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Helicobacter hepaticus is a bacterial pathogen that causes chronic active hepatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hepaticus was constructed and was screened with sera fromH. hepaticus-infected mice. A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified. The gene had high identity with genes coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was expressed in Escherichia coli as a glutathioneS-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusion protein was detected on Western blots probed with sera from H. hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice free of Helicobacterinfection. The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H. hepaticus antibodies in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA that uses a detergent extract ofH. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bilis, or H. muridarum and sera from mice free ofHelicobacter infection were evaluated. Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%). These data indicate that H. hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.
Helicobacter hepaticus is a helical, gram-negative, motile, microaerobic, bacterial pathogen of mice. It naturally colonizes the lower intestinal tract and can cause chronic active hepatitis or inflammatory bowel disease in a variety of immunocompetent mice (9-11, 13, 23, 24, 31, 39, 41) and immunodeficient mice (6, 8, 23, 40, 42). This bacterium is of concern to the scientific community because of its potential to invalidate studies that use infected mice (13). The hepatic and intestinal pathology induced by H. hepaticus can directly affect research involving these organ systems. Infected mice have abnormal liver function tests, as demonstrated by altered serum bile acid concentrations (39) and elevated levels of serum alanine aminotransferase (10, 11). Rectal prolapse, as a sequela to inflammatory bowel disease, may cause mice to be prematurely removed from an experiment. There is also evidence that H. hepaticus infections can disrupt host immune homeostasis. For example, infected mice mount a prompt and vigorous type 1 immune response against the bacterium (24, 42).
The potential for H. hepaticus to adversely affect scientific studies of mice is considerable, because infections in mouse colonies in the United States and Japan are widespread (30, 35). Since infected mice rarely display clinical signs of disease, accurate and inexpensive diagnostic tests to detect H. hepaticus infections are needed to effectively design and manage plans to eradicate this bacterium from mouse colonies. Diagnostic methods currently used to detect H. hepaticus-infected mice include histopathologic examination of silver-stained liver sections (39), culture of feces or livers (10), PCR analysis of liver, feces, or cecal tissue (3, 4, 27, 32), and enzyme-linked immunosorbent assay (ELISA) of serum (25) or feces (29) to detect anti-H. hepaticusspecific antibodies.
Diagnostic ELISAs that use recombinant proteins as antigens to detect antibodies to bacterial pathogens have been described. Several of these recombinant protein-based ELISAs perform with high levels of sensitivity and specificity or function better than immunoassays that use alternative antigen sources (12, 14, 16, 17, 26). Recombinant protein antigens can be advantageous in bacterial ELISAs because they contain defined antigens that potentially have fewer cross-reactive epitopes than other antigen preparations. Also, large amounts of affinity-tagged recombinant proteins produced in prokaryotic or eukaryotic expression systems can be harvested and purified by affinity chromatography. In addition, the availability of recombinant antigens produced from fastidious organisms, such as H. hepaticus, eliminates the need to cultivate the bacteria.
In this report, we describe the identification and cloning of an immunogenic membrane-associated protein of H. hepaticus. This protein was expressed in and purified from Escherichia coli as a glutathione S-transferase (GST) fusion protein and used as an antigen to create an H. hepaticus-specific ELISA. The performance of this ELISA was compared to that of an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. The recombinant protein-based ELISA was highly specific for detectingH. hepaticus-infected mice; however, this assay was less sensitive than the detergent extract-based H. hepaticusELISA.
Bacteria, plasmids, and sera.An isolate of H. hepaticus was obtained from an adult male A/JCr mouse from an endemically infected colony (39, 41) as previously described (25). The isolate was cultured under microaerobic conditions (25) and was identified as H. hepaticus based on its ultrastructural morphology and urease activity and the results of DNA sequence analysis of the 16S rRNA gene (32). E. coli DH5αMCR (Gibco BRL, Gaithersburg, Md.) was used in all cloning and subcloning experiments. E. coli BL21(DE3)pLysS (Novagen, Madison, Wis.) was used to express recombinant proteins. TheBamHI-digested plasmid Ready-To-Go pUC18 (Pharmacia Biotech, Piscataway, N.J.) was used to construct the genomic library, and the plasmid pUC18 (New England Biolabs, Beverly, Mass.) was used for subcloning procedures. The plasmid pGEX-5-X-3 (Pharmacia Biotech), which codes for an N-terminal GST moiety, was used to express fusion proteins in E. coli.
The H. hepaticus genomic library was screened with serum that was produced in adult C57BL/6 mice that were inoculated intraperitoneally with 108 viable H. hepaticusorganisms. Six weeks after inoculation, mice were euthanized, blood was collected by cardiocentesis, and serum was separated, pooled, and preabsorbed against E. coli by pseudoscreening (33). Sera used to evaluate the H. hepaticusELISAs (25) were collected from multiple strains of immunocompetent mice naturally infected with H. hepaticus(n = 44) or free of Helicobacter infection (n = 41) as determined by a fecal PCR assay (4, 32). To further evaluate the specificities of the ELISAs, groups of seven C57BL/6 mice were orally gavaged with 107H. hepaticus, Helicobacter bilis, or Helicobacter muridarum organisms. Sixteen weeks following inoculation, mice were euthanized, blood was collected by cardiocentesis, and serum was separated and stored at −20°C.
Construction of genomic library. H. hepaticusgenomic DNA was isolated from cultured bacteria by using Genomic-tips (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's protocol. Genomic DNA was partially digested with Sau3AI (New England Biolabs), and DNA was separated on NuSieve GTG low-melting-temperature agarose (FMC, Rockland, Maine). DNA fragments in the 4- to 12-kb size range were recovered with the Qiaex II gel extraction kit (Qiagen). Partially digested genomic DNA was ligated into BamHI-digested Ready-To-Go pUC 18 (Pharmacia Biotech), and E. coli DH5αMCR (Gibco BRL) was transformed, plated onto Luria-Bertani agar plates (33) containing 50 μg of ampicillin per ml and 50 μg of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) per ml, and incubated for 14 to 16 h at 37°C.
Screening of genomic library.Transformed bacteria were lifted onto nitrocellulose membranes (Nitropure; Micron Separations Inc., Westborough, Mass.), and bacterial colonies were incubated on filter paper saturated with lysis buffer (0.1 M NaHCO3, 1% Triton-X, and 2 mg of lysozyme per ml) in an atmosphere saturated with CHCl3 (18). The membranes were incubated withH. hepaticus-positive serum diluted to 1:2,000 in phosphate-buffered saline (PBS) (14.5 mM NaH2PO4, 108 mM Na2HPO4, 1.37 M NaCl [pH 7.4]) containing 5% nonfat dry milk (PBS-milk) and then incubated with an alkaline phosphatase-labeled antibody [F(ab′)2 fragment goat anti-mouse immunoglobulin G (heavy and light chains)] (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) diluted 1:2,000 in PBS-milk. The membranes were developed with 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (BCIP–NBT) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Md.). Immunoreactive clones were identified and rechecked for immunoreactivity as described above. Three immunoreactive clones designated 1A, 1B, and B2, that contained plasmids p1A, p1B, and pB2, respectively, were identified.
Subcloning and sequence analysis.Plasmids from positive clones were isolated by using the QIAprep Miniprep kit (Qiagen). To generate an immunoreactive clone that contained a small insert ofH. hepaticus genomic DNA, plasmids p1A, p1B, and pB2 were subjected to restriction endonuclease digestion withHindIII, EcoRI, Sau3AI,PstI, or KpnI and DNA fragments were separated on 1% SeaPlaque GTG low-melting-temperature agarose (FMC). The DNA fragments were excised from the gel and, with an in-gel ligation procedure (33), ligated into pUC18 (New England Biolabs). DH5αMCR bacteria were transformed with the subcloned plasmids and tested for immunoreactivity as described above. Bacteria transformed with the plasmid p1A2, which contains a 3.2-kb HindIII fragment of the plasmid p1A, were immunoreactive. This plasmid, p1A2, was chosen for further study and was sequenced by the dideoxy chain termination method (34) at the University of Missouri DNA Core Facility. The DNA sequences were assembled and analyzed for open reading frames (ORFs) by using GCG software (Genetics Computer Group, Inc., Madison, Wis.) and by inspection. Gene sequences and the predicted translated gene products of ORFs were compared to existing sequences deposited in the GenBank database by using software from GCG, the National Center for Biotechnology Information (2), and the Human Genome Center, Baylor College of Medicine (1, 43).
PCR and subcloning into E. coli expression vectors.The 3.2-kb insert of p1A2 was amplified by PCR in a thermal cycler (2400; Perkin-Elmer Cetus, Norwalk, Conn.) by using the Expand High Fidelity PCR System (Boehringer Mannheim, Indianapolis, Ind.). Each reaction mixture contained 40 ng of p1A2 as the template, a 0.4 μM concentration of the universal sequencing primer M13, and a 0.4 μM concentration of the reverse sequencing primer M13 (Pharmacia Biotech).
ORF 1 and ORF 2 were amplified by PCR in a thermal cycler (2400; Perkin-Elmer Cetus) by using the Expand High Fidelity PCR System (Boehringer Mannheim). Fifty nanograms of the 3.2-kb amplified insert of p1A2 was used as the template, and the annealing temperatures used to generate ORF 1 and ORF 2 were 49 and 53°C, respectively. The oligonucleotide primers used to generate ORF 1 were ORF1Forward (gcggatccggtaccTCTTAAAGGAGATGATGATGATG), which corresponds to nucleotides 276 to 295, and ORF1Reverse (cggaattcccatggCAACTTTATTCCCCATATTG), which corresponds to nucleotides 780 to 761. The primers used to generate ORF 2 were ORF2Forward (gcggatccggtaccAAGGATTCATTATGGAAGCTC), which corresponds to nucleotides 2347 to 2367, and ORF2Reverse (cggaattcccatggTAATGTGAGTTTGCACAGGC), which corresponds to nucleotides 2643 to 2624. The nucleotides of forward primers represented by lowercase letters were added to create restriction enzyme sites for BamHI and KpnI, and the nucleotides of reverse primers represented by lowercase letters were added to create restriction enzyme sites for EcoRI andNcoI. The amplified ORF 1 and ORF 2 genes were digested withBamHI and EcoRI and directionally cloned, in frame, into a GST gene fusion vector, pGEX-5X-3 (Pharmacia Biotech), to create plasmids pGEX-5X-3-MAP18 and PGEX-5X-3-P8, respectively.E. coli DH5αMCR was transformed with the recombinant plasmids and was tested for immunoreactivity by using the immunoscreening procedure described above.
Recombinant protein expression and purification. E. coli BL21(DE3)pLysS transformed with the plasmid pGEX-5X-3-MAP18 produced an H. hepaticus-specific immunoreactive fusion protein, GST-MAP18, that was chosen for further study. E. coli BL21(DE3)pLysS transformed with the plasmid pGEX-5X-3, which contained no foreign DNA insert, was used to express GST for use as a control protein. Proteins were expressed at 30°C, and proteins were purified with glutathione-linked agarose beads by the pGEX vector protocol (Pharmacia Biotech). The recovered proteins were dialyzed overnight at 4°C against PBS through a 12,000- to 14,000-Da cutoff membrane (Spectrum Medical Industries, Inc., Houston, Tex.), and the protein concentrations of the eluted fractions were determined by a bicinchoninic acid procedure (36).
Western blot analysis.Purified GST-MAP18 and GST or sonicated preparations of immunoreactive E. coli clones that harbored plasmids from an H. hepaticus genomic library were subjected to electrophoresis on a sodium dodecyl sulfate (SDS)–12% polyacrylamide gel under nonreducing conditions (22). Proteins were electrophoretically transferred to an Immobilon-P membrane (Millipore, Bedford, Mass.) and blocked with PBS-milk. Western blots were incubated with sera from Helicobacter-free mice or mice infected with H. hepaticus, H. bilis, orH. muridarum diluted 1:400 in PBS-milk. Next, blots were sequentially incubated with biotinylated goat anti-mouse antibody diluted 1:1,000 in PBS-milk and Vectastain (Vector Laboratories, Burlingame, Calif.). Membranes were developed with 3,3′,5,5′-tetramethylbenzidine membrane peroxidase substrate (Kirkegaard and Perry Laboratories).
