text:
MLVA methods for strain discrimination among Mycobacterium tuberculosum strains are disclosed. Nine VNTR loci have been identified from genomic sequences of Mycobacterium tuberculosum strains and primer pairs suitable for amplifying the VNTR by PCR are disclosed. Polymorphisms at these loci were used to resolve genotypes into distinct groups. This sub-typing scheme is useful for the epidemiological study of Mycobacterium tuberculosum and may be applied to the local detection of the pathological causative agent of tuberculosum.
1. A method for subtyping Mycobacterium tuberculosum comprising the steps of:
amplifying VNTR (variable number tandem repeat) loci in Mycobacterium tuberculosum DNA present in a sample, said DNA having a sequence comprising at least two different VNTR loci, wherein the amplifying comprises amplifying the DNA by PCR (polymerase chain reaction) with different forward and reverse primer pairs to generate amplicons;
determining a size of the amplicons by a rate of movement of the amplicons in an electric field; and
comparing the size of each amplicon with a rate of movement in the electric field of a corresponding control amplicon PCR amplified from a VNTR locus for a known subtype of Mycobacterium tuberculosum,
wherein the rate of movement of the amplicons similar to the rate of movement of the control amplicons in the electric field is indicative of a subtype of Mycobacterium tuberculosum in the sample.
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6. A method for subtyping Mycobacterium tuberculosum comprising the steps of:
PCR amplifying at least two VNTR loci from a test sample comprising Mycobacterium tuberculosum DNA to generate amplicons, and PCR amplifying the corresponding at least two VNTR loci from a control sample comprising one or more known subtypes of Mycobacterium tuberculosum DNA to generate control amplicons, wherein the at least two VNTR loci are selected from the group consisting of Mtb-v1, Mtb-v3, Mtb-v4, -Mtb-v5, Mtb-v6, Mtb-v10, Mtb-v15, Mtb-v18, and Mtb-v20;
identifying the amplicons by a rate of movement of the amplicons in an electric field;
comparing the rate of movement of the amplicons to a rate of movement of the control amplicons in the electric field,
wherein the rate of movement of the amplicons in the electric field similar to the rate of movement of the control amplicons in the electric field is indicative of a subtype of Mycobacterium tuberculosum.
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19. A kit for subtyping Mycobacterium tuberculosum comprising:
a DNA polymerase;
buffers and salts suitable for a PCR reaction; and
at least two forward and reverse primer pairs for the PCR reaction, wherein the primer pairs are selected from the group consisting of; SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO:4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; and, SEQ ID NO: 17 and SEQ ID NO: 18.
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This application is a divisional of, and claims priority to, U.S. application Ser. No. 10/624,714 entitled "A High Resolution Typing System For Pathogenic Mycobacterium tuberculosum" filed Jul. 21, 2003, by Paul S. Keim et al., now issued U.S. Pat. No. 7,026,467, which is incorporated herein by reference. This application claims the benefit of priority to U.S. Provisional application Ser. No. 60/397,224 entitled "A High Resolution Typing System For Pathogenic Mycobacterium tuberculosum" filed Jul. 19, 2002, by Paul S. Keim et al., which is herein incorporated by reference in its entirety.
The present invention is generally directed to sub-typing Mycobacterium tuberculosum, the causative agent of tuberculosis, and is more specifically directed to PCR amplification of variable number tandem repeat sequences (VNTR) with primer pairs designed to amplify specifically certain VNTR identified in Mycobacterium tuberculosum isolates. Results of the analysis may be compared to results from known Mycobacterium species to determine the sub-type of the species for epidemiological and diagnostic purposes.
Tuberculosis (TB) is the primary cause of death due to a single microbial pathogen, accounting for 2 to 3 million deaths and an estimated 8 to 10 million new cases per year worldwide (1). It is estimated that one third of the world's population is infected with Mycobacterium tuberculosum. The global epidemic is growing, concomitant with HIV co-infection and the increasing prevalence of drug resistant strains. Genotyping clinical isolates of M. tuberculosum has the potential to significantly impact both clinical practice and public health. Clinically, determining strain relationships can distinguish treatment failure versus potential laboratory cross-contamination, and reactivation of drug-resistant disease versus infection with another strain (24). In the public health arena, determining strain relationships during the course of a tuberculosis outbreak can focus public health resources. However, the most discriminatory M. tuberculosum genotyping method, IS6110 restriction fragment length polymorphism (RFLP) analysis, is time-consuming, technically demanding, and difficult to standardize between laboratories (3). In addition, IS6110 RFLP does not provide sufficient strain discrimination when fewer than six IS6110-hybridizing bands are present. A secondary genotyping method is required in these instances. A number of PCR-based genotyping methods have been developed, but are limited in their ability to reproducibly provide a level of strain discrimination that matches associations defined by traditional epidemiology. In addition, the high degree of coding region sequence conservation between strains of M. tuberculosum virtually excludes the use of more commonly used population genetics methods like multi-locus enzyme electrophoresis, restriction fragment length end labeling analysis, and gene sequencing (12).
Spoligotyping is currently the most widely used PCR-based method of M. tuberculosum genotyping due to the relative technical ease of the protocol and the ability to compare results between laboratories using a standardized nomenclature system (6). However, the discriminatory power of this method is low. In a database of 3,319 spoligotypes from isolates collected in geographically distinct areas, seven spoligotype patterns were found for 37% of all clustered isolates (18). These spoligotype patterns are associated with strains having a much larger variety of IS6110 RFLP patterns. For instance, 18% of isolates in the spoligotype database had the "Beijing" spoligotype pattern, characterized by the presence of spacer oligonucleotides 39 through 43 (18). However, although the W-Beijing family of strains is recognized as one of the most homogeneous families of M. tuberculosum strains (19), the Beijing spoligotype is associated with at least 450 distinct strains as determined by IS6110 RFLP analysis (2).
Following the public release of the complete genomic sequences of M. tuberculosum strains H37Rv and CDC-1551, investigators began the process of identifying repetitive genetic elements to develop higher resolution DNA typing systems. Variable number tandem repeats (VNTR) of 21-111 bp genetic elements have been identified (9, 14, 17, 20, 21). A set of 12 VNTRs, also known as mycobacterial interspersed repeat units (MIRUs), was more discriminatory than IS6110 RFLP when used to analyze 180 M. tuberculosum isolates with 6 or fewer IS6110-hybridizing bands. The combination of MIRU analysis and spoligotyping and IS6110 RFLP analysis has provided maximum specificity (5).
However, a greater discriminatory power for resolution and differentiation of species is necessary for sub-typing Mycobacterium tuberculosum species in order to track sources of infection and ultimately to prevent the spread of disease. Methods and means for determining the genetic differences between M. tuberculosum species with speed, accuracy and with great discriminatory capacity have been sought.
The present invention discloses methods and means for detecting and sub-typing M. tuberculosum species by multi-locus VNTR analysis (MLVA) genotyping system of VNTR identified within the genome of M. tuberculosum In the system, independent, tandemly-inserted repeated motifs in the M. tuberculosum genome are amplified using fluorescently-labeled primers in multiplexed PCRs and electrophoretically separated on polyacrylamide gels for analysis.
