161-0433; Bio-Rad, Hercules, CA). Electrophoretic separation of products was carried out for 60 min at 0.4 mV/cm2 in 1× Tris-borate-EDTA (TBE) buffer (catalog no. 1B70153; IBI Scientific, Peosta, IA). Amplicons were visualized under UV transillumination, and size estimation was made using a TrackIt 1 kb Plus DNA Ladder (PN 10488-085; Life Technologies). After visualization, the remaining PCR mixtures for each clinical isolate gene amplification (∼45 μl), corresponding to rpoB, katG, pncA, gyrA, and rrs (16S) targets, were transferred to a single microcentrifuge tube. Pooled genes corresponding to each clinical isolate were subjected to PCR purification and eluted in 50 μl low Tris-EDTA (TE) (catalog no. 602-1155-010; Life Technologies) using the MinElute reaction cleanup kit (catalog no. 28204; Qiagen) according to the manufacturer's instructions. The concentration and purity of DNA were determined spectrophotometrically using a NanoDrop ND 1000 (Thermo Fisher Scientific, Wilmington, DE).
Ion Torrent library preparation.Bar-coded libraries were generated using the Ion Xpress Plus fragment library kit (catalog no. 4471269; Life Technologies) and the Ion Xpress DNA bar coding kit with adaptors 1 to 16 (catalog no. 4468654; Life Technologies) as described previously (10).
Amplicon shearing.Chemical shearing was performed using 1 to 3 μg DNA containing an approximate equimolar pool of rpoB, katG, pncA, gyrA, and rrs (16S) gene amplicons. DNA shearing was performed in a 50-μl total reaction volume by combining 5 μl Ion Shear Plus 10× reaction buffer, 10 μl enzyme, and 35 μl pooled DNA template (Ion Xpress Plus fragment library kit, catalog no. 4471269; Life Technologies). The reaction mixture was incubated at 37°C for 45 min, the reaction was terminated using 5 μl Ion Shear stop buffer, and the mixture was stored on ice until purification. Sheared DNA was purified using Agencourt Ampure XP-PCR purification beads (PN A63880; Beckman Coulter, Brea, CA) with a Dynal magnetic bead stand (catalog no. 123-21D; Life Technologies) according to the manufacturer's recommendations. Briefly, 99 μl Agencourt beads was mixed with 50 μl Ion Shear reaction mixture, incubated for 5 min at room temperature, placed on a magnetic stand, washed twice with 70% (vol/vol) ethanol, and eluted using 12 μl low-TE buffer (catalog no. 602-1155-010; Life Technologies).
Adaptor ligation.Adaptor ligation was performed in a 0.2-ml low-binding PCR tube (PN PCR-02-L-C; Axygen Inc., Union City, CA) by combining 12 μl sheared amplicon with 1.25 μl ligase buffer, 1.25 μl P1-IA adaptor mix (Ion DNA bar coding kit with adaptors 1 to 16; Life Technologies), and 0.2 μl DNA ligase (Ion Xpress Plus fragment library kit). The mixture was pipetted up and down 5 times and incubated at room temperature (22 to 25°C) for 30 min. Adaptor ligation reactions were purified and eluted in 10 μl low-TE buffer using Agencourt Ampure XP-PCR purification beads (Beckman Coulter) with the Dynal magnetic bead stand (Life Technologies) according to the manufacturer's recommendations.
Nick translation and bar code amplification.Amplicon pools from each patient sample were bar coded using the Ion DNA bar coding kit with adaptors 1 to 16 and Ion Xpress fragment library kit (Life Technologies). To maximize yields, reactions were scaled 2× by combining 40 μl Platinum PCR SuperMix High Fidelity, 4.4 μl Ion primer mix (BC X, where X = bar codes 1 to 16), and 10 μl ligated DNA. Amplification was performed using an ABI 2720 thermocycler (Life Technologies). Thermocycling parameters consisted of 72°C for 20 min and 95°C for 5 min, followed by 10 cycles at 95°C for 15 s, 58°C for 15 s, and 68°C for 1 min. Following amplification, bar-coded samples were purified and eluted in 50 μl low-TE buffer (Life Technologies) using the MinElute reaction cleanup kit (Qiagen) according to the manufacturer's instructions. DNA concentration and purity were determined spectrophotometrically using a NanoDrop ND 1000 (Thermo Fisher Scientific). Equimolar concentrations (∼2 to 3 μg of each bar-coded sample) were combined into a single 1.5-ml nuclease-free microcentrifuge tube and subjected to size selection.
Size selection.The appropriate volume of GelPilot 5× loading dye (PN 1037649; Qiagen) was added to the pooled bar-coded M. tuberculosis library tube and loaded onto a 1% (wt/vol) agarose gel (catalog no. BP1356; Fisher Scientific, Pittsburgh, PA) containing ethidium bromide (0.1-μg/ml final concentration, catalog no. 161-0433; Bio-Rad, Hercules, CA). The bar-coded library was electrophoresed for 60 min at 0.4 mV/cm2 in 1× TBE buffer (catalog no. 1B70153; IBI Scientific, Peosta, IA) and visualized under UV transillumination. Size estimations were determined using a TrackIt 1 kb Plus DNA ladder (Life Technologies). Gel excision was performed under UV transillumination using a sterile scalpel blade, excising out a target region between 75 and 200 bp. Excised agarose gel slices were placed into sterile 1.5-ml microcentrifuge tubes and subjected to DNA purification using the PureLink quick gel extraction kit (catalog no. K210012; Life Technologies) according to the manufacturer's instructions. Concentration and purity values for the bar-coded DNA library were determined spectrophotometrically using a NanoDrop ND 1000 (Thermo Fisher Scientific). The recommended library input for emulsion PCR is ∼140 × 106 to 560 × 106 molecules per 18 μl. This range was achieved by a 1:1,000 dilution using library stock and nuclease-free water.
emPCR.Emulsion PCR (emPCR) was performed in a 1-ml reaction volume using the Ion template preparation kit (catalog no. 4469000; Life Technologies) by adding 582 μl nuclease-free water, 200 μl 5× PCR reagent mix, 100 μl 10× PCR enzyme mix, 100 μl Ion Sphere particles (ISPs), and 18 μl diluted library template. The preparation was mixed thoroughly, followed by brief centrifugation in a microcentrifuge. Emulsion was achieved using the Ultra-Turrax tube drive (Life Technologies). A total of 9 ml chilled emulsion oil (Ion template preparation kit; Life Technologies) was added to an Ion template preparation tube (catalog no. 4467226; Life Technologies). The emulsion tube was placed and locked onto the IKA Ultra-Turrax tube drive and initiated. While the tube was in motion, the entire 1-ml PCR master mix solution was dispensed into the cap port and mixed for 5 min. The mixed emulsion was transferred to a 96-well PCR plate and amplified using an ABI 2720 thermocycler (Life Technologies) using the following thermocycling parameters: 94°C for 6 min, followed by 40 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 90 s and then 5 cycles at 94°C for 30 s and 68°C for 6 min.
ISP recovery and qubit measurement.Ion Sphere particles were recovered using reagents supplied in the Ion Xpress template kit (catalog no. 4469001; Life Technologies) according to the manufacturer's protocol (Ion Xpress template kit user guide, version 2.0, pages 18 and 19). Quantification of recovered particles was performed using a Qubit 2.0 fluorometer (Life Technologies) and an Ion Sphere quality control kit (catalog no. 4468656; Life Technologies) according to the manufacturer's recommendations (Ion Xpress template kit user guide, pages 25 and 26). The optimal amount of template-positive ISPs is between 4 and 50%. Relative fluorescent unit (RFU) values obtained outside this range were not pursued into subsequent ISP enrichment.
ISP enrichment.Enrichment of ISPs was achieved using reagents supplied in the Ion Xpress template kit, Ion sequencing kit, and DynaBeads MyOne streptavidin C1 beads (catalog no. 4469001, 4468997, and 650.01, respectively; Life Technologies) according to the manufacturer's protocols (Ion Xpress template kit user guide, version 2.0, pages 15 to 17).
Ion Torrent 314 chip preparation and PGM sequencing.Ion Torrent 314 chips (catalog no. 4462923; Life Technologies) were prepared and loaded according to the manufacturer's recommendation (Ion sequencing kit user guide, version 2.0). The Ion Torrent PGM was run according to Ion Torrent 314 chip specifications, including a 65-cycle sequencing protocol, use of 18 megaohms purified water, and standard compressed argon gas to drive fluidics through the PGM system. For each sequencing run, up to 16 clinical samples consisting of full-length genes (rpoB, katG, pncA, gyrA, and rrs) can be placed on a single 314 chip. Depending on the number of bar codes used, i.e., 1 to 16, a typical sequencing run produced an average base pair read length of 60 to 70 bp, a total of 250 to 300,000 high-quality (AQ20) reads, and gene coverage of 300 to 500 times.
Phenotypic and genotypic results.Amino acid characterizations of 26 M. tuberculosis isolates by Ion Torrent sequencing of the rpoB, katG, pncA, gyrA, and rrs (16S) genes are summarized in Tables 2 to 6, respectively, and compared to results obtained by Bactec MGIT 960 (phenotypic) and/or Hain GenoType MTBDRplus (genotypic) LPA. Of the 26 M. tuberculosis clinical isolates, 14 (54%) were MDR, 7 (27%) were XDR, and 5 (19%) were susceptible to drugs by Bactec MGIT 960 phenotypic analysis. The Ion Torrent PGM sequencing method showed 100% (26/26) concordance to both phenotypic resistance obtained by MGIT 960 culture (Tables 2 to 6) and genotypic rpoB and katG data obtained by Hain LPA (Tables 2 and 3).
rpoB gene mutations.A total of 10 rpoB amino acid substitutions were identified in the 26 clinical isolates compared to the H37Rv wild-type strain. The common S531L mutation was the most prevalent, but mutations at positions 516 and 526, also known to confer resistance to rifampin, were observed (Table 2). Additionally, mutations were observed within the rpoB open reading frame but outside the 81-bp rifampin resistance-determining region (RRDR) (Table 2). The V194I mutation observed outside the RRDR in one strain is a unique substitution that is likely not associated with rifampin resistance. Five amino acid substitutions were noted in at least one strain beyond residue 900 of the RpoB protein. There were seven strains with an RpoB mutation (6 at position 516 and 1 at position 526) where a wild-type band was absent without a corresponding mutation band according to LPA. In six of these seven isolates, Ion Torrent sequencing revealed an uncommon amino acid substitution (i.e., glycine) within a known mutation site at position 516, where a valine substitution (D516V) is typically known to occur (Table 2). Similarly, in one isolate Ion Torrent sequencing revealed an arginine within a known mutation site at position 526 where tyrosine or aspartic acid substitutions (H526Y/D) typically occur.
katG gene mutations.Four amino acid substitutions were observed in the katG gene product, with S315T, which is known to confer isoniazid resistance, present in all resistant strains (Table 3). Clinical strains harboring R463L, W191R, and N138H mutations were detected by DST (Table 3) and have been previously characterized. A substitution at position 463 (R463L) in the katG product has been previously shown to have no effect on antibiotic resistance and can be used to categorize M. tuberculosis isolates into genetic groups 1 (Arg463) and 2 or 3 (Leu463). Of the 26 clinical isolates assessed, 7 (27%) were members of genetic group 1 as evident by this R463L substitution.
pncA gene mutations.Seven nucleotide mutations were noted in at least one strain among 561 bp comprising the full-length coding region for the pncA gene (Table 4). Nine of 26 strains (34.6%) contained an amino acid mutation conferring pyrazinamide (PZA) resistance (Table 4). In one strain, a silent (synonymous) nucleotide mutation was characterized at a position corresponding to amino acid 195 (C195T). Five strains contained previously characterized amino acid substitutions (C14R, A102V, V139G, R154G, and L172P) known to confer resistance to pyrazinamide. A novel mutation, not previously reported elsewhere, including a termination codon was found in one isolate at residue 122 (Q122Stop) in the PncA protein (Table 4).
gyrA gene mutations.Nine unique mutations were observed in the 2,517-bp full-length gyrA gene encoding subunit A of the DNA gyrase enzyme. Resistance to fluoroquinolones (FQs) was noted only in strains harboring mutations in the quinolone resistance-determining region (QRDR) defined by substitutions in gyrA at positions corresponding to amino acids 88, 90, and 94. A number of additional mutations were also observed in regions outside the QRDR, including two "mixed strain" mutations at positions 549 and 613 in the GyrA protein (Table 5). Mutation at position 95 (S95T) is known to have no effect on FQ resistance but can be used to categorize strains in genetic group 2 or 3. Of the 19 total clinical isolates belonging to genetic groups 2 and 3, 18 (96%) were group 2 and 1 (4%) was group 3 according to assessment of GyrA position 95 (T indicates genetic group 2 and S indicates genetic group 3).
rrs (16S) gene mutations.Four nucleotide mutations were noted among the 1,540 bp comprising the full-length 16S rrs gene. Seven of the 26 (27%) clinical isolates were shown to be resistant to aminoglycosides by DST, and all strains harbored an A1401G mutation known to confer resistance (Table 6). Two other amino acid mutations (C492T and A514C) were observed but have been previously shown to not inhibit aminoglycoside efficacy. A previously uncharacterized G878A nucleotide mutation was observed, but the isolate was shown to be susceptible according to DST (Table 6).