H. hepaticus-specific ELISAs.The GST-MAP18 fusion protein and the control GST protein were diluted to 1 μg of protein/ml in coating buffer (0.1 M NaHCO3 [pH 9.6]), and 200 μl of each solution was added to separate wells of Immulon 2 96-well plates (Dynex, Chantilly, Va.). Proteins were allowed to bind to plates for 48 h at 4°C. The wells were emptied; 300 μl of blocking buffer (PBS with 0.5% nonfat dry milk and 0.05% Tween 20) was added to each well, incubated for 30 min at room temperature, and discarded; and 200 μl of serum diluted 1:100 in blocking buffer was added to duplicate wells coated with the antigen and to duplicate wells coated with the control protein. After incubation at 37°C for 2 h, plates were washed three times in PBS with 0.05% Tween 20 (PBS-Tween). Two hundred microliters of alkaline phosphatase-labeled secondary antibody [F(ab′)2 fragment goat anti-mouse immunoglobulin G (heavy and light chains)] (Jackson Immunoresearch Laboratories, Inc.) diluted 1:7,000 in PBS-Tween was added to each well and incubated at 37°C for 2 h. Plates were washed five times in PBS-Tween. One hundred microliters of phosphatase substrate solution (1 mg of Sigma 104 phosphatase substrate [Sigma] per 1 ml of substrate buffer [2 mM MgCl2, 27.5 mM Na2CO3, 22.5 mM NaHCO3]) was added to each well and incubated in the dark at room temperature for 30 min. The optical density (OD) of each well was measured (Kinetic Reader EL312E; Bio-Tek Instruments, Inc., Winooski, Vt.) and automatically calculated as the OD at 405 nm (OD405) minus the OD490 to correct for optical interference. The final OD405 for each serum sample was calculated as the mean OD405 of the paired recombinant-antigen-coated wells minus the mean OD405 of the paired control protein-coated wells. The cutoff OD405 was set at two standard deviations above the mean for 41 serum samples from mice negative forHelicobacter infection by PCR (4, 25, 32). To evaluate the performance of the recombinant-protein-based ELISA, this ELISA was directly compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen (25).
Nucleotide sequence accession number.The GenBank accession number of the sequence for the 3.2-kb HindIII fragment and the MAP18 gene is AF134212 .
Identification of immunoreactive protein.Immunoscreening of 8,000 white clones from the H. hepaticus genomic library with sera from H. hepaticus-infected mice revealed the three immunoreactive clones designated 1A, 1B, and B2, which contained the plasmids p1A, p1B, and pB2, respectively. Western blot analysis of all three positive clones demonstrated an 18-kDa immunoreactive protein when blots were probed with sera from H. hepaticus-infected mice (Fig. 1). The 18-kDa band was not visualized when clones were probed with sera from mice free of H. hepaticus infection (Fig. 1). Thus, subsequent experiments were focused toward identifying an ORF in the H. hepaticus genome contained in the plasmid p1A, p1B, or pB2 that coded for an H. hepaticus-specific 18-kDa immunoreactive protein. Restriction endonuclease digestion of the plasmids p1A, p1B, and pB2 revealed DNA inserts of approximately 8.5, 9.0, and 6.5 kb, respectively. Subcloning and immunoscreening of these plasmids revealed that a gene coding for an 18-kDa immunoreactive protein was located on a 3.2-kb piece of DNA generated by HindIII digestion of p1A. This subcloned plasmid, p1A2, was chosen for further study.
Identification of an ORF encoding an immunogenic protein.The DNA sequence of p1A2 was determined, and four ORFs that had putative ribosome binding sites and coded for predicted proteins of >7,000 Da were identified. The ORF 1 gene, which coded for a predicted 18,836-Da membrane-associated protein (MAP18), and the ORF 2 gene, which coded for a predicted 7,959-Da protein (P8), were generated by PCR, and the amplicons were directionally cloned in frame into the expression vector pGEX-5X-3 (Pharmacia Biotech) to create the plasmids pGEX-5X-3-MAP18 and pGEX-5X-3-P8, respectively. Colony lifts of E. coliharboring the plasmid pGEX-5X-3-MAP18 were immunoreactive when probed with sera from H. hepaticus-infected mice. Colony lifts ofE. coli harboring pGEX-5X-3-P8 demonstrated no immunoreactivity when probed with sera from mice infected with H. hepaticus. Because ORF 1 was identified as the gene that coded for the 18-kDa immunoreactive H. hepaticus protein that was being sought, ORF 3 and ORF 4 were not evaluated for the production of immunoreactive proteins.
Gene sequence and predicted translated protein of ORF 1.The 459-nucleotide sequence of the ORF that coded for the 18-kDa immunogenic protein of H. hepaticus, ORF 1, is presented in Fig. 2. The gene had a putative ribosome binding site at positions −6 through −11, a TAA stop codon, and a G+C content of 40.3%. The ORF had a 59.8% identity in a 316-bp overlap with an ORF of the H. pylori genome that codes for a putative 18-kDa peptidoglycan-associated lipoprotein (GenBank nucleotide sequence accession no. AE000619 ) (37). The gene also had a 61.8% identity in a 338-bp overlap with a gene coding for a peptidoglycan-associated outer membrane lipoprotein ofCampylobacter jejuni (GenBank nucleotide sequence accession no. U47617 ) (20) and had a high sequence similarity with genes coding for outer membrane proteins of other gram-negative bacteria. The predicted translated protein of ORF 1, MAP18, contained 153 amino acids and had a high identity with predicted outer membrane lipoproteins of other gram-negative bacteria. For example, MAP18 had a 48% identity over 104 amino acids with a predicted 18-kDa peptidoglycan-associated lipoprotein of H. pylori (GenBank accession no. AE000619 ) (37) and had a 49% identity over 107 amino acids with a predicted 18-kDa peptidoglycan-associated lipoprotein of C. jejuni (GenBank accession no. X83374 ). The N-terminal 19-amino-acid peptide fragment of the predicted translated protein of MAP18 (Fig. 2) was a predicted prokaryotic membrane lipoprotein signal sequence (19, 28, 38) that had a 52% identity with the leader sequence of a 20-kDa membrane-associated lipoprotein of H. pylori (GenBank accession no. AE000645 ) (7, 21). In addition, the Ser-Val-Gly-Cys sequence at amino acid positions 15 through 18 is a putative cleavage site for a specific lipoprotein signal peptidase, signal peptidase II (15), with the serine at position 19 serving as a probable outer membrane trafficking signal (44) and the cysteine at position 18 serving as a possible site for the protein to be attached to a membrane lipid (15). Thus, it is likely that MAP18 is a membrane-associated protein of H. hepaticus.
Western blotting of recombinant GST-MAP18.Western blots of affinity-purified GST-MAP18 demonstrated a 44-kDa fusion protein that reacted with sera from H. hepaticus-infected mice but did not react with sera from mice infected with H. bilis orH. muridarum or with sera from uninfected mice (Fig.3). This 44-kDa recombinant protein, GST-MAP18, was composed of the 18-kDa immunoreactive protein ofH. hepaticus (MAP18) coded for by ORF 1 fused with the 26-kDa GST protein affinity tag coded for by the expression vector.
Comparison of recombinant-antigen-based ELISA to detergent extract-based ELISA.The GST-MAP18 recombinant protein was expressed in and purified from E. coli and used as the antigen in an ELISA to detect anti-H. hepaticus specific antibodies in the sera of infected mice. This recombinant-antigen-based ELISA was directly compared to an established H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen (25) (Table1). The sensitivity and specificity of each ELISA were determined by evaluating sera from multiple strains of immunocompetent mice that were documented by fecal PCR assay to be infected with H. hepaticus or free ofHelicobacter infections. Both ELISAs were highly specific (98%); however, the recombinant-protein-based ELISA was 66% sensitive in identifying mice naturally infected withH. hepaticus while the detergent extract-based ELISA was 89% sensitive in identifying these mice. To further compare the sensitivities and specificities of the two H. hepaticus-specific ELISAs, sera from mice that were orally gavaged with H. hepaticus, H. bilis, or H. muridarum were tested. When these sera were evaluated, the detergent extract-based ELISA and the recombinant protein-based ELISA were both 100% specific and 100% sensitive in identifying theH. hepaticus-infected mice. Thus, the recombinant-antigen-based ELISA was less sensitive than the detergent extract-based ELISA in detecting mice naturally infected with H. hepaticus while both assays were equally highly specific.
In this report we describe the identification and cloning of a gene that codes for an 18-kDa immunogenic membrane-associated protein (MAP18) of H. hepaticus. The protein was expressed in and purified from E. coli as a GST fusion protein, GST-MAP18, and was used as the antigen in an ELISA to detect anti-H. hepaticus specific antibodies in the sera of infected mice. This ELISA was directly compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. Both ELISAs performed with high specificity; however, the recombinant protein-based ELISA performed with a lower sensitivity than did the detergent extract-based ELISA in identifying mice naturally infected with H. hepaticus. The decreased sensitivity of the recombinant protein-based ELISA may have resulted from the presence of only a limited number of antigenic epitopes on the single protein antigen and the failure of all infected mice to generate a vigorous antibody response against this protein. Several immunoreactive bands were detected when Western blots of the H. hepaticusdetergent extract antigen were probed with sera from H. hepaticus-infected mice (data not shown), suggesting that this antigen contains more immunogenic epitopes than the recombinant-protein antigen. Thus, the increased sensitivity of the detergent extract-based ELISA may result from its ability to identify several differentH. hepaticus-specific antibodies that are produced in response to infection. The decreased sensitivity of the recombinant-protein-based ELISA may also have resulted from antigenic variation of MAP18 so that antibodies directed against the MAP18 proteins of other H. hepaticus isolates may not recognize the antigenic epitopes of the recombinant MAP18 protein used in our ELISA. Therefore, potential ways to increase the sensitivity of the ELISA are to clone other antigenic variants of MAP18 or to clone other immunoreactive proteins of H. hepaticus and to combine these proteins with GST-MAP18 to create a multivalent antigen preparation for use in an H. hepaticus-specific ELISA.
Our results suggest that MAP18 is a membrane-associated lipoprotein ofH. hepaticus. The gene coding for MAP18, ORF 1, had a high sequence identity with genes coding for outer membrane lipoproteins of other gram-negative bacteria, including a putative 18-kDa outer membrane lipoprotein of H. pylori (37) and an 18-kDa outer membrane lipoprotein of C. jejuni(5), a bacterium from a closely related genus. Also, the predicted amino acid sequence of MAP18 had high identities with the amino acid sequences of these same proteins. In addition, the N-terminal 19-amino-acid peptide fragment of MAP18 contained a prokaryotic signal peptide that predicted that the protein would be trafficked to the bacterial membrane (19, 28, 38). The C terminus of the signal peptide had a putative signal peptidase II cleavage site; signal peptidase II is an enzyme that is involved in cleavage of signal sequences of prokaryotic membrane-transported proteins (15). In addition, the cysteine residue following this cleavage site is a likely point of attachment of membrane proteins to a lipid moiety (15) and the serine at position two of the cleaved sequence has been shown to be an outer membrane trafficking signal in E. coli (44).
In summary we cloned a gene that encodes an immunogenic protein ofH. hepaticus. The protein was expressed as a fusion protein and was used as an antigen in an ELISA to detect antibodies to H. hepaticus in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. Both assays performed with an equally high specificity; however, the recombinant-protein-based ELISA was not as sensitive in detecting infected mice. This report provides a foundation for the further development of recombinant-protein-based immunoassays for the detection of H. hepaticus infections in mice.
- Received 5 April 1999.
- Returned for modification 25 May 1999.
- Accepted 25 June 1999.
- Copyright © 1999 American Society for Microbiology


