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Fluorescent Oligonucleotide Probes for Clinical and Environmental Detection of Acanthamoeba and the T4 18S rRNA Gene Sequence Type
Diane R. Stothard, John Hay, Jill M. Schroeder-Diedrich, David V. Seal, Thomas J. Byers
Diane R. Stothard
Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, 1 and
John Hay
Tennent Institute of Ophthalmology, Western Infirmary, Glasgow University, Glasgow, United Kingdom 2
Jill M. Schroeder-Diedrich
David V. Seal
Thomas J. Byers
The first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of Acanthamoeba are described. Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA). The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific forAcanthamoeba as shown here by its hybridization to growing trophozoites of all 12 known Acanthamoeba 18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (Hartmannella vermiformis and Balamuthia mandrillaris), two human cell lines, and two bacteria (Pseudomonas aeruginosa and Escherichia coli). The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for Acanthamoeba 18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types. T4 is the subgenus group associated most closely withAcanthamoeba keratitis (AK). GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls. GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed. Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of Acanthamoeba in 21 of 24 specimens from the 22 patients. The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genusAcanthamoeba in both environmental and clinical samples. In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.
The genusAcanthamoeba consists of small, ubiquitous amoebae that exhibit a biphasic life cycle consisting of a vegetative trophozoite stage and a physiologically static cyst stage. These amoebae have been isolated from a variety of environmental sources (7) and are associated with human infection (17,24-26, 37, 39). In immunocompromised patients,Acanthamoeba infections include granulomatous amoebic encephalitis (GAE), which is a fatal brain disease, and disseminated infections of various other tissues. In otherwise healthy individuals, the prominent disease is Acanthamoeba keratitis (AK), a potentially sight-threatening eye infection. This disease was discovered in 1973 (19, 27). More than 500 cases of AK had been reported in the United States by 1993 (31), and another 200 cases had been reported by 1997 (25). AK is most prevalent among contact lens (CL) wearers (34). A recent prospective population-based study estimated that the incidence of AK among soft-CL wearers in Scotland was 1 in 6,710 (32). In other countries—for example, India—AK usually is associated with non-CL-associated ocular trauma (33).
Identification of acanthamoebae in ocular and other tissues can be difficult and time-consuming, even for trained microscopists. In histological preparations, these amoebae look very similar to keratoplasts as well as neutrophils and monocytes, and this often leads to false-negative and/or false-positive results. It has been estimated that up to 70% of clinical AK cases are misdiagnosed as viral keratitis (2, 12, 18). Thus, many patients are initially treated with inappropriate drug therapies.
Early detection of Acanthamoeba infection is important because trophozoites, which predominate in the initial stages of infection, are more susceptible to treatment than the subsequent cysts. Calcafluor white (42) and similar fluorescent stains, which often are used to detectAcanthamoeba, stain cyst walls and fail to detect trophozoites. The mean time to diagnosis of AK can average 2.5 weeks longer for non-CL wearers than for CL users (6). This lag time may hamper disease resolution, since several studies have found that diagnosis and treatment within 1 month of onset results in lower morbidity and a better visual outcome (1, 2, 38). Thus, the availability of a rapid, accurate, and relatively simple diagnostic test would significantly enhance the initiation of appropriate chemotherapy.
Several RNA- and/or DNA-based methods are being developed to aid in the detection and accurate identification ofAcanthamoeba in clinical and environmental settings. These include PCR with and without DNA sequencing (16,23, 35, 40), restriction fragment length polymorphism analysis (5, 36), and nucleic acid blotting methods (9). Although these techniques are important diagnostic aids, they involve indirect evaluation of samples. They are unable to distinguish different developmental stages of Acanthamoebaspp. and lack precision in the localization of the organisms in situ. These methods cannot determine whether the majority of amoebae in a sample are cysts or trophozoites; they also cannot discern the density of the infection or the depth of corneal penetration of the amoebae. Traditional histological techniques, in trained hands, often can identify amoebae in situ, but this depends on morphology or immunoreactivity. Furthermore, structural characteristics for identification of acanthamoebae at the subgenus level are unreliable.
The most specific and sensitive technique devised to date for in situ staining is phylogenetic staining, first used by DeLong and coworkers (8) for identification of single cells in samples of mixed species. This technique uses oligonucleotide probes complementary to phylogenetically informative segments of rRNA. Different probes can provide accurate identification of an organism at various levels of taxonomic organization. In addition, specific probes labeled with different chromophores can be used in fluorescent in situ hybridization (FISH) to differentiate the different species or other taxonomic levels in a mixed sample.
The present article reports the development and use of two fluorescent probes. The first identifies all members of the genusAcanthamoeba (genus-specific probe [GSP]); the second is specific for the subgenus group that is most commonly identified in AK infections (sequence type T4-specific probe [ST4P]). We have used these probes for identification ofAcanthamoeba in laboratory cultures and in human corneal scrapings from patients presumed to have AK based on traditional methods of microscopy and follow-up cultures.
Cell cultures and corneal scrapings.The in situ hybridization of the modified universal probe (MUP), GSP, and ST4P described below was tested with cultured trophozoites from 21 strains (Table 1) representing the 12 differentAcanthamoeba 18S rDNA sequence types previously described (35). Cultures were maintained at Ohio State University (OSU) as described previously (4, 10). GSP also was tested with 24 human corneal scraping specimens obtained in a study of microbial keratitis in Scotland (32).Acanthamoeba terricola was a gift from Frederick Schuster, Brooklyn College, Brooklyn, N.Y. Human U937 lymphocytes and human HeLa cells were provided by the OSU laboratories of Ing-ming Chiu and Mark Muller, respectively. The amoeba Balamuthia mandrillaris and the Acanthamoeba strains prefixed by CDC in Table 1 were gifts from Govinda S. Visvesvara of the U.S. Public Health Service's Centers for Disease Control and Prevention, Atlanta, Ga. Hartmannella vermiformis andAcanthamoeba palestinensis, originally from the Cambridge Collection of Algae and Protozoa (see Table 2), were provided by Peter H. H. Weekers, University of Nijmegen, Nijmegen, The Netherlands. The remainder of the Acanthamoebacultures (see Table 2) were obtained from Thomas Nerad at the American Type Culture Collection, Manassas, Va. Bacterial specimens were from laboratory cultures at OSU.
Acanthamoeba strains used in testing the genus- and T4-specific probesa
The corneal scrapings examined here were isolated by two of us (J.H. and D.V.S.) during a population-based longitudinal study of microbial keratitis in western Scotland (32). Scrapings were placed in sterile isotonic saline after being obtained from the eye by an ophthalmologist. Unstained aliquots were examined by bright-field or phase-contrast microscopy. This was followed by culture of samples forAcanthamoeba in a medium described previously (15). One to 19 months after the initial specimen collection, portions of the original scraping samples were delivered to OSU, where they were retested for growth in culture and analyzed with the GSP probe described in this paper.
Pretreatment and fixation of amoebae and other cells for FISH.All solutions, with the exception of culture media, were prepared with diethylpyrocarbonate-treated double-distilled water (DEPC water). Portions of the human corneal scrapings (see Table 2) that had been suspended in sterile saline and stored at 4°C for 1 to 19 months were fixed. One milliliter of a laboratory cell culture or 100 μl of a scraping specimen was spun in a Microcentrifuge (Costar model 10) for 15 s to loosely pellet the cells. The medium was pipetted off, and the cells were resuspended by gentle shaking in 1× phosphate-buffered saline (PBS; 0.13 M NaCl, 7 mM Na2HPO4, and 3 mM NaH2PO4 in DEPC water; pH 7.2). The cells were then loosely pelleted again by centrifugation for 15 s. The wash buffer was pipetted off, and the cells were gently resuspended in 1 ml of cold (4°C) 12% freshly depolymerized paraformaldehyde (PFA). The PFA was made by dissolving 6 g of paraformaldehyde in 40 ml of 1× PBS for 10 min at 60°C with stirring. Then 10 ml of 0.1 M NaOH in DEPC water was added to clear the solution, and the mixture was chilled before being used. The cells were fixed at 4°C for at least 5 h and to a maximum of overnight. After being fixed, the cells were loosely pelleted by a 15-s centrifugation. The PFA was pipetted off, and the cells were washed in 1 ml of DEPC water with gentle shaking. The cells were loosely pelleted again by a 15-s centrifugation. The DEPC water was pipetted off, and the cells from cultures or corneal scrapings were resuspended in ∼100 or ∼30 μl, respectively, of 70% ethanol and stored at 4°C indefinitely.