This study describes the first standardized protocol utilizing Ion Torrent sequencing of full-length genes to characterize MDR and XDR M. tuberculosis strains. The protocol described in this report enables sequencing of entire coding regions implemented in M. tuberculosis resistance, allowing characterization of known mutations and discovery of new polymorphisms.
Rapid analysis of genes associated with drug-resistant strains is a major challenge for successful treatment of tuberculosis. In addition, real-time geographical surveillance of emerging M. tuberculosis drug resistance would facilitate more appropriate antibiotic treatment strategies. Currently, available molecular methods such as the GenoType MTBDRplus LPA offer limited detection capabilities, particularly when novel or uncommon amino acid substitutions are within known drug resistance regions or when undiscovered amino acid mutations impact drug resistance. We have established a simplified Ion Torrent sequencing protocol for rapid characterization of five full-length genes (cumulatively sequencing 11.4 kb per isolate) for determining drug resistance in MDR and XDR strains.
The M. tuberculosis rpoB gene encodes the 1,178-amino-acid beta subunit for a DNA-dependent RNA polymerase enzyme. Mutations within an 81-bp "core region" of the rpoB gene are responsible for approximately 95% of rifampin resistance in M. tuberculosis strains (24). Three of these mutations, yielding substitutions at positions 516 (D→V), 526 (H→Y/D), and 531 (S→L), constitute the majority of mutations within this region. Of the 21 rifampin-resistant strains characterized in this study, 11 (52.4%) carried the S531L mutation, 7 (33.3%) contained an amino acid substitution at position 516, and 3 (14.3%) contained a mutation at position 526 of the rpoB gene product (Table 2). The most prevalent RpoB substitution observed at position 516 is a valine (D516V) (26). Interestingly, Ion Torrent sequencing revealed that 6 of 7 strains contained a rarer glycine residue (D516G) at this position (Table 2). Using LPA, these 6 strains were determined to be resistant, since both mutant and wild-type bands were absent (Table 2). The D516G mutation occurs less frequently than D516V, is not clustered or epidemiologically significant, and has been reported to occur in isolates characterized from China, Africa, and North America (7, 12, 23). Similarly, an uncommon amino acid substitution was identified at position 526 in the rpoB gene product. The most prevalent amino acid substitution reported at position 526 in the rpoB gene product is a change from histidine to tyrosine or aspartic acid (H526Y/D) (24, 26). However, Ion Torrent sequencing revealed that 1 of 3 isolates contained an uncommon arginine residue (H526R) that by Hain LPA was shown to be absent for both wild-type and mutant bands (Table 2). While the absence of wild-type and mutant bands in a sample is interpreted as resistant according to LPA testing (6), there remains ambiguity, since the type of amino acid change is not directly characterized. The absence of wild-type and mutant bands as an indication of drug resistance using the LPA test could possibly give a false-positive result if different amino acid residues in known amino acid positions affect resistance or levels of resistance to a particular drug. This underscores the utility of Ion Torrent sequencing for directly detecting specific amino acid substitutions in positions known to confer resistance, as well as discovery of novel amino acids outside known sites that may impact resistance.
The katG gene encodes catalase peroxidase, an enzyme that converts isoniazid (INH) into the active form. The majority of isoniazid resistance is associated with the katG codon corresponding to amino acid 315 (S315T) (9), although mutations in the promoter region of inhA and nod also contribute to resistance (9, 15). Of the 26 strains assessed, 16 contained the characteristic serine-to-threonine amino acid substitution at position 315 (S315T) conferring isoniazid resistance (Table 3). These sequencing results exhibited 100% concordance with comparisons made using the Hain LPA and culture DST.
Pyrazinamide (PZA) is a synthetic derivative of nicotinamide that has been used as a first-line drug to fight tuberculosis since 1952. Standard DST for PZA is complicated due to an acidic pH requirement in vitro, which inhibits M. tuberculosis growth and complicates accurate phenotypic assessment. PZA resistance is attributed to mutations in the pncA gene, which encodes a pyrazinamidase. However, these resistance-conferring mutations are numerous and consist of amino acid substitutions, frameshifts, and stop codon mutations (13). Seven mutations were characterized from the 26 South African isolates assessed, including one silent mutation, five amino acid substitutions, and one chain termination mutation. The Q122Stop termination mutation (Table 4) observed in one isolate is novel, having never been reported elsewhere. The difficulty in PZA phenotypic assessment and the variability of mutations along the pncA gene highlight the use of Ion Torrent gene sequencing to assess mutations in this hypervariable M. tuberculosis gene.
The primary target of fluoroquinolones (FQs) in M. tuberculosis is DNA gyrase, a type II topoisomerase composed of two A and B subunits encoded by the gyrA and gyrB genes, respectively (2). Amino acid substitutions located within a short region of the gyrA gene known as the quinolone resistance-determining region (QRDR) account for the majority of known FQ-resistant M. tuberculosis strains (22). Substitution mutations in the QRDR at positions 88, 90, and 94 were observed in 10 of 26 sequences from this study (Table 5). Three of the 10 strains contained substitutions at position 94 in the gyrA gene product; two were noted as D94G substitutions, and one was a D94Y substitution. A previous study characterized both D94G and D94Y substitutions and demonstrated that these amino acid substitutions at position 94 give rise to similar levels of FQ resistance (3). Among the strains assessed in this study, the gyrA gene was the most variable, yielding nine amino acid substitutions in the 26 clinical isolates assessed. Furthermore, two of these gyrA codons (corresponding to amino acids 549 and 613) exhibited heterogeneous residues (Table 5), an advantage of performing Ion Torrent sequencing over Hain LPA and DST.
Emerging cases of XDR tuberculosis, defined as MDR cases having acquired additional resistance to FQs, e.g., ofloxacin, and at least one of the second-line injectable drugs, e.g., amikacin (AMK), kanamycin (KAN), or capreomycin (CAP), have become a public health threat in developing countries worldwide (11). The majority of resistance to second-line drugs is associated with mutations in codons corresponding to amino acids 1401 (A1401G), 1402 (C1402T), and 1484 (G1484T) in the 16S rRNA rrs gene (11, 21). Analysis of African M. tuberculosis strains revealed that 7 of the 26 (27%) were defined as XDR as evident by mutation at position 1401 (A1401G) (Table 5). Three additional nucleotide mutations affecting positions 492, 514, and 878 were also discovered (Table 6) in strains from this analysis. G878A is a novel mutation, but the strain carrying it was shown to be susceptible to AMK, KAN, and CAP according to DST (data not shown). The overall cumulative impact of these newly discovered mutations on drug resistance remains to be established.
Previous studies have shown that mutations in the katG codon corresponding to amino acid 463 and the gyrA codon corresponding to amino acid 95 are genetic markers for categorizing strains into epidemiological genetic groups 1, 2, and 3 and that these codons have no effect on antibiotic resistance (16, 18). Group 1 strains are genetic ancestors of group 2 and group 3 strains that link the predominately nonhuman Mycobacterium species (M. microti and M. bovis strains) with human M. africanum and M. tuberculosis lineages (21). As evident by substitution mutations in the katG codon corresponding to amino acid 463 and in the gyrA codon corresponding to amino acid 95, a total of 7 of 26 (27%), 18 of 26 (69%), and 1 of 26 (4%) African isolates characterized in this study were members of genetic groups 1, 2, and 3, respectively. Tracking group 1 organisms is important in terms of M. tuberculosis detection, since several isolates belonging to genetic group 1 lack insertion sequence 6110 (IS6110) (18), a common genetic target for several PCR-based M. tuberculosis detection assays.
The purpose of this article was to describe the development of an Ion Torrent protocol for evaluating genotypic drug resistance using clinical isolates. In this study, we characterized a very limited set of MDR, XDR, and drug-susceptible clinical isolates from two provinces in South Africa. We intend to apply our sequencing methodology to a much larger prospective study using samples collected from Kwa-Zulu Natal Province in South Africa. Evaluation of a larger sample size will further validate the developed methodology and make possible detection of uncommon or novel mutations in the full coding regions of these five genes.
One inherent concern with performing full-gene sequencing, especially from geographically diversified samples, is the discovery of novel mutations that may have no impact on drug susceptibility. Conversely, detected mutations outside known resistance sites may cause complete resistance or an additive resistance when combined with other mutations. Thus, genotypic identification of mutations through full-gene sequencing is the first step in understanding the complete resistance picture. However, genotypic discovery might be complemented and validated by subsequent phenotypic MIC studies.
There is potential for the Ion Torrent instrument and described methodology to be integrated into selected regional and reference settings throughout Africa, India, and China. In contrast to most Sanger sequencers, the Ion Torrent does not utilize (expensive and maintenance-requiring) lasers that typically require modified fluorescence-based, light-sensitive chemistries, and it has a much smaller overall footprint. Furthermore, our described methodology does not require expensive ancillary equipment such as the Agilent 2100 BioAnalyzer, the Ion OneTouch system, ultracentrifuges, or the Pippin Prep Workstation as current Ion Torrent protocols recommend. This is significant, since these instruments and needed accessories and consumables can be expensive, require large laboratory footprints, and often require routine maintenance. Additionally, since our protocol employs an initial PCR amplification for full-length genes (not full genomes), we do not require the DiaGenode Bioruptor Sonication System for DNA shearing. When clinical isolates derived from traditional detection methods require further genotypic drug susceptibility characterization, they can safely be inactivated in PrimeStore MTM for extraction and PCR amplification in a standard molecular biology laboratory setting prior to Ion Torrent sequencing. Using our described methodology for characterization of 5 full-length genes, between 1 and 16 patient libraries can be sequenced and analyzed within 2 days for $75 to $80 per patient; these costs will likely be reduced as the technology matures.
In contrast to the GenoType MTBDRplus or MTBDRsl LPA, the Ion Torrent PGM protocol reported here provides full-length characterization of genes, making possible discovery of new amino acid substitutions that could potentially be missed by LPA, since LPA is limited to only known mutations. Using the protocol reported here, several uncommon amino acid changes in clinical field isolates have been found. Furthermore, the extensive depth of sequence coverage from the Ion Torrent allows for discovery of heterogeneous or mixed-strain genetic populations within an isolate.
The scalability of Ion Torrent sequencing permits expansion to include megabases of additional genes on a single chip. As a proof of principle with five full-length M. tuberculosis genes, our methodology could easily be expanded to include all 16+ genes that currently constitute M. tuberculosis drug resistance. Full-length gene analysis using the Ion Torrent PGM may identify novel mutations that, when correlated to phenotypic MIC testing, could identify new M. tuberculosis resistant residues as well as the cumulative inhibitory effect of multiple mutations.
This work would not have been possible without the efforts of Chris Helm and Susan Graham, who facilitated international planning meetings, travel, and teleconferences between U.S. and South African collaborators. We thank Richard Tamfu for his critical review of the manuscript. We are also appreciative of Matthew (Matt) Keyser from DNAStar Inc. (Madison, WI) for his bioinformatics assistance.
- Received 18 July 2012.
- Returned for modification 9 August 2012.
- Accepted 5 September 2012.
- Accepted manuscript posted online 12 September 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.