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North Hempstead to demolish two vacant structures
The Town of North Hempstead on Friday is to demolish two structures that have been vacant since 2010.
The structures on Sheridan Street will be torn down at 10 a.m. Town officials say the houses, which have been subdivided, have been the site of loitering and create a "hazardous situation."
"Zombie houses are dangerous eyesores and I am pleased that the Town was able to demolish these adjoining structures," said Supervisor Judi Bosworth in a news release. "It will certainly breathe new life into this area and improve the quality of life."
"The Town has the authority to demolish this structure for safety reasons and in order to maintain a quality of living that our local residents in New Cassel deserve," said Councilwoman Viviana Russell in the release. "The demolition of this structure will surely improve the look of the neighborhood and is another step forward in the revitalization of the area."
Town officials will place a lien on the property in order to recover the costs spent on demolishing the house.

subset: commoncrawl


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In Canto Three of his poem John Shade (the poet in VN's novel Pale Fire, 1962) speaks of IPH (a lay Institute of Preparation for Hereafter) and mentions "Terra the Fair, an orbicle of jasp:"
While snubbing gods, including the big G,
Iph borrowed some peripheral debris
From mystic visions; and it offered tips
(The amber spectacles for life's eclipse) -
How not to panic when you're made a ghost:
Sidle and slide, choose a smooth surd, and coast,
Meet solid bodies and glissade right through,
Or let a person circulate through you.
How to locate in blackness, with a gasp,
Terra the Fair, an orbicle of jasp.
How to keep sane in spiral types of space.
Precautions to be taken in the case
Of freak reincarnation: what to do
On suddenly discovering that you
Are now a young and vulnerable toad
Plump in the middle of a busy road,
Or a bear cub beneath a burning pine,
Or a book mite in a revived divine. (ll. 549-566)
According to Kinbote (Shade's mad commentator who imagines that he is Charles the Beloved, the last self-exiled king of Zembla), "How to locate in blackness, with a gasp, Terra the Fair, an orbicle of jasp" is the loveliest couplet in this Canto. "An orbicle of jasp" brings to mind "a perfect image of one orbicular millennium" that Archibald Moon, a character in VN's novel Podvig ("Glory," 1932"), wanted to give in his book on Russia:
Профессором русской словесности и истории был в ту пору небезызвестный Арчибальд Мун. В России он прожил довольно долго, всюду побывал, всех знал, всё перевидел. Теперь, черноволосый, бледный, в пенсне на тонком носу, он бесшумно проезжал на велосипеде с высоким рулём, сидя совсем прямо, а за обедом, в знаменитой столовой с дубовыми столами и огромными цветными окнами, вертел головой, как птица, и быстро, быстро крошил длинными пальцами хлеб. Говорили, единственное, что он в мире любит, это - Россия. Многие не понимали, почему он там не остался. На вопросы такого рода Мун неизменно отвечал: "Справьтесь у Робертсона" (это был востоковед) "почему он не остался в Вавилоне". Возражали вполне резонно, что Вавилона уже нет. Мун кивал, тихо и хитро улыбаясь. Он усматривал в октябрьском перевороте некий отчетливый конец. Охотно допуская, что со временем образуется в Советском Союзе, пройдя через первобытные фазы, известная культура, он вместе с тем утверждал, что Россия завершена и неповторима, - что её можно взять, как прекрасную амфору, и поставить под стекло. Печной горшок, который там теперь обжигался, ничего общего с нею не имел. Гражданская война представлялась ему нелепой: одни бьются за призрак прошлого, другие за призрак будущего, - меж тем, как Россию потихоньку украл Арчибальд Мун и запер у себя в кабинете. Ему нравилась её завершённость. Она была расцвечена синевою вод и прозрачным пурпуром пушкинских стихов. Вот уже скоро два года, как он писал на английском языке её историю, надеялся всю её уложить в один толстенький том. Эпиграф из Китса ("Создание красоты - радость навеки"), тончайшая бумага, мягкий сафьяновый переплёт. Задача была трудная: найти гармонию между эрудицией и тесной живописной прозой, дать совершенный образ одного округлого тысячелетия.
At that time the chair of Russian literature and history was occupied by the distinguished scholar Archibald Moon. He had lived fairly long in Russia, and had been everywhere, met everyone, seen everything there. Now, pale and dark-haired, with a pince-nez on his thin nose, he could be observed riding by, sitting perfectly upright, on a bicycle with high handlebars; or, at dinner in the renowned hall with oaken tables and huge stained-glass windows, he would jerk his head from side to side like a bird, and crumble bread extremely fast between his long fingers. They said the only thing this Englishman loved in the world was Russia. Many people could not understand why he had not remained there. Moon's reply to questions of that kind would invariably be: "Ask Robertson" (the orientalist) "why he did not stay in Babylon." The perfectly reasonable objection would be raised that Babylon no longer existed. Moon would nod with a sly, silent smile. He saw in the Bolshevist insurrection a certain clear-cut finality. While he willingly allowed that, by-and-by, after the primitive phases, some civilization might develop in the "Soviet Union," he nevertheless maintained that Russia was concluded and unrepeatable, that you could embrace it like a splendid amphora and put it behind glass. The clay kitchen pot now being baked there had nothing in common with it. The civil war seemed absurd to him: one side fighting for the ghost of the past, the other for the ghost of the future, and meanwhile Archibald Moon quietly had stolen Russia and locked it up in his study. He admired this finality. It was colored by the blue of waters and the transparent porphyry of Pushkin's poetry. For nearly two years now he had been working on an English-language history of Russia, and he hoped to squeeze it all into one plump volume. An obvious motto ("A thing of beauty is a joy forever"), ultrathin paper, a soft Morocco binding. The task was a difficult one: to find a harmony between erudition and tight picturesque prose, to give a perfect image of one orbicular millennium. (chapter XVI)
A distant northern land, Kinbote's Zembla has a lot in common with Martin's and Sonia's Zoorland in "Glory." As to Archibald Moon, he brings to mind Aunt Maud's verse book open at the Index (Moon, Moonrise, Moor, Moral):
I was brought up by dear bizarre Aunt Maud,
A poet and a painter with a taste
For realistic objects interlaced
With grotesque growths and images of doom.
She lived to hear the next babe cry. Her room
We've kept intact. Its trivia create
A still life in her style: the paperweight
Of convex glass enclosing a lagoon,
The verse book open at the Index (Moon,
Moonrise, Moor, Moral), the forlorn guitar,
The human skull; and from the local Star
A curio: Red Sox Beat Yanks 5-4
On Chapman's Homer, thumbtacked to the door. (ll. 86-98)
On First Looking into Chapman's Homer (1816) is a sonnet by Keats:
Much have I travell'd in the realms of gold,
And many goodly states and kingdoms seen;
Round many western islands have I been
Which bards in fealty to Apollo hold.
Oft of one wide expanse had I been told
That deep-brow'd Homer ruled as his demesne;
Yet did I never breathe its pure serene
Till I heard Chapman speak out loud and bold:
Then felt I like some watcher of the skies
When a new planet swims into his ken;
Or like stout Cortez when with eagle eyes
He star'd at the Pacific—and all his men
Look'd at each other with a wild surmise—
Silent, upon a peak in Darien.
Describing the last moments of Shade's life, Kinbote uses the word "demesne" (used by Keats in his sonnet):
One minute before his death, as we were crossing from his demesne to mine and had begun working up between the junipers and ornamental shrubs, a Red Admirable (see note to line 270) came dizzily whirling around us like a colored flame. Once or twice before we had already noticed the same individual, at that same time, on that same spot, where the low sun finding an aperture in the foliage splashed the brown sand with a last radiance while the evening's shade covered the rest of the path. One's eyes could not follow the rapid butterfly in the sunbeams as it flashed and vanished, and flashed again, with an almost frightening imitation of conscious play which now culminated in its setting upon my delighted friend's sleeve. It took off, and we saw it next morning sporting in an ecstasy of frivolous haste around a laurel shrub, every now and then perching on a lacquered leaf and sliding down its grooved middle like a boy down the banister on his birthday. Then the tide of the shade reached the laurels, and the magnificent, velvet-and-flame creature dissolved in it. (note to Lines 993-995)
Shade's poem is almost finished, when the author is killed by Gradus. Kinbote believes that, to be completed, Shade's poem needs but one line (Line 1000, identical to Line 1: "I was the shadow of the waxwing slain"). But it seems that, like some sonnets, Shade's poem also needs a coda (Line 1001: "By its own double in the windowpane"). Dvoynik ("The Double") is a short novel (1846) by Dostoevski and a poem (1909) by Alexander Blok. The L disaster that happened on Demonia (aka Antiterra, Earth's twin planet on which VN's novel Ada, 1969, is set) in the beau milieu of the 19th century and seems to correspond to the mock execution of Dostoevski and the Petrashevskians on Jan. 3, 1850 (NS), in our world:
The details of the L disaster (and I do not mean Elevated) in the beau milieu of last century, which had the singular effect of both causing and cursing the notion of 'Terra,' are too well-known historically, and too obscene spiritually, to be treated at length in a book addressed to young laymen and lemans — and not to grave men or gravemen.
Of course, today, after great anti-L years of reactionary delusion have gone by (more or less!) and our sleek little machines, Faragod bless them, hum again after a fashion, as they did in the first half of the nineteenth century, the mere geographic aspect of the affair possesses its redeeming
comic side, like those patterns of brass marquetry, and bric-à-Braques, and the ormolu horrors that meant 'art' to our humorless forefathers. For, indeed, none can deny the presence of something highly ludicrous in the very configurations that were solemnly purported to represent a varicolored map of Terra. Ved' ('it is, isn't it') sidesplitting to imagine that 'Russia,' instead of being a quaint synonym of Estoty, the American province extending from the Arctic no longer vicious Circle to the United States proper, was on Terra the name of a country, transferred as if by some sleight of land across the ha-ha of a doubled ocean to the opposite hemisphere where it sprawled over all of today's Tartary, from Kurland to the Kuriles! But (even more absurdly), if, in Terrestrial spatial terms, the Amerussia of Abraham Milton was split into its components, with tangible water and ice separating the political, rather than poetical, notions of 'America' and 'Russia,' a more complicated and even more preposterous discrepancy arose in regard to time — not only because the history of each part of the amalgam did not quite match the history of each counterpart in its discrete condition, but because a gap of up to a hundred years one way or another existed between the two earths; a gap marked by a bizarre confusion of directional signs at the crossroads of passing time with not all the no-longers of one world corresponding to the not-yets of the other. It was owing, among other things, to this 'scientifically ungraspable' concourse of divergences that minds bien rangés (not apt to unhobble hobgoblins) rejected Terra as a fad or a fantom, and deranged minds (ready to plunge into any abyss) accepted it in support and token of their own irrationality. (1.3)