Probes.Three oligonucleotide probes containing a fluorochrome at the 5′ end were synthesized by Amitof Biotech, Inc. (Boston, Mass.). The 21-base MUP (5′-rhodamine-GWATTACCGCGGCTGCTGGCA-3′) was complementary to positions 653 to 633 in the 18S rRNA of Acanthamoeba castellanii Neff (13). The 22-base GSP (5-fluorescein-TTCACGGTAAACGATCTGGGCC-3′) was complementary to positions 957 to 936 in the Neff strain rRNA. The 30-base ST4P (5′-rhodamine-GCTGCCAAAACCAACTGAAAATAGGAGGAC-3′) was complementary to positions 1066 to 1037 in the Neff strain rRNA. MUP is based on the universal probe designed by Giovannoni and coworkers (11), with three modifications: base 14 was changed from K to T, a G between bases 16 and 17 was removed, and a GCA triplet was added to the 3′ end. Probes were resuspended in DEPC water to a final concentration of 1 μg/ml and stored at −20°C. Immediately before hybridization, probes were diluted to 30 ng/ml in DEPC water.
Whole-cell hybridization.Cells fixed in suspension were pipetted onto poly(l-lysine)-coated microscope slides (Sigma, St. Louis, Mo.) in 2- to 5-μl aliquots and air dried. The slides were then incubated in a methanol-formalin mixture (9:1) at room temperature for 20 min. The slides were then rinsed briefly in DEPC water and allowed to air dry. Thirty microliters of a hybridization mixture containing 60 ng of each probe in hybridization buffer (0.9 M NaCl, 20 mM Tris [pH 7.8], 5 mM EDTA, 0.01% sodium dodecyl sulfate) was pipetted onto each dry slide and covered with a 24- by 50-mm coverslip. The slides were immediately transferred to a dark chamber humidified by a sponge soaked in DEPC water and incubated at 50 to 52°C for no more than 2 h of hybridization. Temperatures above 52°C led to a reduction in the fluorescence signal for all probes used.
After hybridization, the coverslips were floated off in DEPC water at room temperature and the slides were washed in wash buffer (30 mM NaCl, 4 mM Tris [pH 7.8], 1 mM EDTA) at 50°C for 20 min in the dark. The slides were rinsed quickly in DEPC water at room temperature and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) for 10 min at room temperature in the dark. The slides were next washed briefly in DEPC water and allowed to air dry in the dark. Coverslips were mounted on dry slides by using 1 drop of Anti-fade Light mounting medium (Molecular Probes, Eugene, Oreg.) and permanently sealed with nail polish. The slides were stored indefinitely in the dark at 4°C. The preparations were examined with an inverted microscope (Zeiss Axioscope, Oberkochen, Germany) fitted for epifluorescence detection with a high-pressure mercury bulb and filter sets CZ902 (for DAPI), CZ 915 (for rhodamine), and CZ 909 (for fluorescein).
Use of MUP with trophozoites.MUP was used here as a control that would stain all organisms tested in the FISH analyses. It was derived from the universal probe, designed by Giovannoni et al. (11), which originally was made to hybridize to all three domains of life: archaebacteria, eubacteria, and eukaryotes. In the present study, however, the probe of Giovannoni and coworkers did not provide a satisfactory signal due to the higher-stringency conditions used. Modification of two sites to be more consistent with the eubacteria and eukaryotes used in the present study and addition of 3 bases at the 3′ end led to a 100% match between MUP and targets and an unambiguously positive red fluorescent signal from all cells tested. For example, hybridization of MUP with B. mandrillaris andA. castellanii is illustrated by the red stain in Fig.1.
–12. In situ hybridization of MUP, GSP, and ST4P with seven sequence types of Acanthamoeba and with its closest relative, the genus Balamuthia.
FIG. 1. One amoeba of A. castellanii V014 (T4) (upper right) and two amoebae of the larger B. mandrillaris V139 are stained red by MUP.
FIG. 2. Same field as Fig. 1, but only A. castellanii V014 is stained green with GSP.
FIG. 3. GSP stained amoebae of Acanthamoebaspecies strain V006 (T1) green.
FIG. 4. A. palestinensis 1501/3c (T2) amoebae were stained green by GSP.
FIG. 5. GSP stained amoebae of A. griffini Panola Mt (T3) (bottom amoeba) and A. castellanii V014 (T4) (top amoeba) green.
FIG. 6. Same field as Fig. 5, but only A. castellanii V014 (T4) is stained with ST4P. Amoebae stained with this probe appear red because it has the same fluorochrome as MUP.
FIG. 7. GSP stained A. lenticulata PD2S (T5) amoebae.
FIG. 8. A. tubiashi OV-15c (T8) amoebae were stained by GSP.
FIG. 9. GSP stained Acanthamoeba species cysts from a corneal scraping.
FIG. 10. Amoebae of A. terricola FS (T9) (the three large amoebae) and A. castellanii V014 (T4) (the single smaller amoeba) were stained by GSP.
FIG. 11. Same field as Fig. 10, but only A. castellaniiV014 (T4) is stained red with ST4P.
FIG. 12. Two sectioned cysts of A. castellanii stained with GSP and showing green amoebae within yellow cyst walls.
Use of GSP with trophozoites.Trophozoites double stained with MUP and GSP were used in tests for determining the specificity of GSP. Representatives from all 12 previously identified sequence types ofAcanthamoeba (35) (Table 1), two amoebae relatively closely related to the genusAcanthamoeba (H. vermiformis[41] and B. mandrillaris), bacteria (Pseudomonas aeruginosa and Escherichia coli), and human cells (U937 and HeLa) were tested for hybridization with these two probes. As indicated above, MUP hybridized withAcanthamoeba and all control cells. GSP hybridized with all acanthamoebae tested but with no control organisms. The differential staining of MUP and GSP is illustrated in Fig. 1 and 2, which include a mixture of one relatively small acanthamoeba of sequence type T4 and two larger Balamuthia amoebae. With the rhodamine filter (Fig. 1), all three amoebae are red, indicating hybridization with MUP. With the fluorescein filter (Fig. 2), only the smaller amoeba is green, indicating that GSP hybridized specifically toA. castellanii. Evidence that GSP hybridizes with divergent sequence types of Acanthamoeba is illustrated by the green staining of amoebae of seven sequence types that had been hybridized with this probe, as shown in Fig. 3 (T1), 4 (T2), 5 (T3 and T4), 7 (T5), 8 (T8), and 10 (T9 and T4). See below for identification of T4 amoebae in Fig. 6 and 11.
Use of ST4P with trophozoites.The specificity of ST4P was tested by using cells doubly stained with GSP and ST4P. Amoebae from each of the 12 described 18S rRNA gene (rDNA) sequence types ofAcanthamoeba (Table 1) plus the bacteria, human cells, and other amoebae described above were used. ST4P successfully hybridized with all acanthamoebae of the T4 sequence type, but not with acanthamoebae of any of the other sequence types or with control cells. The degree of specificity is best observed in Fig. 5 and 6, which illustrate differential staining of the two most closely related sequence types, T3 (A. griffini Panola Mt.) and T4 (A. castellanii V014). In Fig. 5, amoebae of both sequence types appear green with the fluorescein filter, indicating that both types have hybridized with GSP. In Fig. 6, however, only the amoeba of sequence type T4 appears red with the rhodamine filter, indicating that it alone has hybridized with ST4P. The specificity of ST4P also is seen in Fig. 10 and 11, in which staining with this probe and with GSP is compared for T4 (A. castellanii V014) and a representative of a more distantly related sequence type, T9 (A. terricolaFS). Both amoebae are green in Fig. 10, indicating that both hybridized with GSP, but only the smaller A. castellanii is red in Fig. 11 and, thus, was able to hybridize with ST4P.
Use of GSP with cysts from corneal scrapings.Hybridization of GSP with intact cysts from a corneal scraping is illustrated by the green stain in Fig. 9. In addition, Acanthamoebacyst walls autofluoresced either in the presence or in the absence of hybridization conditions. Attempts to quench the fluorescence with a number of different agents failed. Nevertheless, the fluorescein filter set did distinguish between the yellow autofluorescence of the cyst walls and the green fluorescence attributable to hybridization of the fluorescein-labeled GSP to the rRNA and rDNA of the encysted amoebae. This distinction is best seen in sectioned cysts from the human cornea (Fig. 12).
To test the usefulness of GSP with corneal scrapings, we employed 24 specimens obtained from 22 patients, 17 with either confirmed AK or a high index of suspicion of AK and 5 with non-Acanthamoeba keratitis or conjunctivitis (Table 2). Bright-field and phase-contrast microscopy were used with wet films to evaluate the specimens. Fifteen scrapings produced positiveAcanthamoeba cultures at Tennent Institute of Ophthalmology (TIO), OSU, or both institutions. It was observed, however, that wet-film microscopy detected acanthamoebae in only 6 (40%) of the 15 culture-positive specimens.
Comparison of bright-field and phase-contrast microscopy, culture growth, and FISH evaluations of corneal scrapings as follow-up to clinical presumption of AK
Microscopy using FISH with GSP was then used to evaluate the corneal scraping specimens. Acanthamoebae were detected in 12 (80%) of the 15 culture-positive samples. Overall, FISH results were consistent with the positive or negative culture results for 21 (88%) of the 24 specimens. The only discrepancies were three FISH-negative specimens (no. 1a, 17, and 23) that were considered culture positive even though positive results were obtained at only one of the two institutions (see Discussion).
The major advantage of using fluorescent oligonucleotide probes and FISH for identification of Acanthamoeba is that such probes have the potential to simultaneously detect and classify amoebae in situ. FISH provides a means of rapid, unequivocal identification of Acanthamoeba in cases of AK for which clinical diagnosis is putative and the appropriate expertise for identification by bright-field and/or phase-contrast microscopy is unavailable. As we have seen here, even when that expertise is available, the number of false negatives can be relatively high in the absence of FISH unless the observations are backed up by tests for culture growth. However, culture may take a week or more to yield satisfactory division of amoeba, especially if the cornea has been exposed to a range of antimicrobial or antiviral drugs prior to the procurement of the scraping specimen.
Although the examples of GSP and ST4P staining described in this paper have used amoebae growing in culture or in corneal scrapings, we also have been able to detect Acanthamoeba in sectioned human cornea (Fig. 12) (20).