text: South facing Villamartin Plaza Penthouse with private terrace and excellent rental potential. Villamartin offers a wide range of services and this apartment is conveniently situated close to shops, bars, restaurants and Villamartin Golf Course. As well La Zenia Boulivard shopping centre and blue flag beaches of La Zenia are easily reached either by car or public transport. There are 3 further championship golf courses in the area and the property is approximately 50 minutes from Alicante Airport and 40 minutes from Murcia airport.

subset: commoncrawl


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The First Amendment to our Constitution seems to have disappeared. It appears we  lost it on June 27, 2009.
In an LA Times interview with California State Assembly Speaker Karen Bass is asked the question, "How do you think conservative talk radio has affected the Legislature's work?"
The Speaker answers with this gem…
The Republicans were essentially threatened and terrorized against voting for revenue. Now[some] are facing recalls. They operate under a terrorist threat: "You vote for revenue and your career is over." I don't know why we allow that kind of terrorism to exist. I guess it's about free speech, but it's extremely unfair.
Its unfair… Its terrorism. Â Oh yes we don't want competition for these jobs of running the state of California and we certainly don't want any Constitutional Ballyhoo.
Ok for all you Constitutional slackers let's look at the 1st Amendment.
Congress shall make no law respecting an establishment of religion, or prohibiting the free exercise thereof; or abridging the freedom of speech, or of the press; or the right of the people peaceably to assemble, and to petition the Government for a redress of grievances.
It appears that Bass doesn't believe in the right of the people to petition the Government for a redress of grievances; a right guaranteed by one of the most powerful documents ever created.
There's a lot of stupidity and tyranny locked into those few words. The First Amendment guarantees the right to petition the government for redress of grievances, even apart from the "free speech†issues Bass casually discards. Elected politicians are accountable to the people who elect them in a free society. Politicians do not acquire lordly status when they go to the Assembly, or anywhere else. The Hot Air Blog
How the hell is it that our "leaders" don't understand the foundation of the Government they work for? I guess it's because America aka the voters are so caught up in non-important matters as to ignore the particularly important ones.
Let's just look at the last week. TV News Devotes 28 Hours to Michael Jackson; 93% of Cable Airtime. While the media was eating up Michael Jackson's death they failed. The media missed Iran having a major upheaval over a tainted election. Congress is working on the largest tax of all time, a tax that will kill our economy. Obama is attempting to spend Trillions of dollars on social programs like government healthcare insurance. Many important issues are being swept aside by the mainstream media and in turn our 2 second attention spans and a society that is showing signs of missing a moral compass are not monitoring what is important to this country.
No one is watching our political leaders. Our political leaders are as confused as anyone as to right and wrong. Our leaders choose to cheat, lie and do whatever with impunity. We see failure to pay taxes, failure to promises and committments including family bonds. We as a society have lost our ability to choose right from wrong. We have become confused as to what is what. Our societies moral compass is broken. Unfortunately if we don't change, we will find bondage as the country as we are crushed under a massive debt created by a failure to choose good from evil.