Darkbloom ('Notes to Ada'): beau milieu: right in the middle.
Faragod: apparently, the god of electricity.
braques: allusion to a bric-à-brac painter.
According to Van Veen (the narrator and main character in Ada), the real destination of poor mad Aqua (the twin sister of Van's, Ada's and Lucette's mother Marina) was Terra the Fair:
Actually, Aqua was less pretty, and far more dotty, than Marina. During her fourteen years of miserable marriage she spent a broken series of steadily increasing sojourns in sanatoriums. A small map of the European part of the British Commonwealth — say, from Scoto-Scandinavia to the Riviera, Altar and Palermontovia — as well as most of the U.S.A., from Estoty and Canady to Argentina, might be quite thickly prickled with enameled red-cross-flag pins, marking, in her War of the Worlds, Aqua's bivouacs. She had plans at one time to seek a modicum of health ('just a little grayishness, please, instead of the solid black') in such Anglo-American protectorates as the Balkans and Indias, and might even have tried the two Southern Continents that thrive under our joint dominion. Of course, Tartary, an independent inferno, which at the time spread from the Baltic and Black seas to the Pacific Ocean, was touristically unavailable, though Yalta and Altyn Tagh sounded strangely attractive… But her real destination was Terra the Fair and thither she trusted she would fly on libellula long wings when she died. Her poor little letters from the homes of madness to her husband were sometimes signed: Madame Shchemyashchikh-Zvukov ('Heart rending-Sounds'). (ibid.)
In the first line of his poem Priblizhaetsya zvuk... ("A sound approaches..." 1912) Alexander Blok mentions shchemyashchiy zvuk (a heart-rending sound):
Приближается звук. И, покорна щемящему звуку,
Молодеет душа.
И во сне прижимаю к губам твою прежнюю руку,
Не дыша.
Снится - снова я мальчик, и снова любовник,
И овраг, и бурьян,
И в бурьяне - колючий шиповник,
И вечерний туман.
Сквозь цветы, и листы, и колючие ветки, я знаю,
Старый дом глянет в сердце моё,
Глянет небо опять, розовея от краю до краю,
И окошко твоё.
Этот голос - он твой, и его непонятному звуку
Жизнь и горе отдам,
Хоть во сне твою прежнюю милую руку
Прижимая к губам.
After the L disaster electricity was banned on Antiterra. In his Commentary Kinbote quotes Shade's poem "The Nature of Electricity" that appeared in the New York magazine The Beau and the Butterfly after the poet's death:
The dead, the gentle dead--who knows?--
In tungsten filaments abide,
And on my bedside table glows
Another man's departed bride.
And maybe Shakespeare floods a whole
Town with innumerable lights,
And Shelley's incandescent soul
Lures the pale moths of starless nights.
Streetlamps are numbered, and maybe
Number nine-hundred-ninety-nine
(So brightly beaming through a tree
So green) is an old friend of mine.
And when above the livid plain
Forked lightning plays, therein may dwell
The torments of a Tamerlane,
The roar of tyrants torn in hell.
Science tells us, by the way, that the Earth would not merely fall apart, but vanish like a ghost, if Electricity were suddenly removed from the
world. (note to Line 347)
In his poem Nedvizhnyi strazh dremal na tsarstvennom poroge… ("The stirless sentinel dozed at the royal threshold…" 1824) Pushkin says that Napoleon (sey tsar, "this king") vanished like a dream, like dawn's shade:
То был сей чудный муж, посланник провиденья,
Свершитель роковой безвестного веленья,
Сей всадник, перед кем склонилися цари,
Мятежной вольности наследник и убийца,
Сей хладный кровопийца,
Сей царь, исчезнувший, как сон, как тень зари.
In the next stanza Pushkin mentions plamya blednoe (the pale fire) of Napoleon's frowning eyes:
Ни тучной праздности ленивые морщины,
Ни поступь тяжкая, ни ранние седины,
Ни пламя бледное нахмуренных очей
Не обличали в нем изгнанного героя,
Мучением покоя
В морях казненного по манию царей.
In his poem Pushkin mentions "the lindens of Tsarskoe Selo and the towers of Gibraltar:"
И делу своему владыка сам дивился.
Се благо, думал он, и взор его носился
От Тибровых валов до Вислы и Невы,
От сарскосельских лип до башен Гибралтара:
Всё молча ждёт удара,
Всё пало — под ярем склонились все главы.
"From the hills of the Tiber's banks to the Vistula and the Neva, / from the lindens of Tsarskoe Selo to the towers of Gibraltar" brings to mind "from Scoto-Scandinavia to the Riviera, Altar and Palermontovia" on a small map of the European part of the British Commonwealth mentioned by Van in Ada. While Palermontovia blends Palermo (a city in and the capital of Sicily) with Lermontov (the author of "The Demon," 1829-40, and of the prophetical "Prediction," 1830, whose name begins with L), Altar hints at Gibraltar.
The Latin name of Palermo is Panormus. In his essay Panorama Moskvy ("The Panorama of Moscow," 1834) Lermontov calls the Kremlin altar' Rossii ("the altar of Russia") and compares it to phoenix (the legendary bird that is reborn from ashes):
Что сравнить с этим Кремлём, который, окружась зубчатыми стенами, красуясь золотыми главами соборов, возлежит на высокой горе, как державный венец на челе грозного владыки?..
Он алтарь России, на нём должны совершаться и уже совершались многие жертвы, достойные отечества... Давно ли, как баснословный феникс, он возродился из пылающего своего праха?..
Shade's murderer, Gradus is a member of the Shadows (a regicidal organization). In Russia in the Shadows (1921), a series of articles that H. G. Wells wrote after visiting the Soviet Russia, the author of The War of the Worlds (1898) calls Lenin "the Kremlin dreamer."
In the last game of Flavita (Russian Scrabble) that Van ever played with Ada and Lucette at Ardis Lucette's letters formed the word Kremlin:
'Je ne peux rien faire,' wailed Lucette, 'mais rien — with my idiotic Buchstaben, REMNILK, LINKREM...'
'Look,' whispered Van, 'c'est tout simple, shift those two syllables and you get a fortress in ancient Muscovy.'
'Oh, no,' said Ada, wagging her finger at the height of her temple in a way she had. 'Oh, no. That pretty word does not exist in Russian. A Frenchman invented it. There is no second syllable.'
'Ruth for a little child?' interposed Van.
'Ruthless!' cried Ada.
'Well,' said Van, 'you can always make a little cream, KREM or KREME — or even better — there's KREMLI, which means Yukon prisons. Go through her ORHIDEYA.'
'Through her silly orchid,' said Lucette. (1.36)
Van's and Ada's half-sister Lucette commits suicide by jumping from the deck of Admiral Tobakoff into the Atlantic. In his poem Net, ya ne Bayron, ya drugoy… ("No, I'm not Byron, I'm another…" 1832) Lermontov compares his soul to the ocean in which nadezhd razbitykh gruz (a load of broken hopes) lies:
Нет, я не Байрон, я другой,
Ещё неведомый избранник,
Как он, гонимый миром странник,
Но только с русскою душой.
Я раньше начал, кончу ране,
Мой ум немного совершит;
В душе моей, как в океане,
Надежд разбитых груз лежит.
Кто может, океан угрюмый,
Твои изведать тайны? Кто
Толпе мои расскажет думы?
Я — или Бог — или никто!
No, I'm not Byron, I'm another
yet unknown chosen man,
like him, a persecuted wanderer,
but only with a Russian soul.
I started sooner, I will end sooner,
my mind won't achieve much;
in my soul, as in the ocean,
lies a load of broken hopes.
Gloomy ocean, who can
find out your secrets? Who
will tell to the crowd my thoughts?
Myself – or God – or none at all!
The last word in Lermontov's poem is nikto (nobody). Nik. T-o was I. Annenski's penname. One of the essays in Annenski's Kniga otrazheniy ("Book of Reflections," 1906) is entitled Dostoevskiy do katastrofy ("Dostoevski before the Disaster"). In his essay Problema Gamleta ("The Problem of Hamlet") included in "The Second Book of Reflections" (1909) Annenski says that Hamlet is not Salieri:
Видите ли: зависть художника не совсем то, что наша...
Для художника это - болезненное сознание своей ограниченности и желание делать творческую жизнь свою как можно полнее. Истинный художник и завистлив и жаден... я слышу возражение - пушкинский Моцарт. - Да! Но ведь Гамлет не Сальери. Моцарта же Пушкин, как известно, изменил: его короткая жизнь была отнюдь не жизнью праздного гуляки, а сплошным творческим горением. Труд его был громаден, не результат труда, а именно труд.
In Pushkin's little tragedy "Mozart and Salieri" (1830) Mozart uses the phrase nikto b (none would):
Когда бы все так чувствовали силу
Гармонии! Но нет: тогда б не мог
И мир существовать; никто б не стал
Заботиться о нуждах низкой жизни;
Все предались бы вольному искусству.
If all could feel like you the power
of harmony! But no: the world
could not go on then. None would
bother with the needs of lowly life;
all would surrender to free art. (Scene II)
Nikto b is Botkin (Shade's, Kinbote's and Gradus's "real" name) in reverse. An American scholar of Russian descent, Professor Vsevolod Botkin went mad and became Shade, Kinbote and Gradus after the tragic death of his daughter Nadezhda (Hazel Shade of Kinbote's commentary). There is a hope (nadezhda) that, when Kinbote completes his work on Shade's poem and commits suicide (on Oct. 19, 1959, the anniversary of Pushkin's Lyceum), Botkin, like Count Vorontsov (a target of Pushkin's epigrams, "half-milord, half-merchant, etc.") will be full again.
Sun, 12/15/2019 - 11:35
Two Queries
'Orbicular' always brings to mind a description from Melville's Bartleby (a strange cousin of Gogol's Overcoat) where a rich client Astor is described: "I do not speak it in vanity, but simply record the fact, that I was not unemployed in my profession by the late John Jacob Astor; a name which, I admit, I love to repeat, for it hath a rounded and orbicular sound to it, and rings like unto bullion." The phrasing is impeccable in Melville.
Sorry for being away and inactive, but may I query of two things: does Kern and Charski have their full name mentioned somewhere in Gift, or is it just engineer Kern and lawyer Charski throughout?
Alexey Sklyarenko
Mon, 12/16/2019 - 09:44
In reply to Two Queries by Shakeeb_Arzoo
orbicular names: Adam Krug
At first I wanted to mention in my post Adam Krug, the main character in VN's novel Bend Sinister (1947) whose surname means "circle" in Russian and "mug" in German, but decided not to do it so as not to cram my post with superfluous information.
It seems that the full names of engineer Kern and lawyer Charski are not mentioned in "The Gift." Tomorrow I will write a separate post (I hope it will be funny) on engineer Kern who intimately knew the late Alexander Blok.
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text:
Snakes that Swim
Feb 01, 2012
Hi Dear Friends,
Well, be careful what you ask in other countries. The other day we were out in the village and one of the villagers walked by with a large dead snake. We all looked at it as he was taking it home for lunch. It got us thinking about snakes and we had never really asked much about snakes in Cambodia...we knew there were a lot of snakes in Burma.
Anyway after some asking and a little research we found out that every year they harvest about 4 million snakes out of the Tonle Sap Lake..http://youtu.be/9n-FioZU0Iw. where Rick has been known to swim. Also that there are a number of different snakes here. We have attached a couple of you tube videos. Needless to say we won't be swimming in the Lake this year and also we will be a little more careful where we walk. Also is attached a video of a very special Cambodian pet..see below.
So long swimming...
Kids International Development Society
Please add a comment
Posted by Ita on Apr 10th, 2013No cpomalitns on this end, simply a good piece.