VNTR loci located throughout the H37Rv genome have been identified. Isolated nucleic acids having the sequence of VNTRs identified in M. tuberculosum are presented. In an important aspect of the present invention nucleic acids comprising at least 12, 15, 18 or total consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 and SEQ ID NO: 18 and sequences complementary thereto are presented.
In certain preferred embodiments of the invention, these nucleic acids are immobilized on a solid surface and are useful, for example, in the detection of a M. tuberculosum species in an assay employing probes, including, but not limited to, a nano-detection device.
In another important aspect of the invention, primer pairs comprising a forward and a reverse primer are presented for amplification of VNTR located in DNA from a M. tuberculosum species. Primer pairs suitable for PCR amplification of VNTR, by MLVA or by multiplex, may be selected from the group consisting of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, and SEQ ID NO: 17 and 18. Certain preferred primer pairs have, in addition, an observable group whereby amplified product may be detected. Such groups may be, for example, a fluorescent group or a radioactive group.
In yet another important aspect of the invention, a method for detecting a M. tuberculosum species is presented. The method comprises the steps of:
In another important aspect of the invention, MLVA methods are presented for observing polymorphisms at VNTR loci in DNA from more than one M. tuberculosum species to resolve unique genotypes between the species and to allow sub-typing of the species into distinct groups. These MLVA methods provide a convenient and rapid method for strain discrimination in M. tuberculosum. MLVA may be applied for strain discrimination among globally diverse M. tuberculosum isolates.
The method of sub-typing a M. tuberculosum strain comprises the steps of:
In yet another important aspect of the invention, kits are provided for detecting and sub-typing M. tuberculosum species. The kits comprise one or more primer pairs suitable for amplifying VNTR in DNA in a sample of said species and may comprise, in addition, nucleic acids, enzymes, tag polymerase, for example, and buffers suitable for causing amplification by PCR, by MLVA or by multiplex, for example. In certain preferred embodiments of the kit the primers comprise a label whereby amplified VNTR may be detected. In other preferred embodiments of the kit, labeled nucleic acids are provided. Observable labels are preferably fluorescent molecules or radionucleotides. Certain kits comprise in addition primers and reagents for MIRU analysis of a DNA isolate.
MLVA methods are presented for rapid genotyping of M. tuberculosum MLVA targets described herein provide discriminatory power that enhances the ability of present methods to determine rapidly molecular relationships of M. tuberculosum isolates. A system comprising a combination of VNTR and MIRU analyses is provided for rapid genotyping of M. tuberculosum.
This MLVA typing system is a PCR-based method for genotyping M. tuberculosum using VNTR loci identified in the present invention. This PCR-based typing has advantages not present in other PCR-systems: rapid turnaround, amplification with crudely isolated or minute amounts, of DNA, and the ability to use nonviable samples which can be transported across various labs with greater ease than viable ones. The rapid typing system using nine VNTR loci has been used to group a collection of 57 isolates of limited diversity (all had 4 IS6110 bands), and 34 isolates from the Beijing strain family. Smaller repeat motifs, for example <50 bp, as credible additions to the molecular typing of M. tuberculosum may also be used. In the MLVA system fluorescent labeling of primer and size of each allele allow for unique and easy identification of each VNTR. Automated gel analysis allows for a high throughput system that reduces error and time in obtaining results. High resolution electrophoresis and discrete repeats allows for the use of standardized data sets and facilitates central databases. An example of the database obtained is given in Table 4.
The ultimate utility of VNTR loci lies in their diversity. The present invention discloses the use of marker diversity using both allele number and frequency to sub-type Mycobacterium tuberculosum species. VNTR markers that exhibit high diversity values possess great discriminatory capacity for identifying genetically similar strains. Less diverse markers may be applied with greater utility in species identification and the analysis of evolutionary relationships. This demonstrated ability to predict VNTR diversity based upon array size allows the guided selection of marker loci.
MLVA analysis using the VNTRs presented here can also contribute to the MIRUs recently developed by Mazars et al. (Mazars, E., S. Lesjean, A. L. Banuls, M. Gilbert, V. Vincent, B. Gicquel, M. Tibayrenc, C. Locht, and P. Supply 2001. High-resolution minisatellite-based typing is a portable approach to global analysis of Mycobacterium tuberculosum molecular epidemiology as described in Proceedings of the National Academy of Sciences of the United States of America. 98:1901-6)(14), herein incorporated in its entirety. The primers of the present invention may be labeled with the same fluorescent dyes as used in the MIRU set, and may be analyzed on the same electrophoretic run as the MIRUs because of their smaller size and allele composition. MIRU and Mtb markers may be incorporated into the same PCR multiplex reactions. The combination of markers and primers extends the power of the VNTR typing system, without additional time or labor, and increases the discriminatory ability of the present VNTR-typing.
Molecular typing of M. tuberculosum using VNTR may produce a useful molecular clock. The "clocking" of a VNTR is a credible methodology for time-geographical correlation In MLVA, different rates of evolution for each VNTR loci may be observed. VNTR markers that match the type of epidemic we are tracking may be chosen. For example, cases of M. tuberculosum incidence in endemic areas would intuitively have little variation amongst markers of low diversity. The antithesis of that would be that more diverse markers would be poor for establishing phylogeny across a diverse isolate collection, which is akin to similar findings with IS6110 elements (7). However, with MLVA it is possible to pick and choose what markers would be most appropriate for a given epidemic. For example, it is possible to deduce that VNTR loci with a low diversity index across a worldwide set would have an even lower or non-existent value across a local outbreak. This ability to create a wide gamut of markers of sundry diversity is helpful in a geographically limited area where a particular genetic clone may be more prevalent. The intrinsic value of this concept supports the idea that some VNTR are not suitable for possible transmission routes, but better as informers of plausible evolutionary scenarios, and vice-versa (11, 22).
Some VNTR markers demonstrate an affinity between actual genetic distance and the expansion or contraction of a repeat motif. The dendogram demonstrates this cascading trend with Mtb-v1. Within the Beijing collection, any cluster with a 0.11 difference (one allele) from each other had a repeat motif that differed by only one repeat or allele. For example, MLVA-BJ-A and BJ-B differ by one marker score in Mtb-v10. Yet MLVA-BJ-C and BJ-D, which differ from MLVA-BJ-A and BJ-B by more than one marker allele, have an Mtb-v1 allele size of 110, as opposed to 119 in MLVA-BJ-A and BJ-B. This indicates that phylogeny can be determined further by the difference in repeat motifs of any particular marker. Each marker gives two types of data: the raw score of allele size, and a relative allele relationship. This relative relationship is possible with MLVA, where the diversity within one marker can be greater than 0.5, whereas binary data can give a maximum diversity of 0.5 for any one locus.
The dominance of a particular marker allele could prove a useful tool in quickly referencing a clonal dispersion is a helpful tool in phylogeny studies. The analysis of a distinctive allele type is only viable when multiple loci are combined in analyzing the data. By doing so, it is possible to curtail the effect of convergent evolution in the analysis.