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Whether it's customer information, sales records, or analytics, the protection and storage of data is essential to businesses today. When companies need to scale quickly or get more competitive, migrating that data into a new system or application can mean huge efficiency improvements. However, not only is data security at stake, but if a migration process goes awry the errors could be costly. Luckily, by keeping an eye out for these common mistakes you can focus on getting your data migration done successfully.
There are some common missteps in all three phases of a data migration: planning, validation, and execution.
Often we see data migration strategies based around assumptions about one or both systems involved. Your data migration team should begin with a thorough infrastructure audit. Fully take stock of all physical and virtual components, as well as network devices and general layout. Additionally, the team should lay out all relevant dependencies between programs and devices, so you know where the connections are. When a migration fails, many times it can be traced back to an update or change that was made to one part without considering the whole. These mistakes can be avoided by establishing clear leadership and well-defined goals.
Validation is a way to confirm beforehand that the migration plan will work. Basically, this means rigorous testing and in-depth analysis by all departments. Once your IT team has set up the plan and list of goals for a given migration, they can create a checklist for every department to follow. This checklist should ask them to refer back to the old application as well as test the new one. This will help them see if any gaps have been created. For example, if your company is undergoing a database migration, confirm that searches performed in the new system will produce the same information.
The validation and testing portions should consider other variables. How long will this migration prevent users from interacting with your materials? What is or could be the financial impact of going offline during this period? Say a bank has data that needs to merge with a new mobile app or customer-facing interface. What would a customer reaction be to a service outage? These kinds of questions may be easy to gloss over in discussion, but in practice, they represent some of the biggest risks.
Once all internal checklists and pre-migration testing has been completed, it's not as simple as just clicking a button. A migration is all about keeping many balls in the air at once–and keeping your eye on all of them. That means that there will likely be numerous changes and corrections even after the transfer has completed. Data migration testing tools like backwards compatibility verification also sometimes prove to be necessary.
Migrating data to a new system often supports businesses looking to become more efficient and streamlined. But even as it gets more common, stakeholders must plan with care to avoid risks or errors. If you're unsure of how to get started or how to be certain the process has been a success, contact iLAB today.