text:
Agency did the hacking unless the cloned his phone which is most unlikely - he said, someone close to Bezos or his mistress must have done it.
My question is - what is the brother doing with his sisters shared dick-pics?
Angels and Insects?
This message has been edited 2 time(s).


text:
January 23, 2019
Windermere Foundation Surpasses $38 Million In Total Donations Raised
Posted in News from the Foundation by Marilou Ubungen
2018 concluded with another great year of fundraising and giving for the Windermere Foundation, thanks to the continued support of Windermere franchise owners, agents, staff, and the community. Nearly $2.5 million was raised in 2018, bringing our grand total to over $38 million raised since the Foundation's inception in 1989!
Read More >
December 17, 2018
Bringing Warmth And Good Cheer For The Holidays
Posted in Collections and drives by Marilou Ubungen
The winter holiday season is one of our favorites! It is also a busy time of the year for Windermere offices, as many are working with local non-profits to support families who need a little extra help during the holidays. From running clothing drives to hosting free photo events with Santa, our agents and offices are out in their communities helping those in need have a warm and merry holiday.
Read More >
December 03, 2018
A Lifetime of Making a Difference - Joan Tate Allen
Posted in News from the Foundation by Marilou Ubungen
Tis the season when we look back upon the year and give thanks for all that we have and count our blessings. At Windermere, we are thankful for all who generously donate to the Windermere Foundation, and we are grateful to have franchise owners and agents who go above and beyond to make a difference in the communities where they live and work.
Read More >
August 29, 2018
Providing Scholarships and School Supplies to Students in Need
Posted in Collections and drives, Grant recipients, and News from the Foundation by Marilou Ubungen
It's that time of year again! As families prepare to send their kids back to school, Windermere agents have been out in their communities raising money to support programs that provide school supplies and scholarships to students in need.
Read More >
May 10, 2018
Helping Fight Hunger in Our Communities
Posted in News from the Foundation by Marilou Ubungen
Many families do not have access to food assistance programs during the summer. And the national summer meal programs reach only a small percentage of children who need help when school is not in session--around 16 percent. That is why many of our Windermere offices choose to support local non-profits that provide weekend and summer meals to children and families in need.
Read More >
December 18, 2017
Making the Holidays Brighter in Our Communities
Posted in Collections and drives and Events by Marilou Ubungen
Every year, Windermere offices throughout the Western U.S. host events to help make the holidays a little brighter for our neighbors in need. From food drives to fundraisers, here are just a few examples of how our offices are giving back this holiday season.
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May 17, 2017
Windermere Helps Fight Food Insecurity by Funding Weekend and Summer Meal Programs
Posted in News from the Foundation by Marilou Ubungen
Through the Windermere Foundation, many Windermere offices throughout our network support programs that provide weekend and summer meal programs. These programs help girls and boys at risk of hunger get access to nutritious meals outside of school.
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March 02, 2017
Read Across America Day: Supporting Literacy Programs in Our Communities
Posted in Events by Marilou Ubungen
Today is Read Across America Day! Although not officially a national holiday, it is observed across the country by schools and libraries, taking a lead role in promoting the day. You can observe the day by picking up an interesting book and reading it with a child.
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November 14, 2016
Helping Fight Holiday Hunger in Our Communities
Posted in Collections and drives by Marilou Ubungen
The holiday season is a time in which Windermere offices across our network come together to help those in need in their communities. Here are just a few of the events that our offices are involved in this month to help fight hunger.v
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September 02, 2016
Raising money for those in need—one putt at a time.
Posted in Events by Marilou Ubungen
Thanks to fundraisers like these golf tournaments, as well as a variety of other fundraisers held by our offices throughout the year, the Windermere Foundation is able to continue to support non-profit organizations that provide services to low-income and homeless families throughout the Western U.S.
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text:
Community Briefs – September 10, 2018
Tag: Apra Harbor, ASAN, Chamolinian Room, Hee Jae Lee
Military training at FDM, in waters around Guam
ASAN, Guam—The U.S. military will conduct training at the island of Farallon de Medinilla from midnight Sept. 10 to 11:59pm Sept. 17.
Training will also take place in waters near Guam, west of Apra Harbor within a five-mile nautical radius:
• Sept. 10, from 6pm to 10pm
• Sept. 11, from 6pm to 11pm
It is recommended that the public, fishermen, and marine tour operators monitor the Coast Guard broadcast notice to mariners for advisories.
The general location of the training on the island of Farallon de Medinilla will be in a 12-nautical mile radius of the island. (PR)
Cancer survivors, caregivers support meeting on Thursday
All people facing cancer, their family members, and caregivers are invited to the next Commonwealth Cancer Association support meeting on Thursday, Sept. 13, starting at 5:30pm at the Hyatt Regency Saipan's Chamolinian Room. Guest speaker for the meeting will be nutritionist Hee Jae Lee. To RSVP or for more information, call the CCA at 682-0050 or email at firstname.lastname@example.com. (PR)