The most immediate public health application of this typing method will be its use during an outbreak. Determination of strain relationship between infected individuals could help in deducing routes of transmission. This information could save time and money for clinics and health-related professionals involved in eradication of M. tuberculosum. It is anticipated that this methodology will be a constructive addition to the molecular epidemiological methods currently used to track M. tuberculosum.
The following definitions are used herein:
"Polymerase chain reaction" or "PCR" is a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase are used to amplify the number of copies of a target DNA sequence by approximately 106 times or more. The polymerase chain reaction process for amplifying nucleic acid is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference.
"Primer" is a single-stranded oligonucleotide or DNA fragment which hybridizes with a DNA strand of a locus in such a manner that the 3′ terminus of the primer may act as a site of polymerization using a DNA polymerase enzyme.
"Primer pair" is two primers including, primer 1 that hybridizes to a single strand at one end of the DNA sequence to be amplified and primer 2 that hybridizes with the other end on the complementary strand of the DNA sequence to be amplified.
"Primer site" is the area of the target DNA to which a primer hybridizes.
"Multiplexing" is a capability to perform simultaneous, multiple determinations in a single assay process and a process to implement such a capability in a process is a "multiplexed assay." Systems containing several loci are called multiplex systems described, for example, in U.S. Pat. No. 6,479,235 to Schumm, et al., U.S. Pat. No. 6,270,973 to Lewis, et al. and 6,449,562 to Chandler, et al.
"Isolated nucleic acid" is a nucleic acid which may or may not be identical to that of a naturally occurring nucleic acid. When "isolated nucleic acid" is used to describe a primer, the nucleic acid is not identical to the structure of a naturally occurring nucleic acid spanning at least the length of a gene. The primers herein have been designed to bind to sequences flanking VNTR loci in Mycobacterium tuberculosum species. It is to be understood that primer sequences containing insertions or deletions in these disclosed sequences that do not impair the binding of the primers to these flanking sequences are also intended to be incorporated into the present invention.
VNTRs useful in the methods of the present invention for detection and sub-typing Mycobacterium tuberculosum have been identified. Eighty-four regions containing tandem repeat sequences as potential VNTRs from the H37Rv sequence were identified. The genomic location of nine identified VNTRs is illustrated in
Table 2 gives the characteristics of these Mtb VNTRs, namely the VNTR loci identification number, amplicon size, size range, motif times repeat and VNTR motif within gene. Table 3 gives the allelic number and diversity of the VNTR across the data set.
The results of PCR assay of these repeats after electrophoretic separation is given in
In this relatively homogenous set, diversity across the nine loci varied greatly, as measured by allele number range and diversity value range (Table 3). Six of the loci (Mtb-v1, v3, v5, v6, v10, v15) were variable across the collection of isolates having 4 IS6110 bands, and six loci (Mtb-v1, v4, v10, v15, v18, v20) were variable across the Beijing collection. In contrast, three VNTR loci (Mtb-v1, v10, v15) had four alleles and diversity values ranging from 0.14 to 0.59 across all samples.
As observed in Table 3, a larger number of alleles did not always correspond to a greater diversity value. In one sense, allele number represents potential discrimination power while the diversity value represents the realized discrimination in a given set of samples. Both values are useful for understanding the genetic loci and the isolate sets being observed.
When the 34 Beijing family strains were considered separately, the number of alleles and diversity values dropped. Six marker loci were informative within Beijing strains and three (Mtb-v1, v15, v18) were still relatively diverse, with diversity values of 0.25, 0.20, and 0.20, respectively. Markers Mtb-v4, v15, and were more diverse across the Beijing collection than across the four-bander set (Table. 3). Diversity values for collection of isolates having 4 IS6110 bands ranged from 0.0 to 0.3. The diversity values for Mtb-v5, v6, and v20 correspond to only one allele variation, in one isolate, across the entire collection set as illustrated in Table 4.
The diversity values of Mtb-v1, and Mtb-v10 across the combined strain set were greater than for either strain set alone. This was due to differential allele frequency, as opposed to unique allele number, across each data set. The Mtb-v1 size of 116 bp had a frequency of 88% within the set of strains having four IS6110 bands. In contrast, the allele size of 116 bp was seen on one occasion across the Beijing strain set, a 2.9% frequency. For Mtb-v10, a similar pattern of allele biasness can be seen. The allele size of 154 bp is seen in 84% of the isolates having 4 IS6110 bands. In contrast, the allele size of 154 is not present at all in the Beijing strains. Twenty-nine (85%) of the Beijing strains had an allele size of 163, whereas eight (14%) of the isolates having 4 IS6110 bands had an allele size 163. Hence the higher diversity value across Mtb-v1 and Mtb-v10 when the data is combined.
The present invention provides primer pairs for PCR amplification of VNTR in DNA of M. tuberculosum. The primer pairs comprise a forward primer and a reverse primer. Table 1 illustrates the M. tuberculosum Primer Sequences of the present invention. Table 1 also gives the source (location) of the VNTR. The location of each VNTR locus is given by the 5′ of the forward primer in accordance with the H37Rv reference strain.
These primer sequences have herein been assigned SEQ ID NO: as follows:
The polynucleotides of the present invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire long double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector
Methods for using these primer pairs to amplify VNTR loci in Mycobacterium tuberculosum are disclosed herein. Generally MLVA analyses or multiplex systems known to the art may be employed to detect and sub-types Mycobacterium tuberculosum. PCR instruments, for example, thermo-cyclers, used in these amplification methods are commercially available.
Methods for applying MLVA of these VNTR for strain discrimination among M. tuberculosum isolates are disclosed. Polymorphisms at these loci may be used to resolve genotypes into distinct groups. Among all 91 isolates, the nine-marker MLVA identified 17 unique genotypes. 12 unique MLVA genotypes are shown in
As observed in Table 3, a larger number of alleles did not always correspond to a greater diversity value. In one sense, allele number represents potential discrimination power while the diversity value represents the realized discrimination in a given set of samples. Both values are useful for understanding the genetic loci in any isolate being observed.
The amplicon size range for 7 of the loci is 107-290 bp. This size range allows for electrophoresis runs of roughly 2 hours on the ABI377 system (Applied Biosystems Incorporated, Foster City, Calif.). Two markers (Mtb-v15, Mtbv-20) have larger ranges from 278-591 bp (Table. 2). The number of alleles across these nine loci ranged from 2 to 8 (Table 3).
All of the polymorphic markers, except Mtb-v20, contained three or more copies of a tandem repeat sequence in the H37Rv sequence. Of these, seven had repeat motifs of three base multiples (Table 2), consistent with "in-frame" insertion and deletion mutational events. Seven loci were within hypothetical or known ORFs, while two were adjacent to genes (Table 2).
The present MLVA methods provide high species discrimination and sub-typing of Mycobacterium tuberculosum. Spoligotyping differentiated the 91 isolates into nine unique genotypes (
In addition IS6110-based restriction fragment length polymorphism (RFLP) may be used in a sub-typing scheme.
Table 5 illustrates clustering by these three methods.