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Something called The Jack News writes:
A recent lawsuit in the Golden State has brought attention to an interesting, and mostly ignored, problem: California is simply too large of a state. …
But the lawsuit raises an enticing possibility: Has the time come to add more stars to Old Glory by breaking up the union's biggest states?
Splitting Up States
Most discussion about potential new states, focus on the longstanding desires of America's two most populous non-state territories: The District of Columbia and Puerto Rico. Statehood is a divisive question in Puerto Rico, however, and the status of the nation's capital city raises complicated questions of sovereignty that could likely only be solved by constitutional amendment.
Creating new states out of existing ones, on the other hand, has a long history. The framers of the Constitution included a clause specifically allowing for it, with the consent of both Congress and the state or states concerned. Vermont, Kentucky, Maine, and West Virginia were all carved out from existing states using this method.
Consent for West Virginia was, controversially, granted by the "restored" unionist government of Virginia during the height of the Civil War, but the legitimacy of that move has been long-settled. In each of the other cases, state legislatures responded to public demand by giving their consent, and Congress dutifully obliged. By exercise of democratic consent, America's sovereign states can divide themselves, and sometimes they have.
Goldilocks States
Proposed movements to split existing states have a wide history; but not all such proposals have much chance of gaining traction. Adding a new member to the union requires not just the consent of the state being broken up, but also Congress, and Congress has usually had some broad principles in mind.
The first, and most important consideration, is population. As the United States expanded westward across the continent, Congress always required a minimum population before a territory could apply for statehood. Since becoming a state comes with two senators and at least one representative regardless of population, Congress is unlikely to ever approve a state less populous than the current 50thlargest, Wyoming (585,501 as of 2016). This also happens to be reasonably close to the size of the average U.S. House district, which is just over 700,000.
That rules out most of America's territories outside of Puerto Rico and the District of Columbia, since the largest, Guam, has just under 160,000 residents. It likewise makes unlikely proposals like Superior, to make a new state from the upper peninsula of Michigan, or splitting a state like Colorado, which ranks 22nd in population of the states. The resulting states simply would not have enough people to justify the Constitution's minimum federal representation.
The Big Five
Because of this aversion to low-population states, it is probable that only the nation's largest and most populous states would be eligible for partition. Those also happen to be the states were movements for partition have been strongest, and a serious topic of debate over the years.
The five most-populated states and their 2016 population are, in order: California (39 million), Texas (27 million), Florida (20 million), New York (19 million), and Illinois (12 million). All are good potential candidates to carve out newer, smaller states.
California already hosts the oldest and most persistent partition movement, the would-be "State of Jefferson" that aspires to encompass far-northern California as well as adjacent parts of southern Oregon. There is also a persistent split between the central part of the state, centered around the Bay Area, and southern California centered around Los Angeles.
A recent proposal to split California into six states ended up failing and folding, largely because six was too ambitious a number, and the proposed borders too arbitrary. A state split into the three parts, however (with the northernmost possibly picking up part of Oregon) would neatly follow the existing political and cultural divisions.
Jefferson already has a suitable name, and several counties in the region have approved of it in referendums. There are many potential options for naming the other two; but for simplicity's sake let's call them North California and South California. Alternately, geographical features like "Sierra" or "Mojave" could provide each with a fresh start and a distinct identity.
Texas is unique in a lot of ways, but one of the most unusual is an obscure bit of trivia tucked into the joint resolution annexing the then-independent Republic of Texas in 1845. In order to sweeten the deal, Congress offered its pre-approval to for Texas to split itself into as many as five states.
Whether or not that offer still stands is unclear and debated among constitutional scholars. Texas has seen many partition proposals, but none ultimately overcame the state's uniquely strong sense of identity.
The sprawling state encompasses many distinct regions; but the most common proposals have centered around the southwest portion of the state nearest the Mexican border. After the Civil War, it was briefly proposed to create a state named Lincoln from the parts of Texas between the Rio Grande and the Colorado River, roughly dividing the state in half.
A similar modern proposal, could perhaps result in a state of Rio Grande, extending from Brownsville to El Paso, and with its capital in San Antonio. The result would leave the more predominately Latino and Democratic parts of the state, free to govern themselves without being dominated by the rest of Texas's overwhelming Republican supermajority.
Florida is another state where the cultural and political divide is stark between two regions: the predominantly conservative and Republican northern half of the state, versus the more Hispanic and Democratic south centered around Miami.
The idea of division has attracted enough support to be endorsed by county legislators in Ft. Lauderdale, and a handful of state legislators. It's less clear where the border would be drawn. Which side would get Orlando, Tampa, and Cape Canaveral?
Central Florida is a culturally mixed region, politically purple, and home to some of the state's most famous and valuable assets. While residents of the conservative panhandle on the one hand, and liberal Miami on the other, might relish being free of the other, there is no clear dividing line between them. Where exactly to draw the border, would probably end up being a contentious, and potentially unsolvable question.
If a fair border could be agreed upon, America's most famous swing state might instead become one reliably red state and one reliably blue state. A repeat of the 2000 election fiasco, where a few hundred votes decided the state and thus the presidency, would be very unlikely.
New York
The Empire State has always housed two very distinct regions: the metropolis in and around New York City, and the rural bulk of the state's landmass that has become known as Upstate New York.
On both sides of that divide, frustrations run high and proposals for a split have been persistent for decades. Upstaters chafe at the domination the city exercises by having the majority of the population, while residents of the five boroughs have little connection to places like Buffalo and Rochester.
If there is any metropolis that can make the case for its own statehood, it would be New York City's eight million inhabitants. Extending that to include Long Island and the adjacent counties immediately north of the city, would push the population north of ten million.
Which half would get to keep the name "New York" could be debated, but to avoid confusion it would probably be best if that name stuck with the city that shares it. The rural rest of the state, perhaps picking a new name based on geography or the local Native American tribes, could govern itself without being overshadowed by the metropolitan neighbors. The state of Adirondack, or Mohawk, or Erie, or Hudson, could even put the question up to vote. After all, "upstate" would no longer work as a description.
Illinois suffers from a similar dynamic as New York: a single dominating metropolis at one end, split from the predominantly rural and politically conservative rest of the state. Chicago's sense of identity is drastically distinct from the rest of Illinois, to the degree that the city's distinctive flag is a much more common sight on its streets than Illinois's banner.
Partition already has a degree of perennial support, with legislators from both Chicago and "downstate" introducing bills to that effect in the legislature. The political calculus is obvious: Democrats who dominate Chicago could firmly control any new state built around it, and Republicans consigned to permanent minority status in southern and central Illinois would instead have a solidly red state to govern.
It's not just politics, of course. There is a real social and cultural divide between diverse and cosmopolitan Chicago, and the vast stretches of small towns and farmland that occupy the rest of Illinois.
A recent survey even found that Illinois was the only state where a majority of its residents said they would move to a different state if they could. They might just have that option, without having to move at all. Like the Czech Republic and Slovakia, Illinois and Chicago could go through a velvet divorce that leaves both happier.
Two by two in the Senate
In the era before the Civil War, it was common practice to admit new free states and new slave states together in pairs, to maintain the precarious balance of power in the Senate. That is a practice that could be revived in the 21st Century, albeit in the much less troublesome context of partisan politics, instead of human bondage.
Past election results provide an easy and fairly reliable guide to guess the partisan tilt of any new state. Pairing likely-Republican and likely-Democratic states would be necessary to secure bipartisan buy-in at all levels of the decision, and solid majorities in favor in both the state and national legislatures.
The State of Jefferson, for example, would most likely give its two Senate seats as well as its electoral votes to the GOP. Splitting the rest of California in two, would produce two Democratic states from what had previously been one. Thus the net effect would be to add two Senators for both parties.
Florida, currently has one Republican Senator and one Democratic Senator. A split state would, most likely, send two from each party, for a neutral net effect. This same calculations can be played out elsewhere. Two new Republican Senators from upstate New York, could balance two new Democratic Senators from southwest Texas.
That kind of partisan horse-trading might sound unseemly, but it could be the necessary grease to arrive at a good policy result: state governments that are more representative, effectively providing localized laboratories of democracy. More states means more experimentation, and a stronger sense of federalism across the country.
There's nothing magic about 50 states and 100 senators. It's more than time for us to reopen the political debate about state sizes. Admitting six new states into the union, for the first time since 1959, would solve a multitude of state problems without upsetting our current national political balance.
David Blaska suggests the opposite, and has a map to match:
No county has ever merged with another. Now, apparently, Ozaukee and Washington Counties are raising the possibility. Both counties are populous (Ozaukee: 88,314; Washington: 134,296). Both counties are wealthy Republican exurbs of Greater Milwaukee. Which means they recognize the value of a dollar and are not in thrall to More and Bigger Gummint.
WTMJ-4 reports that Washington County, faced with budget troubles, is "thinking out of the box [including] dissolving county lines and completely merging with Ozaukee County.
"I know that if we go down this path, that guys like me don't have a job but I'm good with that," the Washington County administrator said.
His County has already saved $300,000 by merging its health department with Ozaukee and another hundred grand merging with the Waukesha County medical examiner.
The Washington County Administrator sent a letter to four of its neighboring counties, letting them know about their fiscal health status.
Of Wisconsin's 72 counties, 71 had been formed by 1901 when most people moved by horseback. The Menominee reservation was carved out of Oconto and Shawano counties in 1959 to create its own county, in an experiment that failed. It was intended to wean it off reservation status but 35% of the 4,533 population are mired in poverty. Florence County (population: 4,423) and Menomonee (population: 4,232) are the two-least populous counties and have not a single incorporated village or city. For comparison purposes, Dane County is home to 531,273 (2010 Census).
The stickler is that all counties must provide the same array of services: law enforcement, roads, courts, health, general welfare, property records, etc. It's a matter of economy of scale.
No county has ever merged, but they can
State law, specifically Chapter 59.08, provides for "consolidation" of counties, which is put to referendum. Let's explore consolidation in low-population southwest Wisconsin, now divided among Grant (population: 52,214), Iowa (population: 23,654), and Lafayette (population: 16,753) for a combined 92,621. Lafayette was cleaved from Iowa County in 1846. You could argue: leave Grant alone. It's large and fairly populous, but let's go with the three-fer for this example.
County boards would take the lead. But if Lafayette's county board remained silent, for instance, voters could petition for a referendum in their county. If its board still dragged its heels, a judge would appoint five citizens to work out the details with the merger partners. If the referendum on consolidation fails in any one of the counties, the whole deal goes down. Although long state law, no county has ever merged. Instead, most of our counties were cleaved from existing counties — originally just three in 1818: Crawford in the west, Brown in the east, and (encompassing the far north and the Upper Peninsula of Michigan) Michilimackinac.
The hard, cold numbers may argue for consolidation but emotions could scuttle the whole thing. First, what to call the new, consolidated county? Nothing wrong with "Iowa-Lafayette" county unless the Lafayetters insist on "Lafayette-Iowa." You see the problem! But the three-county merger we have imagined includes Grant County. That calls for a completely new, neutral name. "Driftless County"? "Lead County"?
Who gets the courthouse?
But it raises the question even more likely to sabotage merger than the consolidated county's name: which city gets the county seat?
That question is particularly poignant in our three southwest county seats. Lancaster, Dodgeville, and Darlington have glorious old courthouses and, frankly, not a whole lot else going for them. Could some functions continue in each courthouse? The law specifies that the courts be consolidated at the county seat. But could the referendum specify all three as county seats? Our reading of 59.08 does not seem to rule out that possibility.
Existing sheriff's facilities could continue as precinct houses. Dane County has three such sheriff's precincts outside of Madison. If new, modern jails would required, the merged county would need build only one, not three.
We could find no authorization to carve up one county between two neighbors, even though it would seem to make sense in some cases.
i am a resident of one of those indigo-colored counties (or is that periwinkie?), which, I was told upon full-time arrival 29 years ago, was larger than the state of Rhode Island. Since Blaska concedes that maybe Grant County shouldn't be part of Tri-Indigo County, reuniting Iowa and Lafayette counties (the latter was created from the former before statehood), that would create a county larger in land area than Grant County, and yet would be smaller in population.
An outstanding weekly newspaper carried Blaska's column and the editor's brilliant response. (Beginning with the possibly surprising fact that Wisconsin has fewer counties than all our neighbors.) Whether or not you like Blaska's idea generally or map specifically, certainly this state needs fewer, not more, lines between providers of government services. My favorite (if you want to call it that) example is the Fox Cities, which runs roughly from Neenah to Kankauna and includes parts of three counties, four cities, four villages, seven towns and several police departments and fire departments, despite the fact that two houses next to each other in the same neighborhood could be in different municipalities. The Fox Cities is a demonstration of how, if not why, this state has 3,120 units of government, second only to Illinois.
Somewhere in the mists of time is probably evidence that I once proposed merging towns that include cities within the original town boundaries — such as the City of Ripon and the Town of Ripon given that the latter has a police department whose purpose is only to generate town revenue by writing speeding tickets. Of course, people live in towns instead of cities because the former has something the latter lacks in their opinion. Usually, lower taxes.
The tricky subject is getting politicians to voluntarily merge their units of government and thus lose some of their political power. It's not very federalist to mandate, for instance, Blaska's map(re)making. But having 3,120 units of government fits no one's definition of good government.


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The parent of Chili's is launching a diner loyalty program and a line of frozen grocery items — both moves designed to extend the reach of the chain that turns 40 next year.
Executives with Dallas-based Brinker International, which also owns Maggiano's Little Italy, discussed the moves with analysts Thursday.
Brinker, which ended its 2014 fiscal year June 25, said putting aside nearly $40 million to settle a long-playing class-action suit lowered its fourth-quarter profit nearly 40 percent.
Brinker profit was $28.8 million, or 43 cents a share, down from $46.4 million, or 64 cents a share a year earlier.
The class-action case was filed by California workers in August 2004 regarding breaks and meal periods. A preliminary settlement reached Wednesday "seeks to resolve all claims in exchange for a maximum settlement payment not to exceed $56.5 million," the company said in a prepared statement.
The agreement is subject to court approval.Read the Full Article


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Please complete the Model/Photo Release Form as we often photograph our Free Teen Programs and Events, for promotional purposes.