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Consulting Services
Karin Peter Consulting offers "Executive Search" not only in Germany but also in international markets, particularly in the markets of Eastern Europe, Russia, and India.
Our consultation services are supplemented by business consulting (especially for start-up projects) as well as assistance in contract drafting both domestically and abroad.
- Automobile Industry
- Automotive Supply Industry
- Logistics
- Mechanical Engineering
- Agricultural Engineering
- Commerce & Trade


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Goods worth lakhs gutted in fire at Sakchi shopJamshedpur December 16, 2013 No Comments on Goods worth lakhs gutted in fire at Sakchi shop 22
Jamshedpur, Dec 15: A major fire broke out at Furnishing House near Ansari Optical in congested Sakchi Market at around 6 in the evening destroying goods worth lakhs.
The market were the shop is situated is crowded most of time but with the brains working at the right time major incident was avoided and the fire was put out without damaging the shops located near to the furnishing house.
The shop owners and staffs of the area doused the fire by themselves as the fire brigade couldn't make its way into the congested lines of the market.
The hawkers have captured the footpaths meant for the shoppers and have made life difficult for the commuters.
A big incident was avoided but it's a wakeup call for the shopkeepers as well as the hawkers whose shops are situated in the area.


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Background How To Create An Online Course That Sells
Earning money with hobbies such as gardening, writing, Art, and fitness are not in the cards. You put together a professional online class to teach people all about it, unless of course. This Teachable review summarizes one of the options that are impressive for developing a gorgeous site for presenting your classes, from video courses to PDF training. How To Create An Online Course That Sells
We have taken a look Teachable's main Competitors: Udemy and Uscreen. Both of these mentioned have their pros and cons, but it's time to summarize Teachable, another firm. Teachable has fantastic customization tools clean site interfaces, marketing options and analytics.
Teachable's online course creation provides Hosting and security, along with all the learning tools you require, to courses from quizzes to blogs and talk forums. So read on if you want to have a look at our comprehensive Teachable review and see if it is the ideal online course builder for you.
Click here to subscribe to Teachable now
What's Teachable?
For those who aren't already familiar with the platform, Teachable is an online course platform or Learning Management System (LMS). Why do you want these? Because they simplify the online course creation and launch process by allowing you to upload your course content, not worry about the technology and design aspects. Yep, you don't have to stress over using plugins and WordPress for your courses.
I will not go on and on. I will only say this: there are A handful of LMS platforms I urge for online entrepreneurs, and Teachable is one of them. Here is my breakdown:
Which Teachable Features Stand Out?
Taking a look at the user layouts, it's Evident that Teachable is doing something right in terms of its website builder. In addition to that, the pricing looks fairly solid. What are a few of the principal features you can look forward to?
Teachable's Drop and Drag Builder
Drag and drop builders are getting better and And if you look at the user layouts from Teachable it's clear they have done a good job in that region. Not every Teachable user has lots of knowledge in web development, and that's kind of the point.
You can move classes around, organize pricing Packages, drop text and imagery on the site, and immediately develop sales pages (which we'll talk about below).
You have The option to upload lecture and media files (such as audio and PDFs) straight from your computer.
What is also interesting is that you can utilize Your existing site to add the path pages on there. Or, you can begin a site with a domain name by starting from scratch on Teachable. How To Create An Online Course That Sells
Advanced edits
One of the advantages of Teachable is you simply Don't have to be programmer or a coder sell and to create classes online. It enables you to start with a template, upload your content, and start earning money as long as your classes are good enough.
However, it makes sense that some individuals and Organizations might want to customization the Teachable interface further. This would let you move around more complex elements such as button positioning, image dimensions, and the overall branding of your website.
Teachable has something called a Power Editor, Which provides more sophisticated users the opportunity to tap in the code and create those customizations if needed.
Upload All Sorts of Files
Teachable supports the Vast Majority of documents you Requirement to get a course that is decent. Including video, picture, audio, text, and PDFs. You can pull your content from a vast array of sources like Google Drive or Dropbox, syncing up your storage areas with the website.
Learning and Community Tools Like Newsgroups, Blogs, and Quizzes
The quizzes will there be to provide content for Your users. I haven't ever taken a class online where I have not been excited to check my knowledge at the end. Teachable also provides a forum and blog for your website, turning it into more of a community than anything. Should you nurture this community you're more inclined to get some VIP customers who urge you to other men and women.
Ultimately, some of those Teachable plans provide Course completion certificates. This contributes to the feeling of community and provides pupils a feeling of accomplishment when they complete one of your classes. You can achieve this for just some of them or all of your courses. It allows you to customize each one of the certificates and add your personal flair to every one. How To Create An Online Course That Sells
Sales Pages
Landing pages are often crucial for promoting courses. The sales pages are designed by you, then purchase some ads linking on Facebook and Google to these. Customers are more likely to purchase from sales pages, and also you don't confuse people by sending them into the standard homepage.
In Teachable, revenue pages have been provided for you. All the templates have been tested to enhance your conversions, therefore all you need to do is go in there and personalize the template to match your own brand and courses.
What's also cool Is These earnings pages (and all Of the pages on your site) can be converted into a wide variety of languages. This way, global customers have the opportunity to look at your courses and potentially sign up. It is not a promise that these people are going to have the ability to comprehend your videos, but the text-based translations would allow you to open your business to people around the world.
Responsiveness for Seeing on All Devices
This ties into the design component of Teachable. In summary, Google wants to be certain your site is visible and user-friendly all devices, from desktop computers to smaller tablets and smartphones. Therefore, you need to ensure that the media and text are viewable on these devices with no problems.
Luckily, your Teachable content snaps into Place based on the device found. Consequently, if your student is watching your lectures on a phone, they can view everything in the ideal dimensions. The same can be said for computers and tablets.
Click here to sign up for Teachable now
Student Management
One of the trickier parts of running an internet Course making sure they take the courses, is handling students, and giving them the assistance they need.
1 Means to Do this is with the pupil comments module in Teachable. Basically, it lets you create items like surveys, Google Forms, and other feedback tools to get information from the students and see what they dislike or exactly what they'd like to see in the future.
Teachable's support integrations help out with Student management as well. Through Zapier you can link to tools like Zendesk and Olark, bringing you all sorts of alternatives for consumer information and remembering past conversations that you've had with specific students. How To Create An Online Course That Sells
Finally, student list segmentation is given to improve On student-relationships and your marketing. Simply speaking, the segmentation module has choices for sending out targeted emails based on particular criteria. For instance, you might send a welcome email when a student enrolls in your program out. Or, you may think about having a code redemption for those pupils who have not purchased one of your courses in quite a while.
Impressive Marketing Features from Teachable
Advertising features aren't always seen by you with online Course creation platforms. However, Teachable has promotions coupons, affiliate programs, email marketing and more.
You can play around with pricing that is innovative Choices, with options like payment programs, bundled subscriptions, and courses.
Customer Support
As You Might Have noticed at the pricing strategy area, Every program has product service. The skilled and higher Volume Plans provide priority product service, so I am presuming when contacting the corporation, you get bumped to the front of the line.
As for the means of contact, Teachable provides a Live chat on the website along with a system for you to submit your questions. But they route you through the knowledge base until you send a message to them.
Teachable also has a wonderful site and Weekly live workshops for learning and an academy full of classes and tutorials similar, tricks!
Overall, Teachable'service options look great. That doesn't appear to be in the cards, although it would be wonderful to see some phone support. How To Create An Online Course That Sells
What I Like
- Teachable is not difficult to utilize – I will not deny itTeachable is pretty darn simple to use. Subscribe to create a new course and upload your content. Done.
- Teachable is focused on helping you build your new – Teachable is not just setup that will assist you produce a course, it encourages you to build a school filled with classes. They would like you to be effective and make online courses part of your business model.
- Teachable is made for entrepreneurs – When you start working with Teachable, you realize that they understand their audience very well. They know you don't just need a solution you also need assistance with advertising and selling your program. And they have built in features with your needs in mind.
- Plenty of built in advertising and sales tools – Earnings pages. Promo videos. Plus webinars and articles .
- Flexible pricing alternatives – Multi-tier pricing, recurring coupons, bundle courses, and coupon/ voucher codes
- Great alternative for free courses – They have a totally free program. If your classes are free, then it won't cost you a dime.
Areas for Progress
- Can't Drip Content – This is really a characteristic a great deal of entrepreneurs utilize. It has to be an alternative.
- Course Builder is Unintuitive – The section of the program builder where you produce your structure and content is known as a Syllabus. Apart from being a misnomer, the Syllabus is just not intuitive. You need to click around to figure out things. To add content, you click on"New Lecture," whether you are adding a lecture. . .or a quiz, or a discussion. This can be confusing and unintuitive. It could be make more sense to have a dropdown with options to include actions, quizzes, projects, etc..
- Lack of Focus on Learning Elements – I'd love to find out more than just press, text and very basic quizzes. As an instance, the ability to perform live, office hours or synchronous workshops. Or the ability to express. How about course portfolios or projects? All of these are features that other platforms have.
- Integrations -There are not enough of these. They have got the basics but this is. At the moment, there are only six native integrations. For everything else, they rely upon Zapier. For instance, want to integrate the stage? You're out of luck if you don't use Mailchimp. . .unless Zapier is used by you. I would especially like to see integrations with Dropbox and Google Drive.
- Navigation menu – It seems weird. A sub-menu overlays the navigation menu After you click like Website or Courses. I find a tree-style menu or fall down, such as in WordPress.
As you can see, most of the regions of progress have to do with studying elements. That is my main gripe with Teachable: you get the bare bones. You soon realize that they have put nearly all of the work after doing some digging.
One of the trendiest parts of Teachable is that you Do not need to go out to get and cover your hosting. It is all included in the pricing package, which is fine for keeping your site up at all times and fast for users.
However, the best part is that the amount of Students and classes supported by Teachable. It seems like more often than not I stumble until money is paid by you upon services which have maximums for courses and students. That's not true with Teachable–since you can have pupils and courses for many programs. How To Create An Online Course That Sells
In Terms of the pricing, here's what Teachable offers:
Basic — $29 per month for all base features, 5% transaction fees, instant payouts, two admin users, product support, class creator training, custom domain support, voucher codes, trickle class content, incorporated affiliate marketing, integrated email promotion, and third party integrations.
Professional — $79 per month to get everything in the Basic program, immediate payouts, five admin customers, priority product support, graded quizzes, innovative theme customization, advanced reports, Teachable branding eliminated, and course completion certificates.
Business — $399 a month to get everything from the previous two programs, no transaction fees, instant payouts, 100 admin users, priority product support, manual student importing, bulk pupil enrollments, custom user roles.
The reason I enjoy this pricing plan set up Because it lets you test your courses out using the Basic Plan is. You still receive 95 percent of whatever comes in for the courses, although the 5% transaction fees are not ideal.
Recently, instantaneous payouts have been added to all of The plans, which means you get your money upfront.
Basic features:
We mentioned that all base features are included in The Basic program, therefore it makes sense to outline what some of those features are:
No fees should you give away courses for free.
Basic quizzes to test your students.
Chat forums for developing a community around your classes on Teachable.
Student management tools.
A integrated payment processing system to collect all payments securely.
Teachable offers nlimited hosting, students, courses, and video. How To Create An Online Course That Sells
A few notes on pricing:
During my Teachable inspection, I discovered that Teachable does not charge any setup charges or cancellation fees. This usually means that for is transaction fees and the monthly plan you may be incurring. You should also keep in mind that there are likely to transaction fees for payment processing, and this is all dependent on which option you go with.
A free trial is not, however you can configure an Accounts and play with the Teachable backend for as long as you would like. Since it lets you construct your course site and comprehend how it's going to function prior to launching, in my mind, this is much better than a trial. Teachable then starts charging your credit card as soon as you start the website.
The pricing programs are predicated On payments. So, you must pay for a year in advance to get that exceptional pricing. Paying monthly for Teachable means your monthly pricing will increase. If you don't pay for a year beforehand, As an example, the Basic plan goes up to $39 per month.
Who Should Consider Teachable as a Way to Make Money on the Web?
Teachable is great for both YouTubers, bloggers, Publishers, niche experts, authors, and a whole lot more. My Teachable review revealed that as long as you have some type of skill you can sell courses teaching that ability on Teachable.
You can try out Teachable without Punching in any payment information, so that's cool. There's no reason to pay for a strategy until you've got some of your courses made.
The sky's the limit, As soon as a program is made by you. I had Urge trying to get from Teachable's Basic Plan since you can take advantage of the zero transaction fees.
Any sort of class can go on Teachable. For Example, Teachable supports everything to web designers from YouTubers to authors and artists. How To Create An Online Course That Sells
Click here to subscribe to Teachable today