The system of analysis has been applied to the globally prevalent homogeneous W-Beijing family of strains. Two sets of clinical isolates of M. tuberculosum: a set of 34 Beijing strains and another set of 57 isolates having 4 IS6110 RFLP bands. The details of this analysis are presented in the Experimental Section hereinbelow The results of these analyses illustrate that the characterization of molecular diversity with MLVA to the strain-typing of Mycobacterium tuberculosum method can be used for the rapid discrimination and identification of remaining major Mycobacterium tuberculosum species. They also illustrate that the sub-typing scheme useful for the epidemiological study of Mycobacterium tuberculosum may be applied to the local detection of the pathological causative agent of tuberculosis.
Kits are herein provided for use with commercially available PCR instruments to detect and sub-type strains of Mycobacterium tuberculosum. The kits contain one or more primer pairs disclosed hereinabove having SEQ ID NOS: 1-18 for amplifying the VNTR in DNA isolated from a Mycobacterium tuberculosum sample. If the sample is to be multiplexed, the kits may contain a suitable "cocktail" of primer pairs.
The kits may also contain nucleic acids needed in the amplification process. The nucleic acids may be tagged by a suitable marker, a fluorescent probe or a radioactive molecule. Any tag for marking the nucleic acid after amplification and size separation as by electrophoresis or other separation means is suitable. In certain preferred embodiments of the invention, the primer pairs themselves comprise a suitable marker.
The kits may also comprise enzymes, taq polymerase, for example and salts and buffers suitable for causing amplification of DNA by PCR. This kits may also comprise suitable containers and bottles for housing these reagents and or convenient use.
Kits for sub-typing strains of Mycobacterium tuberculosum comprise, in addition, DNA isolated from known Mycobacterium tuberculosum strains. This isolated DNA containing VNTR loci may be used as standards in the sub-typing of the species. In certain instances the kits may comprise in addition primers and standard references for MIRU analysis.
The following experiments illustrate the materials and methods used in genotyping M. tuberculosum isolates by MVLA with the primers of the present invention.
Source of Mycobacterium tuberculosum Strains.
As part of the Centers for Disease Control and Prevention (CDC) National Tuberculosum Genotyping and Surveillance Network, the University of Texas Health Science Center at San Antonio (UTHSCSA) serves as a genotyping reference laboratory for M. tuberculosum isolates collected by the Texas Department of Health (TDH). From 1996 to present, isolates from 4,269 persons with TB were genotyped (4, 15). The Institutional Review Boards of the TDH and the UTHSCSA approved culture collection and analyses (TDH-CDC cooperative agreement, TDH protocol number 980025, and UTHSCSA protocol number 001-6000-105). Two sets of clinical isolates were analyzed. One set consisted of 57 isolates having four IS6110 RFLP bands. The second set included 34 isolates with an identical Beijing spoligotype pattern containing spacer oligonucleotides 36-43 and 14 different IS6110 RFLP patterns.
DNA Isolation
M. tuberculosum isolates were grown on Lowenstein-Jensen media slants (Becton, Dickinson, and Co., Franklin Lakes, N.J.) for four to six weeks at 37� C. under 5% CO2. In a BioSafety Level 3 facility, the bacterial mass was removed using a sterile inoculating loop, placed in a microcentrifuge tube containing 1 ml ddH2O, and heat-killed at 85� C. for 20 min. The cells were pelleted by centrifugation and resuspended in a 10 mM Tris-HCl, 1 mM EDTA buffer (pH 8.0). Cell walls were digested with lysozyme (10 mg/ml), proteinase K (10 mg/ml), and 10% sodium dodecyl sulfate. DNA was extracted using 0.3 M cetyltrimethylammonium bromide and 5 M NaCl, purified by chloroform-isoamyl alcohol separation, and precipitated using isopropanol (Sigma, St. Louis, Mo.).
Spoligotype analyses (10) were done using locally synthesized and biotinylated PCR primers (UTHSCSA Advanced Nucleic Acids Technology Core Facility, San Antonio, Tex.) and commercially available spoligotyping membranes (Isogen, Maarssen, The Netherlands) as previously described (15).
IS6110 RFLP Analysis.
IS6110-based restriction fragment length polymorphism (RFLP) was performed by Southern blotting of PvuII-digested genomic DNA using a 523 bp (base pair) right-handed IS6110 probe (23), the ECL™ detection system (Amersham, Piscataway, N.J.), and BioImage Whole Band Analyzer version 3.4.2 (Genomic Solutions, Ann Arbor, Mich.) as previously described (15).
VNTR Identification and Primer Design
The complete genome sequence of the Mycobacterium tuberculosum H37Rv strain was downloaded from the Sanger Centre. Potentially polymorphic repetitive sequences were identified using the DNAstar software program Genequest (Lasergene, Inc Madison, Wis.). Selection criteria of repetitive sequence were set for nucleotide repeat motifs of more than 8 bp within 100 bp proximity of each other. Primers were designed around 84 repetitive sequences identified using the DNAstar software program Primer Select (Lasergene, Inc Madison, Wash.). Complementary primers were designed around interspersed repeats to minimize risk of mobile DNA targets. Fifteen of these repetitive sequences were found to be polymorphic, i.e., VNTR loci. Nine loci were chosen for use in these analyses (Table. 1)
PCR Amplification of VNTR Loci
PCR reactions were performed using a total volume of 10 μL. All PCR reagents used were obtained from InVitrogen (Madison, Wis.) unless otherwise indicated. Each PCR reaction mix contained the following reagents and concentrations: 1�PCR buffer (20 mM Tris-HCL [pH 8.4], 50 mM KCL); 2 mM MgCl2, 200 uM of each four deoxynucleoside triphosphates (dNTPs), 0.4 Units of Platinum Taq Polymerase (Gibco-Life Technologies) per uL, 0.2 uM forward primer, 0.2 uM reverse primer R110, R6G, or Tamara phosphoramide fluorescent labeled oligonucleotides (Perkin Elmer Biosystems), 1 uL DNA template, and ddH2O bringing the total volume to 10 uL. Each 10 ul PCR mix was then denatured for 5 minutes at 95� C. Following the denaturing, the samples were cycled 35 times through the following program: 95� C. for 20 seconds, 55� C. for 25 seconds, and 72� C. for 20 seconds. These 35 cycles were followed by a final extension at 72� C. for 5 minutes and the samples were then stored at −20� C. until genotyped. Reactions that failed under the above conditions were then repeated with 2 ul of DNA in a 10 ul reaction; all other concentrations and conditions remaining the same.
Detailed automated genotyping methods have been described previously (Keim, P., L. B. Price, A. M. Klevytska, K. L. Smith, J. M. Schupp, R. Okinaka, P. J. Jackson, and M. E. Hugh-Jones. 2000. Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. Journal of Bacteriology. 182:2928-36), herein incorporated in its entirety. Briefly, fluorescent labeled amplicons were sized and scored using the ABI software program Genotyper.
Statistical Analysis
Genetic distances were determined by calculating the percentage shared alleles pair-wise among all isolates. The clustering method used was unweighted pair group method arithmetic average (UPGMA). UPGMA clustering analysis was performed using the software program NTSYS (16). There was no missing data across the nine VNTR loci and 91 isolates. The diversity index of each VNTR marker was calculated using [1-Σ (allele frequencies)2](25).