In these studies, successful culture of a specimen at both TIO and OSU provided the highest level of confidence that viable acanthamoebae were present in an original scraping. However, successful culture at either one of the two institutions also was considered strong evidence that amoebae were originally present in the scraping. This is because the amoebae in the scrapings we examined were encysted. In our experience, negative culture results due to failure of excystment are relatively common whereas positive culture results due to accidental contamination with acanthamoebae are relatively rare. Thus, in the present study, the observance of an occasional failure of culture growth for scrapings that included cysts was not surprising. However, obtaining positive cultures from scrapings that did not originally include amoebae was highly unlikely. Therefore, in our analysis, all specimens giving rise to cultures at TIO, OSU, or both institutions were considered to have included acanthamoebae in the original scrapings. In some cases, positive cultures might have been obtained from samples with very few amoebae. Because FISH was performed on the original samples, small numbers of cells could have resulted in negative FISH results. On this basis, FISH with GSP detected Acanthamoeba in 80% of the culture-positive samples, and FISH and culture results were consistent for 88% of all samples. The comparable values are slightly higher if the FISH results, which were all obtained at OSU, are separately compared with culture results from each of the two laboratories. In this case, FISH detected amoebae in 86% (12 of 14) of culture-positive samples, and FISH and culture results were consistent for 92% (22 of 24) of all samples tested at OSU. At TIO, the values were 92% (11 of 12) and 92% (22 of 24), respectively. If comparisons of FISH and cell culture results are limited to the 20 specimens for which positive or negative culture results were identical at TIO and OSU, there was 100% agreement. Thus, FISH results were most consistent with culture results when there was no ambiguity in the latter.
The data indicate that FISH with GSP clearly improves the accuracy of microscopy for detection of Acanthamoeba. This method produced no false positives and only three false-negative results among the 24 scrapings. The most likely explanation for the false negatives is that viable amoebae, although present, were too sparse to be picked up by FISH. This problem probably could have been overcome by the use of a more effective method of cell concentration. The FISH result for specimen 1a (Table 2) appears to be a false negative since amoebae were successfully cultured at OSU and because specimen 1b, obtained from the same eye on the next day, was culture positive by all methods used, including FISH.
The FISH results for scraping specimens 17 and 23 also appear to be false negatives because amoebae were cultured successfully at either TIO or OSU and because PCR results to be discussed elsewhere (31a) indicated the presence of acanthamoebae in both scrapings. Specimen 17 is a special case. The clinical diagnosis was bacterial keratitis, and the acanthamoebae detected by wet-film microcopy and cell culture were identified as transients present in the scraping. This result emphasizes that detection ofAcanthamoeba should not be the only criterion used for a diagnosis of AK. Finally, the FISH result for scraping specimen 8 appears to be a true positive because the presence of acanthamoebae also is supported by positive cell culture and PCR results at OSU.
Two important advantages of FISH are the unambiguous identification of the organisms and the relative rapidity with which results can be obtained. Identification with FISH can be accomplished in 1 to 2 days, whereas cell culture time is variable and can take up to several weeks. It is recommended, however, that all scrapings, especially those that give negative FISH results, be cultured. Any amoebae that grow can then be tested by FISH with GSP to determine whether they are acanthamoebae.
The availability of a large number of DNA sequences for the nuclear small-subunit rRNA gene (35) has made it possible to design cytological stains that are specific for the genusAcanthamoeba and for separate lineages within this genus. We plan to develop a set of probes that could be used to identify the amoebae at the subgenus level. ST4P, which identifies 18S rDNA sequence type T4, is the first of these probes to become available. The specificity of this probe has been demonstrated for trophozoites in this study but remains to be demonstrated for cysts. We focused on this sequence type for the design of a probe mostly because of its apparent importance in AK. Our previous sequence studies (35) plus 18S rDNA sequences obtained for corneal scrapings used in the present study (31a) indicate that 29 of the 30 AK isolates we have examined to date have T4 sequences. The only exception is a T3 isolate (22), and this sequence type is very closely related to T4. Another reason for concentrating on the T4 probe is because we have 18S rDNA sequence information for many more T4 strains than for any other sequence type. The greater amount of information available about sequence variation for this lineage permitted the design of a more robust probe. ST4P hybridizes to a region of the 18S rRNA gene that differs among the sequence types. Thus, this region may be a good target for other probes designed to be specific for other sequence types. However, more information on sequence variation in most of these other types is required before a complete set of robust sequence type-specific probes can be developed.
The FISH protocol was designed to be both specific and sensitive. The cells tested included axenic cultures of trophozoites and corneal scraping specimens that mostly included cysts. GSP worked very well with either developmental stage, but ST4P was used only with multiplying amoebae. Amoebae actively growing in cultures gave the brightest signal, most likely because of a high rRNA content in the cytoplasm. However, amoebae that have been stored in isotonic saline for 1 to 19 months, as was the case for some of the corneal scraping specimens, also were detectable. The fact that this technique works on stored samples is important because there can often be some delay between the collection of a scraping and its analysis in a specialized laboratory. The success of FISH with older stored samples is not surprising because the amoebae were present as cysts. It previously has been shown that encysted acanthamoebae can remain viable in CL saline for 14 to 90 days (3). In the present study, however, cysts remained viable during refrigeration in isotonic saline for at least 19 months, as shown, for example, by scraping sample 18 (Table 2), which was collected 3 December 1994 and still produced a culture at OSU in early July 1996.
The probes were designed to hybridize with 18S rRNA in addition to the large number of rDNA copies in the nucleolus in order to maximize the number of intracellular targets. Probe specificity at the level of sequence type was tested only with trophozoites because interpretation of results from cysts might be complicated by the autofluorescence of the cyst walls. It should be possible to alleviate this problem by using colorimetric detection of a nonfluorescent tag such as digoxigenin, which has been used with probes forAcanthamoeba 26S rRNA (21).
The probes have a number of potential applications in addition to those tested in the present study. For example, they can detect amoebae in deparaffinized tissue sections (Fig. 12). Also, Paillasson and coworkers (29) have recently developed a procedure for in situ hybridization of FISH probes to RNA in living human cells. The cells were permeabilized with streptolysin O and probed without fixation. They then were detected with a fluorescence-activated cell sorter. We recently proved a linkage between a patient with AK, the CL storage case contents, and the patient's home water supply (22). Thus, it might be possible to use FISH probes in a fluorescence-activated cell sorter format for prophylactic studies of water from treatment plants, home water supplies, or other sources.
Although this report has focused on AK,Acanthamoeba also is responsible for GAE. Two of the specimens tested in this study, Acanthamoeba healyi V013 and A. castellanii V006, are isolates from patients with GAE. This disease has been increasing in incidence due to its association with AIDS (14). Hawley et al. (14) recently reported the ability to identifyAcanthamoeba in cerebrospinal fluid via culturing. Nearly all reported cases of GAE have been fatal, but early diagnosis, possibly using FISH on cerebrospinal fluid, might improve the odds of successful treatment.
The level of specificity required in probes will depend on their use. In some cases, it may only be necessary to distinguish between organisms that are known to cause a particular infection and those that do not. For studies of biodiversity, a more varied set of probes might be needed. The large collection of 18S rDNA sequences now available at GenBank plus the sequence alignments available on the Internet (3a) should be consulted by those wishing to design other probes. In addition, since cysts can be the major component of clinical samples, methods of suppressing the autofluorescence of cyst walls or the use of nonfluorescent markers for the genus- and sequence type-specific probes will be explored.
The molecular biology studies described in this report were funded by Public Health Service grant EY09073 from the National Eye Institute.
We thank our colleague Gregory Booton for helpful discussions and assistance with the figures.
Received 30 November 1998.
Returned for modification 18 February 1999.
Accepted 28 April 1999.
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Journal of Clinical Microbiology Aug 1999, 37 (8) 2687-2693; DOI:
You are going to email the following Fluorescent Oligonucleotide Probes for Clinical and Environmental Detection of Acanthamoeba and the T4 18S rRNA Gene Sequence Type