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Algo IT Solutions is a leading custom software and application development company with offices in Ontario Canada. We understand the importance of customized web and mobile application design and development. We work with a diverse clientele to develop customized multi-platform applications and help integrate technology in their business or bring their ideas come to life. Our focus on quality and innovative strategies to provide software application development services are beneficial to corporate and hybrid environments.
A new age company, We are apt at early adoption of new technologies for our clients to always stay ahead of the curve in their go-digital strategy. Our Services offering spans across Mobile & Web App, Digital Transformation, Digital Marketing, IoT & Wearable Apps, Creative Designs and many more.


text: The work of Yuki Matsuda is perhaps best-known with his footwear brand Yuketen, however the Japanese designer has also put his love for detail in Monitaly. The name of the brand comes from the name of his daughter Monica and his love for Italy. All his products, just like the footwear from Yuketen, are with a heritage feel and high quality construction. The Monitaly Jackets are inspired from the 1940's heavy wool motorcycle jackets.


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Liberal Minyan Shabbat Morning Service
Please click here to RSVP to receive Zoom info, which will be sent out Friday before 5pm.
At our Liberal Shabbat morning services we draw on our inherited traditions in a consciously modern context. Using the Reconstructionist prayer book, Kol Haneshamah, we pray old and new liturgy in both Hebrew and English. We read Torah in a triennial cycle, and there is always a discussion of the week's parashah. Healing prayers are offered at every Torah service, and there is always an opportunity to say Mourner's Kaddish at the end. The service ends at 12:30 pm and is followed by a kiddush lunch. To sponsor a Kiddush, please email Ruth in our Development Office, email@example.com. CBST celebrates Shabbat morning services in a variety of styles. Each is an opportunity to mark smachot (happy occasions), observe lifecycles, and commemorate losses. We engage with prayer and Torah, and provide the means for spiritual and communal connection.
Prayerbook Dedication Opportunities
**MAKE SURE that when you join us via Zoom, that your "display name" is your actual name; to make sure that our services are protected from those who might disrupt them, no participants will be allowed entrance who use a generic (like "iPhone/iPad") or fraudulent names (like "Judy Garland") as their display name.
Please take a moment to review below some important information about how to use Zoom, and the proper etiquette for participating in Zoom Meetings.
Zoom Instructions & Etiquette
Zoom is a free technology for calling and videoconferencing. You don't need a personal Zoom account. It's available on your desktop computer, iPhone, iPad, Android, or Mac. Overall Guides: Zoom has step-by-step instructions on how to use it here; This is also a good guide to using Zoom; This is the simplest video on how to "join a meeting."
- Please "arrive" 10 minutes early for any Zoom call so that we can start on time.
- Please note: Zoom works better on a computer (laptop or desktop). Using Zoom via phone will limit your capacity to participate.
- Access to some events may close 10 minutes after the start time, so if you arrive after that you will not be able to get in. Please check the program details.
- Please note: most CBST Zoom programs are video recorded. By participating, you authorize your consent to be video and audio recorded. If you do not want to be recorded, please mute yourself and turn off your video.
- On most CBST Zoom programs, the meeting leader will control participation. You may be muted upon entry into the program, or on and off throughout the program. By default, please remain on mute unless you are asked to unmute, or you are umuted by an admin.
- Any offensive content will not be tolerated and any offending participant will be removed from the program immediately by an admin.
Video/Microphone: If your computer has a camera, you can use video (or audio only). If you want to talk, you must have a microphone or call in on the phone.
Basic Requirements and Directions: Directions if you want to use Zoom on Windows (PC) or Mac; You can download the app here for your computer.
On Your Phone:
- On your iPhone or Android, download the app from the App Store/Google Play--search for Zoom Cloud Meeting and download the app.
- Once you are on the Zoom class/conference/meeting--with or without video, on your phone, tablet, or computer: Make sure you are in a relatively quiet place. If you talk to someone else when you are unmuted, we can hear you. Consider earphones or headphones. In a large group, we will mute you.
- If you want to "raise your hand" to be recognized, hit *9 (star 9).
- To unmute or mute yourself, press *6 (star 6).
Advanced Instructions
- Here are directions for how to "raise your hand" on different platforms.
- Here are directions on how to use the spacebar to unmute in large groups.
United States


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Parents At Lunch Day
The Parent at Lunch Program: Each month parents have the opportunity to join their child (or children) at lunch! The sign up for the lunch is via the Google documents link here. The program is limited to 8 spots per grade and the office must be able to verify your CORI status for you to attend. The name you sign up with on the Google Doc should match the name on your license EXACTLY. In order to attend PAL, you must have an approved CORI on file with Alcott. CORI's are valid for 3 years and each organization/school is required to have their own CORI. When you complete the CORI form, the school must also view a photo ID such as a driver's license. Back to School night is a good opportunity to complete the form. Once a grade is full, we use a wait list for the next month. Names on the wait list have priority for the next month's lunch. For more information about the program, please click here.


text: The Marcus Foundation Northside Hospital (Atlanta) Mizuno Kubota Gwinnett Medical Center Goddard School For Early Childhood Development Gas South City of Stockbridge City Club Of Buckhead Childrens Healthcare of Atlanta Dunkin Donuts United Health Group Quest Piedmont Hospital worldpay Sibley Heart Center Chicos Clothing Aetna As well as hundreds more! What can we do for your company? CONTACT