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v1 with primer pairs SEQ ID NO: 1 and SEQ ID NO: 2. 5′ Beginning location of VNTR: 55,533. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v3 with primer pairs SEQ ID NO: 3 and SEQ ID NO: 4. 5′ Beginning location of VNTR 241,464. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v4 with primer pairs SEQ ID NO: 5 and SEQ ID NO: 6. 5′ Beginning location of VNTR -241,464. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v5 with primer pairs SEQ ID NO: 7 and SEQ ID NO: 8. 5′ Beginning location of VNTR 241,464. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v6 with primer pairs SEQ ID NO: 9 and SEQ ID NO: 10. 5′ Beginning location of VNTR 1,112,923. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v 10 with primer pairs SEQ ID NO: 11 and SEQ ID NO: 12. Beginning location of VNTR 2,604,155. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v15 with primer pairs SEQ ID NO: 13 and SEQ ID NO: 14. Beginning location of VNTR 3,238,462. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v 18 with primer pairs SEQ ID NO 15 and SEQ ID NO 16. 5′ Beginning location of VNTR 4,227,024. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
This example illustrates the amplicon produced during the amplification of VNTR locus Mtb-v20 with primer pairs SEQ ID NO 17 and SEQ ID NO 18. Beginning location of VNTR 23,693. (All accession numbers are according to the H37Rv M. tuberculosum sequence done by the Sanger Institute.)
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
Patent Citations
Non-Patent Citations
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Smoked Turkey and Gnocchi Salad
19 Sep 2011
|1⁄4 Pound (About 110 Gram, Dried, In Shapes, Not Dumplings)
|Olive oil
|45 Milliliter (3 Tablespoon)
|Smoked turkey
|8 Ounce, flaked into pieces 1 1/4 - 1 1/2 inches long (225 Gram)
|Sliced button mushrooms
|165 Milliliter (2/3 Cup)
|Chopped fresh chives
|15 Milliliter (1 Tablespoon)
|Freshly ground black pepper
|To Taste
|Balsamic vinegar
|1 Teaspoon (5 Milliliter)
|Extra virgin olive oil
|2 Teaspoon (10 Milliliter)
|Avocados/1 large sized
|2 Small, quartered and sliced
|Smoked mozzarella cheese/Bruder basil
|1⁄4 Pound, cut into 1/2 inch cubes (110 Gram Natural Smoked Cheese)
1) In a pan, cook the Gnocchi shapes in the boiling salted water, until tender. Drain and rinse the pasta in cold water, then drain again and keep aside.
2) In a large frying pan, saute the turkey pieces, mushrooms and chives in the olive oil, until the turkey is lightly browned.
3) Season with the black pepper to taste.
4) Stir in the vinegar and olive oil, stir continuously and cook, until the liquid has reduced and thickened.
5) Adjust the seasoning, toss thoroughly with the pasta and cook for 10 to 15 seconds.
6) Turn off the heat, stir in the avocado slices and cheese, then allow the dish to stand for 2 to 3 minutes before serving.
7) Serve the Smoked Turkey and Gnocchi Salad warm, or at room temperature.
Recipe Summary
Difficulty Level:Medium
Course:Main Dish
Preparation Time:5 Minutes
Cook Time:25 Minutes
Ready In:30 Minutes
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Smoked Turkey And Gnocchi Salad Recipe

subset: commoncrawl


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[New York Post] Which socialite was caught in a compromising position » Continued
[New York Post] Which socialite was caught in a compromising position » Continued
[PopBitch] While most people know this rock guitarist for being married to a society beauty, which of his New York-based school friends remember him chiefly as the boy who shat in her swimming pool when they were kids?
Rock Guitarist:
His Wife:
His School Friend:
[Out] Which socialite sisters were livid when they showed up at an event wearing the same couture outfit?
(It was sort of like the "Paris Original" number from How To Succeed In Business Without Really Trying, but not quite as hilarious.)
[Blind Gossip] We have LOTS of fascinating information for you about the married pregnant socialite who has recently been thrust into the headlines!
She has a long and » Continued
[New York Post] Which globetrotting socialite refuses to allow her outrageously lavish home to be photographed despite groveling overtures from a top fashion editrix?
BG Note: Editrix = a female editor
[New York Post] Which socialite is the butt of jokes for waiting patiently at every event for party photographers to find her, which they seldom ever do?
[Village Voice] Which daughter of a late socialite used to lube herself from head to toe in an attempt to seduce a Siberian husky? (The maid would find the daughter all scratched up and the dog » Continued
[VillageVoice] Which thirtysomething blonde socialite is using her diet pills at such an alarming rate that her doctor » Continued
[NewYorkPost] Which younger brother of an infamous socialite appears in a » Continued
[NewYorkPost] Which married socialite was recently spotted in a compromising position with a man not her husband on a fire escape at a fashion party?
Copyright © 2015 BLIND GOSSIP - All Rights Reserved


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Ernest Hemingway, exemplar of all things craggy and masculine, was the inspiration for this complex fragrance—but don't expect notes of rum and tobacco. This is a cultured fragrance for a cultured man. Papa would approve.
I've never purchased any scents above what you might find at Target or Walgreens. That being said, this product has a very nice smell that lasts for a long time. I could even smell it after I had taken a shower the next day. I don't find it overpowering, but it is definitely noticeable.5


text:
You spend all morning arming yourself for the day—the Shield Mirror just adds that final kick of confidence. Designed by Alan Wisniewski for Umbra in the shape of a family crest, this beveled mirror hangs slightly away from the wall to create a dramatic shadow effect.
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text: Chiles Poblanos in a Cream-Cheesy Sauce (Rajas con Crema) Mark Thompson March 22, 2014 Recipes For being a meatless dish it is very filling and just plain delicious. Many families in Mexico use this as their 'Friday' food during Lent. Typically poblano chiles are not spicy, but every once in a while a spicy one can sneak in there, so a taste test might be in order before serving this meal to children. It can be served as a main meal in tacos or as a dip with corn chips. [gmc_recipe 21902] The following two tabs change content below.BioLatest Posts Mark Thompson Mark is the Editor-in-Chief of So Good Blog. Latest posts by Mark Thompson (see all) Secret Menus You Need to Try Immediately - June 12, 2015 Jerky Club of the Month Review – Monthly Jerky - April 15, 2015 The Food Revolution in Poker - April 10, 2015 Leave a Reply Cancel Reply Your email address will not be published. Name* Email* Website Comment Current day month ye@r * Leave this field empty * Notify me of follow-up comments by email. Notify me of new posts by email.


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Just shy of his teenage years, Brendan Doyle remembers being at a few of the water stops along the course of the 1990 Ocean State Marathon.
His older brother Patrick was there, too.
Earlier in their lives they were two young to really witness the impact that their father had at the 26.2-mile race that traversed over the Newport streets. On this day, though, it was different.
First-hand, the oldest sons of the late Bobby Doyle, got to see their dad in action.
PAWTUCKET ŌĆō Andrew Miller is proof that itŌĆÖs never too late to turn things around. At the seasonŌĆÖs onset the lanky lefty was viewed as the ultimate redemption project, a pitcher who had spent the better portion of his career mixing promise with underwhelming results.