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Tag: New Books
A Special Event at KP Library / Learning Commons
On January 26th and 27th, English classes excitedly piled into KP Learning Commons to greet an author, and talk to him about his recently published novel. What made this event very special, was that the author was non other than our very own, Mr. Blenman! Yes, that's right, Mr. Blenman, Inter-A teacher and wrestling coach,… Read More ›
Summer Reading Preparation
Summertime for voracious Kwantlen Park readers can be a little tricky. Classes are winding down and the school library will soon be closing its doors for the summer. If you aren't already involved in Surrey Library programs, now is the time! Surrey Library cards are free and the librarians are only too happy to give… Read More ›
More New Books!
The Fine Art of Truth or Dare by Melissa Jensen This is a straight-up, sweet romance. Readers of YA romance will delight in this book. Ella is involved in a live at first sight romance; however, the object of her desire, an artist Edward Willig, has been dead since the late 1800s. Reluctantly, Anna does… Read More ›
New Books!
We've recently received a huge crop of great new books for the Creative Commons. Here's a taste of the newest titles… Eleanor & Park by Rainbow Rowell In a word, Eleanor & Park is adorable. Set in the mid-1980s, Eleanor is the new kid in town. She comes from a less than stable home life… Read More ›
Carrie Mac
B.C. novelist, Carrie Mac, is a unique voice in the world of Young Adult literature. On Friday May 10, Kwantlen Park Creative Commons hosted Mac as part of their student-author dialogues. Carrie Maac visited our school as part of the International Writers' Festival Outreach Program. Mac wowed and inspired the students and teachers with her humour, wit, and honesty. Background and Inspiration Carrie… Read More ›
Just In: Ichiro by Ryan Inzana
Ichiro lives with his mother in New York City. His father is killed during the war in the middle east and Ichiro`s mother decides that they should move to Japan to be closer to her family. Ichiro and his mother stay with his Grandfather Sato. Grandfather Sato teaches Ichiro about a land and culture… Read More ›
Just In: The Diviner by Libba Bray
Naughty John, Naughty John, does his work with his apron on. Cuts your throat and takes your bones. Sells 'em off for a coupla stones… Our story begins with Evie, who is living in the roaring 1920. She has been banished from her small town Ohio home to live with her uncle in… Read More ›
Just In: Lost Voices by Sarah Porter
This is not your average mermaid story. While these mermaids have shimmering fins and powerful tails, they are cruel, manipulative, and violent. Luce is a fourteen year old girl living in Alaska. She is brutally attacked and is left to die by the water. She falls into the icy waters expecting to die a quick… Read More ›
Just In: The Infects by Sean Beaudoin
Nero lives an unconventional life. He has a fractured family (his delinquent dad called `The Dude` and his socially inept sister) and must work to support them. He works the night shift slaughtering chemically enhanced chickens to help feed his family. An unfortunate mishap lands him with a sentence of three to six months… Read More ›
Just In: This Dark Endeavour by Kenneth Oppel
This Dark Endeavour is a book about Frankenstein, but not like any you`ve ever read before. This is a gothic story about Victor Frankenstein before he created his horrifying Creature. Victor and his twin brother Konrad are sixteen. The are living at 1700`s, a time when science and technology was mysterious and dangerous. Konrad becomes… Read More ›


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Since Uber raises the company value to billions, the term "Uber" has become synonymous with any kind of business that uses an online platform to deliver on-demand services. We hear about "Uber for repair service," "Uber for haircuts", and even "Uber for everything" …That is the latest technology trend.
In this article, I would like to criticize a new coming business "Uber for Tech Support" or "Uber for IT support". Is it their time yet? Will people accept this kind of services? What are the challenges?
I have to admit I want this kind of online service. My computer keeps running slowly, and my Windows could not upgrade to latest version Window 10. However, it is inconvenient to go service center where people may ask me to leave the laptop with them. I have to travel all the way to the shop, leave the PC there, and make another ride to pick it up. Another thing I have no idea what the supported person is doing with my personal data.
But if you ask me to get online support from a stranger. I would like to say "No." The reason is simple. IT support is not like other Uber traditional business where the work result is obvious. For example, Uber for Car, a driver drives you to the place quickly, nicely and cheap…you are happy and rate them at five stars. Or Uber for repair service, when they make your air conditioner work in a short time and at an inexpensive price you give them five stars.
However, for IT support, the result is not that obvious. Your problem seems to be solved, but you never know internally there may be:
– A virus or spyware is installed on your device; your PC may already become a zombie just wait for a signal to start attacking other PC.
– Your personal email or image may already be stolen.
– The spyware can steal your credit card not immediately but maybe on another day.
And many more possibilities. These things can happen not only to an inexperienced user but also to an expert if they do not keep an eye on the operations of the support person.
It seems there is only one choice for me, which is to use the service from people I know such as friends, relatives or from a well-known company like IBM, Geek Squad, etc. But I really love the Uber model, it not only gives customers a competitive price but also offer more kinds of services. Just imagine the services are provided by IT experts all over the world who have different expertise in different areas. You could ask someone to do any crazy thing (such as turn your Laptop become super secure Laptop, which NSA could not break). You can ask them to do at your time and at your price. Which I believe the traditional online support business could not provide.
So I would like to raise the question again: Is it the time of "Uber for IT support" yet?
In my own opinion, it is not their time yet unless there is a technology that can protect the user from the IT support person, prevent them steal the data, install a virus or spyware then the Uber model for IT support may work. I have gone through the remote access solution from a big vendor such as TeamViewer, LogMeIn or MSP Anyware — none of them can provide this feature yet. If I am wrong, prove it to me.
I am proud to introduce our company named ELINKGATE. We have developed and patented technology that can prevent IT support person copy user data and amazingly, the solution can work at preboot environment of BIOS level so reinstall a new OS and any kind of boot up problem can be solved remotely. Three advanced features they offer over current remote access solution:
- Simple: there is no pre-installed software required. They provide a USB dongle; users just need to plug the USB to PC and press a button. Anyone can use this to call for support – even my father, who is in his seventies.
- Smart: the solution can work for all Operating System and even the Bios level.
- Safe: the solution can protect user data from copied by IT support person.
However, there is a con : The user has to buy the USB dongle before getting the support. But I believe this should not be a problem if they can make the device inexpensively and provide easy access to buy it.


text: With a diverse team backed by a vast network of resources throughout Northeast Oklahoma, we are able to secure surety bonds for you or a family member without hassle or extended wait times. We offer some of Delaware County's most flexible and affordable repayment terms, and we'll construct an individualized plan that works for you and your budget. Our first priority is to obtain a quick and stress-free release, whether for you, a family member, or a client. We don't believe in making you jump through hoops, and we won't try to haggle terms. We strive to help you experience the peace of mind that you deserve after an arrest.


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Are We Experiencing a "Tiny House" Revolution?
Images by Tammy Stroebel at rowdykittens.com
Tiny house communities could quickly become a huge "thing" in the USA.
Well, so say researchers at Kansas State University. Why? To start with, tiny house villages are eco-friendly, they invite a sense of community, and they urge healthy and balanced way of living. There are also big health benefits to be gained from living in them, apparently. Additionally, they're a secure and inexpensive housing choice for the "masses". So, some "experts" are wishing that tiny house villages will spread across the country very soon, according to the The Wichita Eagle.
Tiny residences, residences that measure under 1,000 square feet, do not comprise a lot of the property market now. Since 2015, just one percent of house buyers intended to reside in a so-called tiny home. But, however, the Kansas State scientists think this might quickly shift.
Inside a Tiny Home
It is clear that there are a whole lot of advantages to living in teeny-weeny homes (less cleaning for one!). Nonetheless, zoning legislation across the USA have frequently impeded the development of tiny house areas – a common occurrence it would seem.
The standards and guidelines set in several locations inhibit tiny homes- generally regarded as mobile homes– because, to name a few factors, they could be seen by neighbours as being "low class." Naturally, this seems to us at the Save the Earth Cooperative just a "tiny" bit unhelpful and counter-intuitive to real progress in providing affordable homes for all – especially the vulnerable. This is, after all, the 21st century!
However, experts believe that the growing interest – both offline and on social media – for these tiny residential properties will certainly boost how people see them. Perhaps because of this, tiny houses seem to conjure up different images in relation to class.
They seem to dodge association with "Trailer-Park" type homes, which the social stigma seems to be pointed at. They are typically seen as middle or upper-middle-class and there is a plan to study specific tiny villages in order to figure out how they can harness the trend and use it to provide affordable housing options to those that need it. Sound good to you?
Would you live in a tiny house? Or would you help build a tiny house community for the homeless?
Please leave your comments and share with your friends.
Just how the Save the Earth Cooperative works. slogan : Save the world and be good to yourself
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Most homes in the Bay Area are built with plaster.
Plaster was an economical material to use during the early part of the 19th century and gives many homes a vintage, old world feel. Unfortunately, most homes in the Bay move when earthquakes rock and roll through the area, causing plaster to crack. Because we are craftsmen, we are able to repair cracks, matching the myriad styles and textures found on interior walls.
For large patches where walls and ceilings have cracked due to lots of movement, we use drywall to create a smooth canvas on which to apply plaster. We can skim for a smooth surface, match your existing texture, or apply a new texture, based on your design goals.