Yesterday, I had a photo jam with Justina at Caitlin's home. The photo shoot was for Woolly Pocket, but I managed to sneak a few shots of Caitlin's casa because I need to show you how beautiful it is. Look at those lush greens and swinging French doors and windows! DIVINE. Ugh, it makes me wanna move out of my downtown loft and into this oasis. Guess we'll be looking for a new home come October!


text:
HomeNewsPoliticsU.S. Condemns WHO for Excluding Taiwan from World Health Assembly
U.S. Condemns WHO for Excluding Taiwan from World Health Assembly
Secretary Pompeo: Director-General Tedros' Cave to China 'Further Damages WHO's Credibility and Effectiveness' During Pandemic
World Health Organization (WHO) Director-General Dr. Adhanom Tedros Ghebreyesus delivers his address to the opening session of the 146th Session of the WHO Executive Board on February 3, 2020. (Photo: People's Pundit Daily/PPD)
Washington, D.C. (PPD) — Secretary of State Mike Pompeo issued a blistering condemnation of the World Health Organization (WHO) for excluding Taiwan from the World Health Assembly. He made clear the administration believes WHO Director-General Dr. Adhanom Tedros Ghebreyesus caved "under pressure" to China.
"The United States condemns Taiwan's exclusion from the World Health Assembly," Secretary Pompeo said in a statement. "At a time when the world continues to struggle with the COVID-19 pandemic, we need multilateral institutions to deliver on their stated missions and to serve the interests of all member states, not to play politics while lives are at stake."
The World Health Assembly is the decision-making body of WHO. At the annual event in Geneva, Switzerland, delegations from all WHO Member States hatch out specific health agendas prepared by the Executive Board.
"The main functions of the World Health Assembly are to determine the policies of the Organization, appoint the Director-General, supervise financial policies, and review and approve the proposed programme budget," the WHO states.
The 73rd World Health Assembly is scheduled to be held virtually from May 18 – 19, 2020. The 147th session for the Executive Board will be held virtually on May 22, 2020.
Taiwan, which China refuses to acknowledge as anything other than a satellite state under Beijing, has been among the most successful nations to contain the coronavirus (COVID-19) pandemic. It's astonishing given the origin of the outbreak was Wuhan, China.
"WHO's Director-General Tedros had every legal power and precedent to include Taiwan in WHA's proceedings," Secretary Pompeo stated. "Yet, he instead chose not to invite Taiwan under pressure from the People's Republic of China (PRC)."
"The Director-General's lack of independence deprives the Assembly of Taiwan's renowned scientific expertise on pandemic disease, and further damages the WHO's credibility and effectiveness at a time when the world needs it the most."
The U.S. credited Taiwan's success to it being a "transparent, vibrant, and innovative" democracy rather than a totalitarian regime such as China. Beijing has been criticized by the West for mishandling the pandemic. Intelligence gathered jointly by the Five Eyes suggests China misled the world about the pandemic, and enlisted the help of the WHO to do so.
The PRC's spiteful action to silence Taiwan exposes the emptiness of its claims to want transparency and international cooperation to fight the pandemic, and makes the difference between China and Taiwan ever more stark. Taiwan is a model world citizen, while the PRC continues to withhold vital information about the virus and its origins, deny access to their scientists and relevant facilities, censor discussion of the pandemic within China and on Chinese social media properties, and casts blame widely and recklessly.
Secretary of State Michael Pompeo
The WHO has come under intense criticism for promulgating Chinese propaganda. In early April, President Donald Trump announced a hold on U.S. funding to the WHO pending an investigation into the organization's handling of the pandemic.
A Five Eyes intelligence report claims China enlisted the help of the WHO to downplay the threat to the virus and cover for their response and its origins. It's unclear whether that funding will resume at the prior limits or if the U.S. will match the contributions from China.
UPDATE: President Donald Trump tore into the WHO response to the pandemic in a letter to Director-General Tedros. He gave the WHO 30 days to make "substantive improvements" before the temporary freeze on U.S. funding to the WHO becomes permanent and membership is reevaluated.
"it's clear the repeated missteps by you and your organization in responding to the pandemic have been extremely costly for the world," the president wrote. "The only way forward for the World Health Organization is if it can actually demonstrate independence from China."
This is the letter sent to Dr. Tedros of the World Health Organization. It is self-explanatory! pic.twitter.com/pF2kzPUpDv
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Hey! I found this information for you: "U.S. Condemns WHO for Excluding Taiwan from World Health Assembly". Here is the link: https://www.peoplespunditdaily.com/news/politics/2020/05/18/us-condemns-who-for-excluding-taiwan-from-world-health-assembly/. This is the website: https://www.peoplespunditdaily.com Thank you.