LINCOLN ŌĆö As Max, an orphaned pit bull, used his teeth to rip bark from a tree just outside the Wellington Road shelter, Animal Control Officer Louann Noreau laughed, then uttered, ŌĆ£I think he was a logger in his past life.
ŌĆ£You know, he just loves anything to do with trees,ŌĆØ she stated early Tuesday afternoon. ŌĆ£He'll climb them, swing from tree branches. If there's a tire swing around, he'll jump through the tire and swing his legs to gain momentum. He's just like a little boy.
Susanna J. Kelley-Magill
JOHNSTON - Susanna J. "Susan" Kelley-Magill, 93, of Johnston formerly of Pawtucket, died Monday at Briarcliffe Nursing Home. She was the wife of the late Francis L. Kelley and Robert A. Magill. Born in Pawtucket, she was a daughter of the late Andrew and Antonina N. (Spiec) Kuc.
She was employed as a secretary at Lincoln Junior-Senior High School for 12 years prior to her retirement in 1980. She had been a volunteer at Memorial Hospital and a member of St. Aidan's and St. Teresa's Senior Citizens.
John W. Richardson, Jr.
Ronald J. McCormack
PAWTUCKET - Ronald J. McCormack died suddenly on Wednesday June 15, 2011 at home. He was 56 years old. He was the beloved son of Joan W. McCormack of Pawtucket, nephew of Patricia Whitaker of Franklin, MA and had several nieces and nephews. He was a life long resident of Pawtucket and a 37 year State of RI employee. He had attended Woodlawn Baptist Church. There are no calling hours and the funeral will be private.
Mary V. O'Malley
This may sound like an altruistic plea, but baseball and wooden bats belong together. Those governing American Legion baseball in this state are inclined to agree, as board members have decided to eliminate the usage of aluminum in favor of a wooden-bat league.
This landmark change takes effect for the 2011 summer session, which is currently under way. Gone is the ringing ŌĆ£ping!ŌĆØ sound that ensues whenever an aluminum bat strikes a baseball. Expect to hear a ŌĆ£crack!ŌĆØ at your local Legion ball field, a natural emanation that figures to add great theater to this season.
Dave Adamonis Jr. knows his younger brother BradŌĆÖs golf game as well as anyone.
Growing up in Cumberland, the two siblings, a mere four years apart in age, spent endless hours on the links during their childhood years and beyond. Although more than 1,000 miles separate them now, they still keep in close contact, talking about golf and their personal lives.
ŌĆ£Even though Brad lives in Florida,ŌĆØ Dave said, ŌĆ£we still talk to each other every day.ŌĆØ
PAWTUCKET ŌĆö Perhaps 30 minutes before the festivities of Tolman High School's Commencement, Dr. Linda Gifford, a guidance counselor, stood inside the James W. Donaldson Gymnasium offering congratulations to members of the Class of 2011, boys clad in red caps and gowns, the girls in white.
Suddenly, Mckenzie Hosford tapped on her shoulder, and Gifford spun around. When the elder saw Hosford, she immediately gave her a massive hug.
ŌĆ£It's been quite a journey,ŌĆØ Hosford told her.
ŌĆ£With a happy ending,ŌĆØ Gifford smiled back.
PAWTUCKET ŌĆö Hundreds of firefighters filled the sidewalk along Roosevelt Avenue from Exchange Street to Slater Mill Wednesday, protesting a proposal to privatize the city's rescue service.
Inside City Hall at the time were companies who submitted requests for proposals to do the rescue work. They were there to attend a ŌĆ£mandatory meetingŌĆØ with city officials to discuss their bids.
Robert Neill, president of the city's firefighters union, IAFF Local XXXX, said the picketing was timed to ŌĆ£discourage those peopleŌĆØ and make them aware that the might ŌĆ£run into a possible labor dispute.ŌĆØ
H. Jim Knight
PAWTUCKET ŌĆō H. Jim "Steve" Knight, 71, passed away February 18th. He was the son of the late G. Tom Gallagher and Mary (Jackson) Knight; step-son of the late Renaldo Knight; husband of Carolyn J. (Illuzzi) Knight; father of Lisa McEnery, Lori Cavallaro, Lynn Masse and Jeffrey Knight; grandfather of 9.
Relatives and friends are invited to attend a Memorial Mass on Saturday at 10 a.m. in The Church of the Presentation of the Blessed Virgin Mary, 1081 Mineral Spring Avenue, North Providence. Arrangements by PONTARELLI-MARINO.
PAWTUCKET ŌĆö Valedictorian Noelle Cabana said she used to feel saddened by the fact that she never got to meet the late Jackie Walsh, and that the teacher passed away before the completion of the arts high school that was her dream. ŌĆ£But then I realized that she has touched us in ways we canŌĆÖt even recognize,ŌĆØ Cabana told her fellow classmates, as they sat together at Tuesday nightŌĆÖs commencement exercises for the school that bears WalshŌĆÖs name.
CUMBERLAND ŌĆō One of three suspects arrested in a violent home invasion and weapons theft on Feb. 23 has been indicted on a string of charges by a Providence County grand jury.
PAWTUCKET ŌĆö Andrew Miller looked very much like a pitcher who doesnŌĆÖt belong pitching every fifth day in Triple-A Tuesday night.
With Wednesday signifying the first of two reported opt-out dates ŌĆö the second occurring Aug. 5 ŌĆö MillerŌĆÖs timing couldnŌĆÖt have worked out any better. The lanky lefthander delivered his finest outing in a PawSox uniform, allowing one run while striking out 10 Charlotte Knights in 5 1/3 innings. He received a no decision as Pawtucket fell in 11 innings, 4-2.
PAWTUCKET ŌĆō To say that the Pawtucket Red Sox are looking forward to having Ryan Kalish back in the lineup would be a vast understatement. It appears, though, that the outfield prospect still needs more time.
On Tuesday PawSox manager Arnie Beyeler revealed that Kalish is fighting through ŌĆ£a neck issue, a minimal thing that keeps hindering him a bit.ŌĆØ The first signal to Kalish that something didn't feel quite right occurred while rehabbing his right shoulder in Fort Myers, Fla. earlier this month.
PROVIDENCE --- They split their regular-season series by winning lopsided games on their home fields. They battled to a 1-0 duel in the winnersŌĆÖ bracket semifinals, and they shared some Division I championship history by playing a one-sided game that was decided by a dozen runs.
On Tuesday night, Cumberland High and Mount St. Charles Academy battled each other in a winner-take-all showdown for the ages, a nine-inning duel that saw the Clippers emerge with their programŌĆÖs first state title.
Claire A. Dubuc
SEEKONK - Claire A. (Dulude) Dubuc, 81, passed away at home on Monday, June 13, 2011. She was the beloved wife of the late Maurice J. Dubuc.
Born in Pawtucket, a daughter of the late Ernest J. and Martha (Hebert) Dulude, she resided in Seekonk for 52 years. Claire enjoyed volunteering at the Knights of Columbus Hall, South Attleboro.