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Mathew's Stardom 5 Star GP Red Stars Results & Review: Day 2
Red Stars Day 2! Does Hana Kimura keep her early momentum going?
Red Stars Day 2! Does Hana Kimura keep her early momentum going?
Welcome back to Stardom's 5 Star Grand Prix as we now look at the second day of the tournament.
We got one making their debut in the tournament today and some others who are looking to either stay undefeated or avenge their first-day loss.
We had an average day one and let's see if this day will be any better as we…dive right in.
Star Rating System:
- 0 Stars: Dave Meltzer
- 1 Star: Vince Russo
- 2 Stars: Tony Schiavone
- 3 Stars: Eric Bischoff
- 4 Stars: Bruce Prichard
- 5 Stars: Jim Cornette
5 Star Grand Prix Red Stars Block Match
Momo Watanabe vs. Avary
Review: Our first match to start it off for my block is Momo Watanabe taking on something who is making their Grand Prix debut. Avary. I'm not familiar with Avary but I know she's a wrestler from Australia and has been wrestling since 2016, so I'm curious to see how she'll fare against Stardom's ace. Momo lost the first day against Hazuki and is now looking to get a win here. Will Momo be able to get a victory or does she take two losses in a row to have herself in a bad start?
So, how did Avary do in her Grand Prix debut? Honestly, I thought she was rather impressive as she was chaining well, sold her moves right, was mostly crisp, and has a nice look on top of it off. Momo being her first opponent was the right call since she was able to use this chance to showcase some of her skills better to look forward to her future matches. Avary applies the Cattle Mutilation with a beautiful bridge as Momo would have her foot on the rope to break the hold. Avary had another advantage as she had Momo hanging on the ropes and hits her with a Double Stomp for a two count. Momo was able to hit a Penalty Kick to get her down and called for the Tequila Sunrise but Avary was struggling until Momo kicked her arm and hits the Peach Sunrise for the victory! Solid match and nice debut for Avary, hope to see more from her.
Now on https://t.co/q8dHnF58vW! It's night 2 of the 5★STAR GP 2019 and Momo Watanabe faces Avary! pic.twitter.com/jV8ZMSuYY1
— We Are Stardom (@we_are_stardom) August 21, 2019
Rating: Eric Bischoff
5 Star Grand Prix Red Stars Block Match
Natsu Sumire vs. Hana Kimura
Review: Our next match is Natsu Sumire taking on Hana Kimura. These two are no strangers to each other as Hana used to be apart of Oedo Tai and Sumire could use this time to get a little bit of revenge for betraying the unit. Natsu Sumire lost to AZM on the first show, so a win here could give her that boost and Hana could go undefeated since she defeated Tam Nakano and could get another win. Will Hana be undefeated or does Natsu ruin that?
Sumire didn't have time to do her entrance as she just bolted into the ring to kick Hana off the top ropes and began to attack her before the bell even rung, you can tell that she is pretty pissed off here. To me, this was a much better improvement for Sumire since she was able to have a much better match here and it does look like she's going into serious mode throughout the whole tournament, but we'll see how long she keeps that part up. This match was a lot more physical than I anticipated it to be and man, Hana threw some very heavy forearms around the middle of the match here while holding her head still but that was what made it exciting because you could just feel the tension between the two and they sold it the right way, by kicking the crap out of each other. Hana was able to generate heat from the crowd when it looked like she was about to hit Sumire with a forearm but would spit in her face instead as the crowd booed her for it, love it. Sumire had Hana in the Dragon Sleeper and it looks like she could be out for the count as she wasn't responding, so Sumire went for the pin but Hana would barely kick out! Sumire would get her up for the DDT but Hana would reverse it to apply the Hydrangea and Sumire taps out, leaving Hana undefeated!
Rating: Eric Bischoff and a quarter
5 Star Grand Prix Red Stars Block Match
Mayu Iwatani vs. AZM
Review: Our next match-up is AZM taking on Stardom's Icon, Mayu Iwatani. AZM was able to get her first win in her Grand Prix debut when she was able to defeat that grandma face, Natsu Sumire. Mayu, however, she didn't have good luck as she lost in her first match against Saki Kashima and looks to get a win back on the board if she wants to win back-to-back with the tournament. Will AZM take care of another 'grandma' or will Mayu put the child in her place?
Mayu doing two back to back fast-paced matches here with everything going on and of course, she delivers again with AZM. Much like Sumire's performance today, AZM did just as well here when she fought Mayu and was even able to keep up with Stardom's Icon in this match but then again, AZM is a rising star in the making when she gets older. While they didn't do too much in the match, it was still enough to make you wanna be interested in it and is still very satisfying with the time length they gave us as well. AZM hits a beautiful La Mistica this time around as she applied it tightly onto Mayu to try and get her to tap out but Mayu's foot was on the rope, so she had to break the hold. AZM quickly goes to the top rope to hit a Double Stomp and then a Suplex for a two count. This is where it gets a little crazy at the end since it's the both of them rolling each other up with flash pins to see who is going to get the other and nothing but reversals, and it's beautiful. AZM almost had her a couple of times but Mayu was able to reverse one more and held her legs down tight to get her first win of the tournament! Very fun, plenty of reversals, satisfying five minutes!
Now on https://t.co/q8dHnF58vW! Mayu Iwatani takes on AZM in Red Stars action! It's the 5★STAR GP 2019! pic.twitter.com/4CooKzZ4MR
— We Are Stardom (@we_are_stardom) August 22, 2019
Rating: Eric Bischoff and a half
5 Star Grand Prix Red Stars Block Match
Hazuki vs. Tam Nakano
Review: Our final match for my blocks on this show is Hazuki taking on Tam Nakano. Hazuki was able to get a win by defeating Momo Watanabe while Tam suffered a loss to Hana Kimura. If Hazuki could get a win here, she'll be tied with Hana for first place but if she were to lose, she'd be tied with the majority of the block except for Avary and Natsu Sumire. Will Hazuki be undefeated or does Tam pull an upset?
soon as that bell would ring, Hazuki attempted the Hazukistral and Tam quickly kicked out of it and it looks like Hazuki is hitting her big moves right away as she hits the Codebreaker right after that to get Tam down on the mat. Much like Tam's last match, her opponent got most of the offense to create heat and that is what Hazuki does here despite being one of the crowd favorites, but Tam still got some support and had an earlier comeback. Tam was almost able to apply an Armbar after catching her to flip her over impressively but Hazuki wouldn't let her get it all the way in and attempts the Hazukistral one more time as Tam shifted her weight to roll her up as Hazuki barely kicked out! The match is getting more interesting as time moves on as Hazuki would have Tam's legs up as if she was going for the Shattered Dreams and hits a Codebreaker in that position but Tam still kicked out of it. Hazuki was calling for the finish as she lifted Tam up but Tam fought out of it to roll her around for a Spinning Back Kick to take Hazuki down to the mat and Tam capitalizes on this opportunity by hitting the Tiger Suplex with a bridge to pin her and Tam gets her first win in the tournament! Damn solid match by both of these two women here and my only complaint is that it could've used another minute or two to elevate it more and bring it home better, but I am still very much satisfied with the match and the outcome.
Rating: Eric Bischoff and three quarters
Overall: None of these four matches on my block were bad at all and I'm very happy to have that outcome. It's a shame one of them is still gonna be on my least favorite despite being good, but rules are rules. Much better night compared to day 1 and I hope the quality improves from here on out to make it much more exciting. Our next show is a doubleheader as they'll be doing a noon and night show for Sunday and we'll cover both as our next part soon as they're all uploaded!
Favorite Match: Hazuki vs. Tam Nakano
Least Favorite Match: Momo Watanabe vs. Avary
Score: 7.5/10
Red Stars Standing:
- Hana Kimura (2-0) (4 Points)
- Hazuki: (1-1) (2 Points)
- Saki Kashima: (1-0) (2 Points)
- AZM (1-1) (2 Points)
- Mayu Iwatani (1-1) (2 Points)
- Momo Watanabe (1-1) (2 Points)
- Tam Nakano (1-1) (2 Points)
- Natsu Sumire (0-2) (0 Points)
- Avary (0-1) (0 Points)
Let us know what you think on social media @ChairshotMedia and always remember to use the hashtag #UseYourHead!
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Andrew's G1 Climax 30 B Block Results & Match Ratings: Day 2


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Editor's Note: The views set forth below undoubtedly reflect the probable positions of the majority of the members who oversee the confirmation of the Administrator of OIRA. Notwithstanding the merits of the arguments presented therein we believe the social entrepreneurial skills of a nominee out rank all other considerations. Recognizing that if one is to be successful in the regulatory space an individual must have a footing in an established discipline, how many of the game changing events that lead to the establishment of OIRA were dependent primarily on economic or legal skills? We are not convinced that the skills necessary to establish the most significant institutional feature of the regulatory state differ from those necessary to operate it on a sustainable basis.
The following quotes are drawn from the Washington Post article on the nomination of Professor Neomi Rao to be the head of OIRA.
Rao seems to have impressive legal credentials. But this job, it seems to me, ought to require someone with considerable training and experience in economic analysis and statistics. It's not clear how well-qualified she is on that front. Ultimately, whether a regulation is a good idea or not is not a question of Chevron deference or other legal questions, but of empirical analysis. I hope she understands that.
There are plenty of people within OIRA with that expertise. The key qualification for an effective OIRA administrator is really the ability to understand the broader legal environment and how to make sure regulatory policy is conducted consistent with the Administration's priorities. …more
4/11/2017 1:50 PM EDT [Edited]
OK, but doesn't understanding "the broader legal environment" more or less mean understanding the permitted scope of regulations, and the required procedures around putting them in place, or eliminating them? That's important, sure, but understanding their actual effects – beyond sloganeering – and being able to advocate intelligently, for or against, looks pretty vital to me, and something that needs more than just reliance on a staff report. I mean, if a non-lawyer technocrat were appointed and you argued that legal expertise was needed I could say, "Gee, aren't there staff attorneys?" Why is one thing and not the other the "key qualification?" ….more


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Hi-Tek Making A Beat From Scratch
For your viewing pleasure…Legendary Producer Hi-Tek making a beat in the Studio!
For your viewing pleasure…Legendary Producer Hi-Tek making a beat in the Studio!
50 Cent reunited with G-Unit at Summer Jam 2014
In this video Dave goes over 5 mixing plugins that every audio engineer should get to know. These plugins can improve your mixes and get your sound to the next level without breaking the bank. Credit: Pensado's Place on Youtube
Here is a very useful tutorial for producers who are using Maschine to sequence their beats. Credit: Maschine Masters Youtube Channel
We've been hearing a lot recently about Bitwig, a new player in the Digital Audio Workstation (DAW) market. If you haven't heard about Bitwig here is a quick overview (taken from the Bitwig Website) Bitwig Studio is a dynamic software Read More
Multi-task. Work on 2 to 3 beats at once, and switch back and forth between them. Finish them one at a time, and don't rush your music. The point of this life hack is to work on beats in "batches". Read More
1. "Can I get a free beat/studio time?" 2. "Let's collaborate." Most producers have heard this one 1,000+ times and already know what it really means: working for free. 3. "Work with me. I'm really well known in my town Read More


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I always feel like there's an excitement in the air this time of year, a freshness that's just waiting to be plucked. The new year is pregnant with possibility and is just waiting for us to snatch it up. In celebration of that, here are some revisited prompts to get your creativity and inspiration going. Enjoy!
Creative Commons love to http://www.flickr.com/photos/bartmaguire/ for the photo! Thanks!


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September 5, 2019
Press Release: Particle Dynamics announces new Innovation Center!
ST. LOUIS, Sept. 5, 2019 /PRNewswire/ — Particle Dynamics today announced the completion of a new, state-of-the-art Product and Process Innovation Center located within its US-based, St Louis facilities. The completion of the strategic expansion marks a key milestone in Particle Dynamics' transformation…More…
January 29, 2019
Particle Dynamics posts another successful FDA inspection!
Particle Dynamics is pleased to announce another successful FDA inspection which again resulted in no FDA Form 483 observations. The January 2019 inspection was the second unscheduled FDA inspection of Particle Dynamics facilities in one month. This multi day inspection…More…
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