redpajama_set_name: RedPajamaCommonCrawl


If you are an Amazon Prime member (free to try!), check this out! You can now shop for your everyday products and groceries right at Amazon.
Prime Pantry boxes hold up to 45 lbs of household products, so you can fit a lot of items in there! You fill it with as little or as much as you want then you pay a flat shipping rate.
As I was browsing around, I noticed the prices weren't that bad considering you don't have to go anywhere to get them! PLUS, there is a whole area of coupons for your Prime Pantry purchases.


Today Lydia sold her dolls on FB to raise money for her Compassion child. I'm so proud of her. I'll have to post her American Girl Doll outfits too!
We had our last Gathering of the school year. This picture is missing quite a few students and families. It's bittersweet to know that this was the last time we will see some of our students. We were even blessed to have two mothers here – one from Saudia Arabia and the other from Mali.
On Tuesday, my friend Krista and I went to San Francisco for my Lyme Dr appointment. We took an early flight and bought a day pass to the Delta Club. We took an Uber to the doctor, ate at a farm to table for supper and walked by the port. The red eye was a little brutal but it was worth it.
Today was a big day with bowling, tour of the courthouse and Classical Conversations year end celebration.
Ava loves chicken wings, climbing trees and frosting cookies.


text:
Two Men on Different Paths Discover the Same Road with Food For The Poor
COCONUT CREEK, Fla. (June 13, 2019) The expression "it's a small world" personifies an encounter between Ed Raine, a former Boston businessman, and Kevin Carges, a deacon from New York.
"We met 10 years ago on a mission trip to Haiti with Food For The Poor, and many in the group were deacons or priests," recalled Raine, now Executive Vice President of Food For The Poor. "It was an incredible experience and I well-remember Kevin; he was compassionate and connected well with the children. That mission trip also made a deep impact on me. When you experience true poverty you can't unsee what you saw – it changes you."
After 30 years in the corporate world, driven by a desire to make a difference, Raine decided he was ready to make a change. He joined Food For The Poor in October 2017 and oversees many aspects of organizational development and strategy. Raine discovered in April 2019 that the deacon he met a decade ago is one of the charity's most dedicated supporters as Carges prepared for a trip to Jamaica.
"The best relationships Food For The Poor can have are the ones that you see grow over time, and this is a wonderful shared experience," Raine said.
Moved by what he saw on the 2009 mission trip, Carges founded the charity Eight 4 World Hope at St. John Fisher College with the help of six members from the college Class of 1984.
His Rochester, N.Y., based-organization has made a difference in the lives of hundreds of schoolchildren in Jamaica through Food For The Poor.
It's a mission that holds a special place in Carges' heart and is something he hopes members of his organization will appreciate.
"Life-changing is how I would describe how it's been for me over the years, and this is how I'll say it will be for those on this trip with us for the first time," Carges said. "I hope when they return home they will go back with an understanding of what it means to give back. The things that you see and the people you meet will stay with you forever."
Carges has built, repaired or expanded seven schools in Jamaica, since his first mission trip to the Caribbean country with Food For The Poor in 2006. Carges and nine members of Eight 4 World Hope, including his son Andrew, were in Jamaica and saw how their donation has transformed Alexander Basic School in Old Harbour, St. Catherine. The previous building had severe leaking, no electricity, a limited water supply and a kitchen without appliances. The staff and students thanked Eight 4 World Hope with a sweet celebration of songs and cake.
The Eight 4 World Hope group also visited several other schools and additional Food For The Poor projects during their five-day mission trip. They also helped a single mother and her three sons paint their Food For The Poor home in Wynter's Pen, Spanish Town.
After a group tour of Food For The Poor-Jamaica, they went to the boardroom where Carges was surprised with a video conference call from Raine in Florida. Raine had a staff member present Carges with a small-scale version of the life-sized sculpture of "Jesus the Beggar." The sculpture is a dominant feature in the lobby of the charity's Florida headquarters, and is the center piece in the group photo with Carges and Raine in 2009.
"We all have choices to make and you've made the choice to make a difference, and that is what it's all about," Raine said. "Jamaica is a very special place and the work you're doing there is very special. Please know it's much appreciated, and I want to sincerely thank and congratulate you for all that you do on behalf of the poor."
This was the first time the two men had seen one another since the 2009 mission trip to Haiti, and Carges was clearly moved by the expression of gratitude.
"I cannot do any of this by myself, and I have to thank all of those who are willing to give of themselves," Carges said. "I cannot say thank you enough for making a difference in this world."
Ed Raine, (L), Executive VP of FFP and Kevin Carges, (R), Eight 4 World Hope Founder.