PAWTUCKET ŌĆö You could see it on the tired but happy faces of Peaches, Chloe, Kevin, Charlie, Tinkerbelle, Oso, Lucy and Willow Spitfire: the cityŌĆÖs new dog park is going to be a howling success.
On Saturday morning, the main proponents of the dog park, including City Councilors Albert Vitali, Mark Wildenhain and Christopher OŌĆÖNeill, Economic and Cultural Affairs Officer Herb Weiss, Animal Control Director John Holmes, Sheryl Rennick and her daughter, Mackenzie, and others joined Mayor Donald Grebien for a ceremonial ŌĆ£leash cuttingŌĆØ to officially open the venue.
PAWTUCKET ŌĆö Yesterday marked the 164th day of the year, the date of the great Lewis and Clark expedition and the graduation of 195 students from Charles E. Shea High School.
At 6 p.m. the atmosphere in Pawtucket was one to be remembered. Although the weather was overcast and gloomy, not a face in the crowd of soon-to-be graduates showed it. Students were overjoyed at the expedition that they were about to embark on, the moment that would forever be a piece of their past: their high school graduation.


text:
Press release from CNW Group
/R E P E A T -- Aimia to hold Annual and Special Meeting of Shareholders Friday May 4, 2012/
Thursday, May 03, 2012
/R E P E A T -- Aimia to hold Annual and Special Meeting of Shareholders Friday May 4, 2012/10:00 EDT Thursday, May 03, 2012MONTREAL, April 24, 2012 /CNW Telbec/ - Groupe Aeroplan Inc., now carrying on business as Aimia (TSX: AIM) will hold its Annual and Special Meeting of Shareholders on Friday, May 4, 2012.Date:Friday, May 4, 2012 Time:11:00 a.m. ET Location:Centre Mont-Royal2200 Mansfield StreetMontreal, QuebecCanada, H3A 3R8 By Telephone:1-888-231-8191 and 647-427-7450Please allow 10 minutes to be connected to the conference call.Webcast: English: http://www.newswire.ca/en/webcast/detail/954827/1022541French: http://www.newswire.ca/fr/webcast/detail/954829/1022545This is a listen-only audio webcast. Windows Media Player is required to listen to the broadcast. Replay:An archived audio webcast will be available at http://www.aimia.com/English/Investors/Presentations-and-Events/Events/default.aspx for ninety days following the original broadcast. Note:A slide presentation intended for simultaneous viewing with the webcast will be available the morning of May 4, 2012 at http://www.aimia.com/English/Investors/Presentations-and-Events/Presentations/default.aspx. Media Briefing:Media are invited to attend, but only shareholders may ask questions during the meeting. A briefing for media will be held immediately following the meeting.About AimiaGroupe Aeroplan Inc., doing business as Aimia ("Aimia"), is a global leader in loyalty management. Aimia's unique capabilities include proven expertise in delivering proprietary loyalty services, launching and managing coalition loyalty programs, creating value through loyalty analytics and driving innovation in the emerging digital and mobile spaces. Aimia owns and operates Aeroplan, Canada's premier coalition loyalty program and Nectar, the United Kingdom's largest coalition loyalty program. In addition, Aimia has majority equity positions in Air Miles Middle East and Nectar Italia as well as a minority position in Club Premier, Mexico's leading coalition loyalty program and Cardlytics, a US-based private company operating in merchant-funded transaction-driven marketing for electronic banking.Aimia is a Canadian public company listed on the Toronto Stock Exchange (TSX:AIM) and has over 3,400 employees in more than 20 countries around the world. For more information about Aimia, please visit www.aimia.com. For further information: Media JoAnne Hayes 416-352-3706 firstname.lastname@example.com Analysts Trish Moran 416-352-3728 firstname.lastname@example.com


text:
I have it: 12 I had it: 2 I want it: 34 My signature: 1
Designers » C » Creed Woody Chypre « Groups
I have it: 12 I had it: 2 I want it: 34 My signature: 1
Cypres Musc, launched in 1948, belongs to the Creed Private Collection, which consists of perfumes originally created for famous clients. The Private Collection scents, when available, are offered in large 250 ml tinted glass flacons with ground glass stoppers, covered in natural hide and sealed with gold thread.
Cypres Musc is a wonderful fresh, woody and green scent. The notes include bergamot, cypress, oakmoss, musk and amber. The nose behind this fragrance is Henry Creed Fourth Generation.
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Now that I finally got a full flacon of this precious juice I HAVE to rewrite this review. Initially your confronted with a smokey citrus blast, I pick up hints of musc, cypres, mint most likely neroli as well. The opening is just breath taking and will fill up a whole house with its smell. As the mid notes come to the surface i get a small amount of ginger, some amber with the musc becoming more prominent and cypres lending it's spicy woodiness. I get a lot of ammonia scent,not sure where its coming from but its there. The base is what is so mind blowing. The musc just smells so perfect, you get hints of freshly cut wood, but nothing overwhelming. It is actually amazing how well it is composed. 90% of the time wood finishes are very sharp but this is so pleasant. Its like walking in a new home. I don't think any fragrance I have worn is as pleasant and beautiful as this. Amazing.
LOL! Toilet freshener, Pine sol, maybe even that little paper pine scented car fragrance you hang on the rearview mirror?
I can see why people would say that!
But ya know what? It's a Damn good freshener, like what you'd find in the finest hotels and mansions of the rich and elite!
Seriously though, those are over simplifications of this regal, outdoorsy scent.
It's a cool, green, and musky cypress experience.
Makes me think of an Ancient Greek Hero walking through the rocky, cypress lined landscape in the dead of winter.
if someone is having this, i would like to swap..please send me a message
This was the personal perfume of Aristotle Onassis....i would like to smell it indeed!!!
I would love to know the identity of the "famous client" for whom Creed CYPRES MUSC was created. Could it perhaps be the director of my old girl scout camp?
Upon application, I was shocked by the smell of this potent little liquid. Was it Pine Sol? No, more complex than that. And also less clean. What was this oh-so-familiar fragrance from my long-forgotten past?
And then it came to me in a flash: CYPRES MUSC smells *exactly* like the latrines at summer camp! The mingling odor of Pine Sol-saturated planks of wood along with the horrific mass of human excrement in the deep dark wells of makeshift bathrooms found hidden away in the midst of the beautiful Rocky Mountains.
Although it was nice to recall the not-always-so-halcyon days of my childhood, I won't be adding CYPRES MUSC to my collection. Who would want to smell like this?
Become a member of this online perfume community and you will be able to add your own reviews.
This page contains information, reviews, perfume notes, pictures, new ads, vintage posters and videos about Creed Cypres Musc fragrance but we do not warrant the accuracy of information. Trademarks and logos belong to respected companies and manufacturers and are used solely to identify products and companies. If you have more information about Creed Cypres Musc, you can expand it by adding a personal perfume review. Fragrantica has a unique user-driven classification system and you may classify Cypres Musc by Creed. Click on the appropriate options on the fragrance classification form below the perfume picture. Also, you can find links to 3rd party websites/Internet stores, but Fragrantica has no access or control over those websites. We do not make guarantees nor accept responsibility for what you might find as a result of these links, or for any future consequences including but not limited to money loss. User reviews of Cypres Musc by Creed represent the views of the credited authors alone and do not reflect Fragrantica's views.