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Dar es Salam camp in Chad is home to more than 7,000 refugees from Nigeria. Each person here has fled horror and violence in their home country. Recently, I met two residents of the camp: 10-year-old Ibrahim and 38-year-old Adamu Yahaya, whose courage and resilience exemplifies that of so many of the people here.
In Nigeria, Ibrahim has been living with his grandmother, after his father died of illness and his mother drowned trying to flee when Boko Haram attacked a neighboring village last year. On the morning of January 3, 2015, tragedy once again entered Ibrahim's life when his small town came under attack. He was forced to flee his home alone, leaving behind his grandmother who was unable to make the journey.
The same morning, not far away, Adamu woke up to the sound of gunshots, and together with his wife and three children, fled in a hurry.
Ibrahim finally arrived in Chad, in the border town of Ngouboua. "A woman took me with her, but after a few days, she said she could not afford to feed me anymore. She took me to another man I knew from my village, but he also said that he could not look after me. Finally, I came to the camp, alone again. "
Ibrahim eventually ended up at the Child Friendly Space set up by UNICEF in the refugee camp. Community workers and psychosocial counsellors deployed by UNICEF gathered the community leaders to see if they could recognize Ibrahim. A woman in the crowd recognized him and informed the counsellors that one the boy's neighbors was also in the camp. That neighbor turned out to be Adamu.
Adamu, who is now Ibrahim's guardian, told me about the day they met at the camp: "They came to pick me up, I remember, it was a sandy afternoon. I saw Ibrahim, his face lit up. The community workers asked me if I could take care of him and I said yes immediately. You know, we all went through the same experience, that's why I feel responsible for him."
In turn, Ibrahim smiled and added: "When I was in Doro, I was friend with Adamu's children, we were neighbors. Now, when I feel safe, it's like a new family to me." UNICEF community workers pass by his tent regularly to monitor his health and well-being.
The last time I met up with them, Ibrahim was about to head to the Child Friendly Space where his friends were waiting for him – the day's activity was drawing – his favorite. I joined Ibrahim at the drawing session and was greeted by around thirty children who could not wait to get started. Their drawings show the horror of the conflict but they also describe what they miss from home: their friends, their clothes, their toys or simple things, like eating pineapples. They are just children – but where are their childhoods?
Ibrahim told me that he had never been to school before because the one of his village was closed. More than 300 schools have been severely damaged or destroyed in northeast-Nigeria since 2012 as a result of the conflict. Over 1.4 million children like Ibrahim have been forced to flee the conflict in and around Nigeria. This figure is just growing every month and it made me wonder: How many children will continue to draw the horrors they have seen? How many children will not go back to school this year?
Earlier this month, on 15 September, Ibrahim went to school for the first time and started the journey to discover letters and numbers.
"When I arrived, I did not want to go to school, I was scared. But this year I'll go and I hope to learn to read and write" he said.
After speaking with Ibrahim and Adamu, I stayed for some time in the Child Friendly Space, chatting with the children and looking at their drawings. I realised how challenging it is to bring hope out of despair. Only education can offer them the skills they need when they grow up to build a better future for themselves and their communities.
Badre Bahaji is Communication Officer working for UNICEF in Chad.
The Child Friendly Space in Dar es Salam refugee camp provides recreational and psychosocial activities to more than 1100 children. 174 unaccompanied or separated children have been registered through the Child Friendly Space. According to OCHA, in Chad only, more than 77,000 people, most of them children and women, have been displaced by violence since the beginning of the conflict in northeast Nigeria. Despite insecurity, UNICEF works with its partners to provide life-saving support to the most vulnerable children and women. However, nearly 70 per cent funding gap prevents children from receiving emergency assistance.


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The plane had touched down nicely and I was very pleased that the flight had gone so smoothly. I had generally only flown on American and Canadian flights in the continental United States. I did not trust American pilots given the crash record and the amount of plans that fell out of the sky from the '70s all the way into the '80s so it was good to know that someone else was at the helm. I watched it taxi through the runway and continued to do the talbiyyah with all the other believers on the flight.
The only break was once it had docked to the raised staircase and we gathered our things. Although my ihram was thick, I braced myself mid-way through the plane for the cold gust of wind that I thought would come from the outside. I saw Khalid and the other attendants and thanked them a great deal for how good they had made the flight with their hospitality. Khalid looked at me and in characteristic Egyptian wit remarked, "Nevermind me. You just go and pray for me. That's what will help me on the flight back!"
Making my way down the steps, I felt no gust, no piercing wind. Instead I was presently surprised to feel weather reminiscent of the West Coast and so the balmy weather felt welcome to me. Large airport buses pulled up, lowered to the ground but with large seats plush for comfort. As soon as the doors opened, our group split into smaller teams and boarded each of the handfuls of buses that had appeared. I stood up once I saw the sign reading, "Seats Reserved for Ladies and the Elderly Only."
I did not feel any fatigue anyway and wanted to look around at this massive complex that was the Jeddah airport. I had spied one unbeliever from the corner of my eye but it would be the last time I would see those under the threat of the eternal curse of Allah for some four weeks. Once at the terminal, we had to come in the door where we had four stops to make. The first stop was a standing area where all of us were separated into lines, military officers with gun belts staring at us cautiously.
Each one of us was huddled into a separate line and told to wait in single file order. Different soldiers of different ranks charged up and down the booths in front of the line, making sure all the documentation was ready, the stamps prepared and each booth occupied. One guard walked daringly into my face (three feet), usually what we call punch radius on the West Coast. Seeing that he was amicable looking, I only looked at him back in the face, while I saw others sheepishly looking down. He seemed surprised that I held his gaze rather than look away.
"You're not Pakistani," he remarked matter of factly in very clear Arabic, his voice slightly raised. "Yes," I agreed, taking a quick scan around the room and seeing a sea of Asian faces in the group. I think I was the only non-Asian in the posse of intendees of hajj. "What are you?" He demanded to know and stood facing me. "I have a United States passport."
This was the standard line of reasoning I was used to and so I was prepared. "No, no. What are you? That's not a race." Now I knew what he meant. He was trying to make sense of this whole thing, who I was and how I was related to all of these other believers. "Well, I'm Egyptian." "Right, okay. So you're on hajj with all of them." He looked at the passport, read my name, printed in the passport in Arabic and then English under it and looked up slightly bemused.
He uttered my name to one of the attendees and I had to leave the single file line that I was in and go to another. What –is-going-on I wondered loudly in my head. How am I already in trouble and I just got off the plane. I thought back if there were any previous convictions or anything I had not considered, litter that I had thrown down when I was young and hadn't rescued or the guy just thought I looked shifty.
Finally I found myself at a desk with a man that resembled my younger cousin Ridwan and had to stifle a laugh that almost came seeing this look-alike in an authority position. I would have jumped behind the desk and yanked Ridwan from his position and sat in his seat if it was him, but this was another man. His name tag read Mansur. After only three questions, he gave me back a well stamped passport, took a few prints from my fingers and did not use the retina scanner.
I was told to head to the next check point. Well, I still have my passport, I happily consoled myself. The second stop was a seating area in which scores of people were sitting on chairs, long couches or some seated on the floor. Signs hung up all over the place indicated the dangers of swine flu, meningitis, malaria and even a small one on AIDS.
Some members of the group had swept forward before me and were seated already, so I chose a seat next to them. Awesome. Soon I heard the footsteps of more military men, stepping from place to place with surgical masks and checking passports. I was next to be checked and he snatched my passport and looked me in the face, studying my features. He read out my name, checked for the stamps from the desk and then handed it back and nodded. As for the passports of the Pakistanis, he snatched them, looked inside and then slapped them back in their hands, almost throwing them. I knew I would see more of this before my journey was over.
All of us were accounted for so the Imam of our group led us out. Lagging behind the people, I saw a large contraption that people were stepping in and out of and a consecutive set of beeps issued from it. I wondered what they were doing until one man, by his accent a Tunisian, got inside. The machine bleeped with bass and treble alerts and startled everyone in the room. "I don't have it! I don't have it! It's just a warm day! It's just a warm day!"
He wept while being led away and others filled his place in the line to the mechanical checker. Someone in the line told me this was some sort of system used to check for swine flu. The Saudi establishment took the threat very seriously, to the point that most military were wearing these surgical masks. Anyone who seemed like their temperature was outside of accepted standards was detained and not allowed to venture further. Stop three was the terminal itself where the people were waiting for the buses that would later carry them to Makkah, may Allah ennoble that city; but before then we had to have our passports checked yet again.
This time more than eight stamps were put in my passport and I got the usual look, but someone gave me a stare that lasted for more than the usual time. What? I thought in my head. What do you want? Soon he looked away and I was waiting for the luggage cart, which slumped by on a three wheel device pretending to be a luggage carrier.
Once we did the compulsory bag counting, checking and counter checking, then a head count we were fine. I just watched one couple trying to reason with a member of the counter staff at stop three. "No, no. I'm an American. Where is the luggage? The luggage?" He made the sign for luggage with his hands, his wife then trying to speak Urdu after him. The counter people looked at them dumbly and shook their heads, saying to them in Arabic. "Go pray, hajji. It's time to pray." So much for the international language rhetoric, I chuckled under my breath.
Once outside of the safe confines of their comfort zone, especially those of the rank and file American, it must be pretty disconcerting to realise that not every one is dying to learn English. And what is more, those that might know it aren't too interested in having to speak it in their country when you have come to visit them.
In mid thought, I received greetings from seven foot Nigerian men going by. Suddenly, five feet eleven inches didn't seem so big. Not only were they large, but well muscled. I'm glad I'm on their team, I thought. "Wa Alaikum us-Salaam," I returned the greeting. Our walk to the waiting area (stop four) began and once there the wait began. The severe heat, the wait and all the other conditions crept up on some of the people that had truly not expected it.
The seats were nice, but only few were available as takers were quick, especially the Turkish. Once seated, the wait was on from 3:30am-11:00am. I saw that there were some people that needed water and soon Allah brought about someone who had water in his suitcase. The chilled two litre bottle of water somehow kept its' coolness on the flight over. Coolness and freshness were the grateful compliments of everyone that sipped from the container after they had first praised Allah.
"Brother, are those people getting ready to pray?" I felt a sharp but careful nudge in my rib cage by brother Khubaib. "Huh? Oh, they're praying. Subhanallah, they're about to pray Fajr!" I dashed over with him to the wudu' area and was soon purified and ready for prayer. The recitation behind the imam felt so nice, simple yet heartfelt.
Many of the brothers that I prayed next to did not have ihram yet as they spent everything they had just to get there. They would have to get one once they arrived in Makkah and go out of the miqat and come back into it; but they all seemed so grateful. I counted people from Ghana, Nigeria, Burkino-Faso, Gambia, Mauritania, Algeria and others among their ranks. We took in the whole atmosphere of this spread out terminal, complete with electricity powered canopies instead of a roof. They could be expanded or contracted by the pressing of a button.
Faces in our group showed apprehension, some were already tired; but this was no time to be weary! It was only the beginning of the journey. Relief swept the camp once our buses arrived but there were the usual organisational problems and teething issues (this just showed me that the Arabs never recovered since forfeiting the khilafah in the 656 AH disaster in Baghdad). The sisters had air conditioning, but mysteriously, the side the brothers were on did not.
I took my second opportunity and went to sleep, brother Mumtaz shaking his head and grinning at me from the front. He did not know how I could sleep in this heat, but I was certainly going to make a try of it. Within moments my head had slammed against the window next to me and I was now in the darkness of heat sleep.
And with Allah is every success,
brother in Islam,
al-Hajj Abu Ja`far al-Hanbali