I watched this episode in a hope that it will be the second last episode & things will start to wrap up but looks like that's not going to be the case because the director has still not given up on the fact that this drama is making no sense at all. Talk about a drama being deprived of character development, character evolvement, purpose of the story, purpose of the conversations, in short everything! This drama is clearly past that stage where the writer could rely on the philosophical dialogues to keep the interest of the viewers alive but it looks like Amna Nawaz Khan is still not getting the point.
When we signed up to watch this drama, we were looking forward to the story of Mahnoor, Haadi & Uzma, & more than that we were looking forward to see the ending the character of Mahnoor will get, but ever since Amna Nawaz Khan took over as a writer, her confusion, her cluelessness & the loss of direction is seen all over this drama especially after the 20th episode mark. It is so obvious that Khalil ur Rehman Qamar wrote this drama while making the characters of Mahnoor, Haadi & Uzma the focal point of the story but when he decided to leave, Amna Nawaz Khan failed to understand his thought process & thought that she will be able to pull it off by continuing the drama as a writer, but she failed miserably & I repeat, she failed miserably. Amna Nawaz Khan all of a sudden brought the side characters of Anoushay, Anoushay's Daadi, Gaiti, Mehtab, Aftab & Waqar in the limelight, while completely forgetting the fact that the story was never about them & it was about entirely different characters & the purpose of the drama was never to show the politics within the family of politicians or it was never to show how lifeless, spoiled & batameez an immature girl like Anoushay can be. Amna Nawaz Khan thought that she will be able to pull this drama off as a writer by pushing the main characters in the background but she was sadly mistaken!!!
Mahnoor spoke to Waqar & everytime I see Mahnoor making an attempt to arrange her temporary Nikkah makes me laugh out loud at her pathetic-ness & desperation. Mahnoor still fails to see that she is not a victim in this situation but just because Haadi lacks some backbone & Aneena's mind is too tangled up in her own philosophy that both of them fail to knock some sense into her mind that it is not Mahnoor who is the victim here, in fact she has victimized everyone around her. Mahnoor conveniently blamed Haadi & even Aneesa for being rude & for not being considerate enough but then has she ever thought about the fact that what did she do when they were considerate & when they loved her? Haadi reacted like Haider for a second & I thought finally he has reached his limit & will make a decision now but his mind got diverted seeing the plate of confectioneries that he forget about all of his problems!
Uzma's philosophy about love is firstly something that I don't agree with & it was so lame that I rolled my eyes so hard that they're hurting now. I don't think I have ever rolled my eyes while watching any drama as much as I have done while watching Zara Yaad Kar because it is quite amusing & lame at the same time that the dialogue writer, whoever he or she may be has relied so much on the falsafa that even the falsafa itself is confused about the falsafa that it churns out. Time & again we are told that Haadi loves Mahnoor, that is why he is putting up with everything but then I guess the writers of Zara Yaad Kar have forgotten that love brings happiness with it & any love that doesn't make the one in love happy, then the love dies quickly & as we see Haadi is not happy with Mahnoor & the way she treats him like a doormat doesn't make him happy either, so where is this love shove coming from bhai???
This episode was just a waste of time & the preview of the next episode left me baffled & infuriated because it suggested that the drama is not going to end next week too. I am not sure what has gone into the heads of HUM Channel's heads because it looks like even they are unable to deal with this torture & their lack of interest in this project is evident because looks like Sanam is replacing it next week. I am not sure what is happening because if this is what they had to do, then why didn't they cut this drama short & ended it this week? The story, the characters & the purpose of this drama is something that is lost & is not making much sense, but the way HUM TV is dealing with this project shows that even they are not interested in this drama anymore. I am not sure when is it ending because I was told that 27th episode will be the last one but it doesn't seem to be the case. I so wish they had ended this drama today because there is practically nothing left in this drama. Please share your thoughts about this episode of Zara Yaad Kar.


text:
Dramatic Sunday in Zandvoort
Sunday August 20th, 2017 Saturday August 26th, 2017 Jens Jensen
It was quite a dramatic Sunday ind Zandvoort when Round 12 of this years DTM Championship was run.
There was a melee already in the qualifying session. Yesterday's winner, Timo Glock, felt impeded by Edoardo Mortara, and instead of taking it to the stewards, he decided to take a revenge himself and braked hard in front of Mortara and blocked him for the rest of the lap, while shouting "Fucking idiot" over the team radio, and later gave Mortara the middle finger in the pits. That kind of behaviour wasn't acceptable for Race Control, so they disqualified Glock from the Qualifying and gave him a 3000 Euro fine for unsporting behaviour.
Gary Paffett would start the race second to last, since he had used unregistered tires in the Qualifying.
At the front of the field, it was once more a BMW party, with Augusto Farfus on Pole Position ahead of Marco Wittmann and Maxime Martin. They were followed by a line of Audi drivers with Jamie Green, Rene Rast and Mike Rockenfeller, before BMW man Bruno Spengler jumped into P7 ahead of the two Mercedes guys, Maro Engel and Robert Wickens.
Tom Blomqvist went off the circuit on the Formation Lap and had to dive into the pits to get a new door. He was awarded with a drive-through penalty, since he had taken a shortcut on the way back to the pits.
That wasn't the only drama at the start of the race, since Maxime Martin stalled on the starting grid, and everybody behind him had to avoid him. Luckily everybody made it past, and he could start the race and second-to-last.
Marco Wittmann took the lead at the start, and at the end of the first lap we saw Farfus, Spengler, Lucas Auer and Paul Di Resta do their mandatory pitstop
Photo: DTM
Bruno Spengler made a jump start that gave him a 5 seconds pitstop penalty.
On the second lap we had both Robert Wickens and Jamie Green pitting at the same time, and on the way out of the pits they were very close at crashing into each other.
Augusto Farfus tried a hopeless overtaking manoeuvre on Jamie Green, but other than some slight bodywork damage, nothing ells happened and both could go on.
Wickens and Green continued their battle on the track where Green tried to overtake Wickens at the chicane. There was a contact between the two car going into the chicane, and Wickens then decided to fight back and turn around Green on the exit of the chicane. Green lost several positions and Wickens got a puncture after the collision.
It was all arms and elbows in the middle of the field, when Tom Blomqvist, Maro Engel and Rene Rast came into contact with each other. That resulted in so much damage to Rast's car and he had to park the car in the pit.
There was no damage to Engel's car and he made a super overtaking manouvre on Blomqvist on the following lap, round the outside in Turn 1.
On lap 13 we saw Wittmann drive into the pits and leave again, still leading all the other guys who had been in for their stop.
Mattias Ekström made a good outbraking manoeuvre on Blomqvist into Turn 1, and Nico Müller went past too.
Mike Rockenfeller came out right behind Marco Wittmann after his pit stop, and were shadowing him for the rest of the race.
Engel was on the road for a great result in P3, when he had to go to the pits with a puncture, and ended up outside the points.
Lucas Auer tried to make an aggressive overtaking of Augusto Farfus, and the two cars came into contact with each other. A few laps later, Auer had to go to the pits with a puncture.
The second half of the race was much smoother, and Marco Wittmann could take an easy victory – at least, so he supposed. He was disqualified post race, since there was too little fuel left in the tank.
That gave Mike Rockenfeller the victory ahead of Loic Duval, who had driven a fantastic race from starting P13, while Mattias Ekström took third. He was followed by the fourth Audi with Nico Müller. Gary Paffett was best of the Mercedes in P5, ahead of BMW trio Maxime Martin, Timo Glock and Augusto Farfus.
Mike Rockenfeller
This result meant that Mattias Ekström was back in the lead of the Championship, ahead of Rene Rast and Mike Rockenfeller, who made a great leap into P3 with today's victory. Timo Glock is the best non-Audi in P4, just ahead of teammate Maxime Martin, while Lucas Auer still is the best Mercedes. It was an expensive weekend for Auer, since he didn't score any points in any of the races. Jamie Green in 7th make it 4 Audi's in the Top-7.
Audi has a big lead in the Constructors Championship ahead of BMW and Mercedes, while Audi Sport Team Rosberg leads the Teams Championship ahead of Audi Sport Team Abt Sportsline and BMW Team RBM.
The next round of the Championship will be held at Nürburgring on September 9th and 10th.
Manthey Racing wins in the rain
Neel Jani joins Faraday Future Dragon Racing for Season 4
Thursday August 25th, 2022 Jens Jensen
Rast switches brand
Rene Rast will switch to a different car brand for the 2023 season. The 35-year-old German...
DTM WEC
Monday November 30th, 2020 Jens Jensen
Audi changes focus
There is a lot of changes coming in the Audi concern. The German car manufacturer made...
DTM Formula E IMSA Rally WEC
Monday April 27th, 2020 Jens Jensen
Audi ends DTM commitment after 2020
After many months of speculations, it's now official: Audi Sport will pull out of DTM following...


Image Title: Island With Cabinet Access Behind Stools Also Love The Idea Of Within Open Kitchen Prepare 17. Post Title: Open Kitchen Island. Filename: island-with-cabinet-access-behind-stools-also-love-the-idea-of-within-open-kitchen-prepare-17.png. Image Dimension: 650 x 431 pixels. Images Format: jpg/jpeg. Publisher/Author: Domenic Fritsch. Uploaded Date: Tuesday - January 15th. 2019 10:47:22 AM. Category: Architecture. Image Source: decoist.com. Trendy Display 50 Kitchen Islands With Open Shelving Within Island Ideas 11. Open Kitchen Designs The Advantages Of Islands And Shared Regarding Island Ideas 8. 60 Kitchen Island Ideas And Designs Freshome Com With Open Plan 16. This Is What Practical Kitchen Planning Looks Like Open Throughout Island Ideas 13. 39 Kitchen Island Ideas With Storage DigsDigs Regarding Open Inspirations 18. Trendy Display 50 Kitchen Islands With Open Shelving Regarding Island Inspirations 2. Mahoney Architecture Open Houzz What S With The Kitchen Island Throughout Ideas 0. 18 Practical Kitchen Island Designs With Open Shelving Pertaining To Design 7. 60 Kitchen Island Ideas And Designs Freshome Com With Open 1. Trendy Display 50 Kitchen Islands With Open Shelving Regard To Island Ideas 4.


Aaron, I'd like to introduce you to a small part of the world of a Florida poet, Lola Haskins. She's written nine books of poems with another due out this year from Anhinga Press. She's written all sorts of prose pieces, too. At the moment, she's working on nature poetry growing out of the considerable time she spends in kayaks and in the woods.
Lola and I are alike in that we like to collaborate with other artists. She's worked with visual artists—like photographers, painters, and printmakers. Her most recent theater piece was "Swan Song" at the Hippodrome State Theater in Gainesville, Florida, with seven dancers, an actor, and a violinist.
But the book I want to focus on today is Forty-four Ambitions for the Piano. It's not her newest—that would be Still, the Mountain, from Paper Kite Press—but 44 Ambitions is so intensely interesting, it's stayed in my mind ever since I read it a few years ago. This book resulted in a performance piece also, with composer James Paul Sain and pianist Kevin Sharpe, in a performance called "Scattered Voices" at two different composers' conventions, and a collaboration with composer Paul Richards on a song cycle based on the poems in this book.
Now this one has surprised me in a different way. I thought it would be about blasting out, but no, it's about holding back. What's held back in this poem? The loudness. It's a tour-de-force, too. The poem is short. It holds back words, which is part of where it gets its impact.
An unpainted sky crossed by branches.
four small red horses that gallop away.
Isn't that an astounding little thing? Rest as the dream of horses that gallop away when we wake up.
heart gets up and struts away.
on, which burn the skin like sun.
The thing about Lola's poems is that they put us in a place of utter experience. They do not ask to be explained.
He was born with the fingerpads of the blind.
and how long before, and who.
and he felt the bruises still on the keys.
He was born with the ears of a dog.
as her cheeks sagged just barely more.
earth seemed so incomparably sad.
He was born with his life in his hands.
of his piano as if it were flesh.
with his leg, his sleeve.
What to say? How could the intense sensitivity of an artist be better shown?
Here's one about a major chord. I won't try to explain the difference between a major and a minor chord. Musicians will know this. Just to say that a major chord has an exact feel to it, no dissonant sounds.
with a curl between finger and thumb.
door, no matter how dark the room.
into the mirror, he begins it now.
That exact chord. I'm pretty jealous of how she managed to make me feel that compulsive exactness.
This one is called "The Uses of the Metronome." If you've ever practiced piano, you know the metronome sitting up there on the piano, ticking away, insisting you keep the right time. It feels like a policeman until you begin to get better and you relax and bend the sound a little, which is where the real music that has attitude can happen.
and you fly, you fly.
red with years of leaves.
you could walk there in an hour.
having chosen to go by water.
Again, anything I say to add to that is just wasted breath. As I listen to these poems I think about the kinds of poetry there are, and how what all good poems have in common. They wake up our senses, they startle us into seeing the same old tired world new again. To do that, they have to drag our rational mind, kicking and screaming, into territory IT would call irrational, silly, nonsensical. But our emotions recognize that territory as the real truth. That's why, I think, we go "Ahhhh," when we hear a good poem. For a moment, that teacher-mind, or critic-mind, that wants to explanations, has to shut up.
Because there is no black flower.
So that their hands can reach out of earth.
Because this is not practice.
not to talk with their mouths.
on the far, dark, shore.
Perfect. The poet is Lola Haskins.