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Christmas Boot Camp 2012
8 years ago
Posted by Chrissy at 9:13 PM
She is quite sweet looking! Good luck to you...hope your day (and hers) improves
She is adorable. I have very similar sentiments when I look at H. Good thing you're so darn cute!
You make me laugh so darn hard!! I love you man! She so reminds me of your little sis. She is totally adorable.
In the end...you'll be glad that you let them live. Trust me on this one.


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Hello and Welcome To The Jennifer Steele Blog!
In Today's Blog………..
Vegetables, Mother's Day Tech gift Ideas & Dogs eating with people! All in the blog today! Happy Friday & I'll see you back here on Monday!
Jennifer Steele, M-F 7p-12a, 101.5 K-HITS
**Hear It First On My Show! You can listen on-line at www.1015khits.com!
**Show Stuff!
11 tech gifts for Mother's Day to make her life easier!
This Mother's Day, go beyond the flowers and sweets and really treat mom to a tech gift that will make her life easier. After all, she deserves nothing but the best.
From a savvy photo and video storage system to smart home upgrades, these gadgets are not just helpful, they're thoughtful. We even did the work for you and rounded up the best Mother's Day tech gifts that are guaranteed to bring a smile to her face. Read more here!
NYC restaurant's dog menu includes $26 salmon for "dieting" canines!
NYC's dogs can eat better than most humans with a new menu at Manhattan restaurant The Wilson. The restaurant's culinary director Jeff Haskell was inspired by the meals he creates for his own English bulldog, Stella, Forbesreports. More here!
7 songs. 1 theme.
That pretty much sums it up!
Each weeknight at 7:00, I play 7 songs that fit some theme. What's the theme? Listen at 7 to find out and listen along. The funnest part is trying to guess which songs we might play that fit the theme!
Have an idea for a theme? Click Here!
Follow Me At:
Me & My Dog Jack!


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The Zebra Pit offers a wide selection of resources both on and offsite. As we grow and change and take on new readers, I wanted to take the time to explain where you can find some of the best resources on health and wellness for spoonies the web has to offer, gathered by the ZP.
Potato Bacon Bean Soup
Soup's On! This delicious potato bacon bean soup is a hearty, delicious meal for lunch or dinner, is low FODMAP and low Histamine with some modification.
Savory Sausage Tarts
This beautiful tart is low FODMAP, low histamine and full of healthy, delicious, affordable ingredients. Makes for a great Sunday brunch item, for family, for special company or anytime!
Quinoa Breakfast Bowl
This delectable bowl is packed with protein, fiber, vitamins and minerals to start your day off right while it's gluten-free, low FODMAP and low histamine, so it's safe for people with gastroparesis, IBS, IBD, MCAS, MCAD and other GI and mast cell disorders.
Melt in Your Mouth Meatballs
This old recipe has gone through a few reincarnations, including the use of my own homemade Italian sausage to eliminate the use of high FODMAP ingredients (mainly garlic and onion). Try as I might, I just can't seem to make Italian fit my low histamine diet requirements, but this does work well for IBS, Gastroparesis … Continue reading Melt in Your Mouth Meatballs


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Gran, Gramps, 'n BeansieLet's see if this works.
Style Copy Attempt
Various Character Concepts
Main Characters: Beansie, Gramps, and Gran
Environment Concepts
Software Used
Sketchbook Pro Adobe Photoshop
April 13, 2018
Category:Characters, Concept Art, Games
I was contacted by Jolly Crouton Media to see if I would be able to "copy" the art style that they had already been using since the current artist was taking too long to produce concept art.
Unfortunately, my style isn't quite the same, I thought I got pretty close. The first image is that attempt.
I did go ahead and do some additional art for them in my style to give him another perspective and they were really excited about my environments.
Last I heard the project was on a hiatus and I am waiting to work on more environments for the team.1


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Posted: Nov 10, 2018
Toccoa, GA - The JWU Women's Basketball Team took on Toccoa Falls College on Saturday, coming home with a 78-34 victory.
The Wildcats (1-2) were led by Justice Wallace with 16 points. Kayla Kent (13), Ineesha Hankerson (12), and Kayla Rogers (11) also reached double figures for JWU. JWU held a 62-31 rebounding advantage over the Screaming Eagles (1-4). Chase Muhammad led the team with 13 rebounds and with 8 assists.
Head Coach Jennifer King on the game: "It was a great team win. Everyone contributed and we were able to get some of our younger players some valuable game experience."
JWU hosts Johnson University (TN) on Nov. 13 at 6:00 pm.


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Vibrant Shawl | Mustard & Turquoise
$ 44.00
One of our most popular warps, the mustard and turquoise do amazing things together! This is our Ochos weave, very supple. Wear it to liven up any outfit while providing a bit of warmth, or wear it like a blanket scarf, too!. The interplay of the two colors creates a special visual effect as well! Handwoven of 100% cotton, the shawls are finished with double hand knotted fringe.
68" L x 26" W
2" fringe
Recommend hand wash to prevent tangling of the fringe.


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A New Gate A New Cycle of Time Keith Miller
Paul prayed in Ephesians 1:17–18 that our hearts would be illuminated by the Holy Spirit—the Spirit of wisdom and revelation in the knowledge of Him—who gives us understanding of the hope of our calling. This is very powerful. I believe there is a fresh call for the deeper things of God in this season. It's kind of like stepping into mom's house and smelling fresh bread. You can't wait to get into that kitchen, break that bread, put butter on that bread, and partake of that fresh bread. That's the kind of aroma or fragrance of the deeper things of God that He's releasing to those who will have hearts open, eyes to see, and ears to hear (see Luke 24:30–31, 45; Matt. 13:11).