text:
package io.github.lonamiwebs.stringlate.classes.resources;

import java.util.Comparator;

import io.github.lonamiwebs.stringlate.classes.resources.tags.ResTag;

public class ResourceStringComparator {
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    public static final int SORT_STRING_LENGTH = 1;

    public static Comparator<ResTag> getStringsComparator(int sortMethod) {
        switch (sortMethod) {
            default:
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                    @Override
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                        return (x < y) ? -1 : ((x == y) ? 0 : 1);
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        }
    }
}

redpajama_set_name: RedPajamaGithub


text:
Baer sells Carson City property
Local | May 15, 2007
Max Baer Jr. has sold his property at 4209 S. Carson St., formerly Wal-Mart, for $8.5 million to Carson Southgate LLC, a new firm in Bellevue, Wash., according to broker Gary Johnson of NAI Alliance in Reno.
"The company purchases buildings like these and puts new tenants in them," Johnson said.
Baer, who once had plans to build the Beverly Hillbillies Casino & Mansion on the property, paid $4.3 million for it in August 2003. The property includes 12.8 acres and a 119,000-square-foot building.
Money from the sale will be used to purchase about 23 acres in the proposed Riverwood commercial development for his Douglas County casino project, Baer said.
"I'm trading a building with paving, utilities, ingress and egress for a pile of dirt in the hopes this location will be superior because of what's going on around it," Baer said.
A Kohl's department store and Sportsman's Warehouse are two of the anchors that have made applications to county officials to build in Riverwood.
The project includes a 40,000-square-foot gaming area with 800 slots and 16 tables, a showroom, cinema complex and five-story, 240-room hotel in almost 300,000 square feet, according to project spokesman Don Smit.
Variances will be required for a proposed 200-foot flameless oil derrick and readerboard. Two 12-story, 240-room hotel towers and convention space are scheduled for future phases and will also need approval from the Douglas County Board of Commissioners.
Harmon Zuckerman, principal planner for Douglas County, said the applications have been completed and are being processed, but staff is not submitting a recommendation at this time.
The special use permit, major variance for height and zoning map amendment will be considered by the Douglas County planning commission June 12 and the zoning map amendment will be considered by the Douglas County Board of Commissioners July 5, he said.
Resistance from Carson city officials blocked development of the casino in Carson City, which would have been a tremendous benefit for the people there, Baer said.
"The people of Carson City have been very kind and I have nothing but respect for them," he said. "But it appears Douglas County will be getting the revenue, brand, and any retail I can help to establish in Douglas County. I'm sorry that it's at the expense of the people of Carson City. They deserve better."
Joe McCarthy, Carson City's economic development and redevelopment manager, said Carson Southgate LLC will completely refurbish the building and bring in tenants. If successful, they will boost energy for the rest of the businesses at Southgate Mall.
"Our community is terribly underserved in a lot of retail areas," McCarthy said. "We're very strong and significantly above state average with respect to auto sales, but far below the state average for general merchandise sales, relative to where we should be."
A previous agreement with Raley's Supermarket and Drug Center prohibited the sale of groceries within a given perimeter, thus preventing Wal-Mart from expanding its inventory.
Carson City leaders made unsuccessful efforts to keep Wal-Mart in Carson City, including attempting to broker a deal with Raley's and offering several Carson City sites, but the retail giant relocated to Douglas County, taking with it an estimated $1 million annually in sales taxes.
Acting on Baer's behalf, long-time friend and California developer John King purchased the former Wal-Mart property and signed it over to Baer in August 2003.
Plans included a 30,000-square-foot casino with 800 slot machines,16 gaming tables, a 240-room hotel and restaurants, all encompassing the "Beverly Hillbillies" theme.
Standing in the way of the project were the covenants and restrictions governing Southgate Mall which prohibit development of a theater, bowling alley, nightclub or other place of recreation or amusement.
For months, Baer negotiated with the other two Southgate owners, Glenbrook Co. and J.C. Penney, but no agreement was reached.
In a letter written to Baer in March 2002, Carson City Supervisor Shelley Aldean, president of Glenbrook Company and manager of the shopping center, agreed to amend the provisions that would keep him from converting the existing Wal-Mart into a hotel-casino, but the agreement was never amended.
Susie Vasquez can be reached at svasquez@recordcourier.com or 782-5121, ext. 211.


text:
DJ Symphony to announce record label and release date for "I Love America"
Home » Hip Hop News » DJ Symphony to announce record label and release date for "I Love America"
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By The Hip Hop Writer
Hip Hop Vibe Staff Writer
Currently, DJ Symphony is touring the world, but he will soon be returning to the United States. When he returns, DJ Symphony will have much news to discuss with his fans. Serving as the official DJ for Raekwon, of the Wu-Tang Clan, DJ Symphony has been making his rounds for years. While fulfilling his obligations to Raekwon, DJ Symphony has become one of the most-popular DJs in the game.
DJ Symphony has dominated the mixtape scene, delivering such projects as MMG vs. YMCMB, Dipset vs. Wu-Tang, and many more. Going along with his popular mixtapes is his Radio Invasion online radio station. Aside from his own mixtapes, the workload for DJ Symphony has increased, as he has prepared mixtapes for a host of other artists.
But, upon DJ Symphony's return to the United States, he will announce the name of his own record label. DJ Symphony has worked alongside many of the most-influential hip hop artists in the game, many of which have been a part of the most-legendary labels in the game. With all of this experience surrounding him, it was only a matter of time before DJ Symphony created his own label.
DJ Symphony is preparing to announce his new record label and the release date for his I Love America mixtape.
Related Topics:dipset vs. wu-tang, dj symphony, i love america, mmg vs. ymcmb, united states, wu-tang clan
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