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University of Washington defensive tackle Danny Shelton talks about his journey to the NFL Draft and his excitement for the future.
NFL Media's Omar Ruiz reports from the Cleveland Browns facility on the quarterback battle, and also if the Browns are planning to draft another quarterback in the first round.
Running back Jim Brown left the game at his peak and still sits atop the record books. Making his mark on and off the field, he is a true legend.
Joe Haden joins Marshall Faulk to talk about the Super Bowl matchup, quarterback Johnny Manziel, and his work with the Special Olympics.
Cornerback Joe Haden talks about his switch from quarterback to cornerback, the best football coach in Ohio and his favorite Katy Perry costumes.
Cornerback Joe Haden once again proved that he is one of the top defensive players in 2014 NFL season.
Joe Thomas shows his appreciation for the sacrifice given by the United States Military.
Solomon Wilcots breaks down how the Cleveland Browns are pressuring Cincinnati Bengals quarterback Andy Dalton.
Solomon Wilcots breaks down how Cleveland Browns quarterback Brian Hoyer and the running game is shredding the Cincinnati Bengals defense.
Solomon Wilcots breaks down the Cleveland Browns drive that culminated in a Isaiah Crowell TD run.


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A Warming World
NASA Goddard Institute for Space Studies, NASA
Discuss this Resource»
Learn more about Teaching Climate Literacy and Energy Awareness»
Grade Level
Climate Literacy
About Teaching Climate Literacy
About Teaching Principle 4
Climate change vs. climate variability and patterns
Other materials addressing 4c
About Teaching Principle 5
Observations are the foundation for understanding the climate system
Other materials addressing 5b
Benchmarks for Science Literacy
Learn more about the Benchmarks
Explore the map of concepts related to this benchmark
Scientific investigations usually involve the collection of relevant data, the use of logical reasoning, and the application of imagination in devising hypotheses and explanations to make sense of the collected data.
Notes From Our Reviewers
The CLEAN collection is hand-picked and rigorously reviewed for scientific accuracy and classroom effectiveness.
Read what our review team had to say about this resource below or learn more about
how CLEAN reviews teaching materials
Teaching Tips | Science | Pedagogy |
Technical Details
Teaching Tips
- A good lecture aid for talking about increasing global temperature and linking it to real-world historical events.
About the Science
- On the first graph, an orange line shows long-term trends by averaging temperatures over a five-year period. Purple error bars represent the uncertainty on measurements.
- On the second graph, the observation is that despite differences in the ways the scientists develop their averaged temperature records, the four different and well-known temperature records show remarkable agreement.
- Comments from expert scientist: Resource is a very effective presentation.
- It gives details behind unusual years.
About the Pedagogy
- The NASA temperature graph includes hyperlink points on the graph providing text information on historical events that provide context for students on why sudden changes in temperature sometimes occur (volcanic eruption, El Niño, La Niña, etc).
- These very effectively show how natural events can affect Earth's global temperatures for short periods of time within the context of global change trends.
Technical Details/Ease of Use
- The visualization is clear and easy to read, and the links work, though you get a flickering of the descriptions of some specific points if you pass over them with the mouse.
Related URLs These related sites were noted by our reviewers but have not been reviewed by CLEANWarming World from NASA Climate: http://climate.nasa.gov/warmingworld/
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These outstanding features set the standard for grilling performance.
Dual-Tube™ Burners
Dual-Tube™ burners are designed to ensure a consistent flame from front to back, providing even and efficient heat distribution at the cooking surface. Made from high quality stainless steel and backed by a 5 year warranty.
Side Burner
Our high-powered side burner allows you to do more. Create mouth-watering side dishes, soups and sauces while you grill.
Cast Iron Cooking Grids
Our cast iron grids are reversible. One side is pointed to give you perfect grill marks and the other side is grooved to capture juices for continuous basting while cooking. With a little care, they will last a lifetime.
Stainless steel Flav-R-Wave™ cooking system provides superior heat distribution, protects the burner and vapourizes drippings for great barbecue flavour.
Cook Box
Depending on the model you choose, the deep and durable cookbox is made from either dye cast aluminum, porcelain enameled steel or high-grade stainless steel. From convection cooking to searing heat, no matter what Broil King model you choose, they all provide excellent heat retention and long lasting durability.
Sure-Lite™ Ignition
The Sure-Lite™ ignition system offers you peace of mind knowing your barbecue will start with the push of a button.
Deluxe Accu-Temp™ thermometer
Our easy to read stainless steel thermometer has a probe that reaches deep into the oven, giving you a more accurate temperature reading.
Wheels and Casters
The Wheels on the Broil King® line-up of grills are built for easy maneuverability. With minimal effort you can slide your grill wherever you want in your backyard or on your patio. However, with the flip of a switch the heavy-duty locking swivel casters keep your grill stationary and stable.
We use high quality stainless steel for many components. All other steel is zinc coated and further protected by an epoxy paint that is baked on through a powder coated process.


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Weleda Skin Food Cream -- 2.5 oz
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A unique cream to be used on the face and body. Ideal for skin that is dry, flaky, and in need of extra care. Skin Food is great for feet, dry elbows, cuticles and chapped lips. Skin food may also be used to moisturize skin on the face. For a special hand treatment apply cream generously and put on cotton gloves overnight. Contains pansy extract, chamomille, calendula and a precious essential mix of lavender, rosemary and sweet orange oil.
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Is there a doctor in the orchestra?
Amanda Holloway: In the latest in a long line of classical-music-related injuries, America's leading conductor, the portly James Levine, tripped and fell on stage during a standing ovation for a Boston Symphony Orchestra concert.
G2 Comment & features p2
Why is Davina's chatshow such a disaster?
Somewhere between flogging hair dye and fronting her eponymous BBC1 chatshow, Davina McCall, or her agent at least, has lost any shred of credibility she ever had. By Gareth Maclean.
Comment & features p3
French students are out on the streets to protest about a new employment law - even though it means more jobs for themMarcel Berlins: For a moment, it was like old times. I was in Paris a few days ago when hundreds of students occupied the Sorbonne and the riot police were called out in excessive numbers to turf them out.
Comment & features p5
Behind every great male writer ...This week Dan Brown, author of The Da Vinci Code, revealed that his wife Blythe helps him write his bestsellers. It puts him in good company, writes Hadley Freeman.
Comment & features p6
Invisible cityChongqing is the fastest-growing urban centre on the planet. Its population is already bigger than that of Peru or Iraq, with half a million more arriving every year in search of a better life. And yet so frequently is this story repeated in China, that outside the country its name barely registers. Jonathan Watts spends 24 hours in the megalopolis you've never heard of.
Comment & features p8
Too lazy to chew?
Yoghurt sales are up by half - but only the liquid kind. Are we now too idle to do anything other than slurp? Lawrence Booth grabs a straw to find out.
G2 Comment & features p14
List of the dayQuirky detectives
Comment & features p29
A chocolate croissant and a facelift, pleaseLucy Mangan: You can now have lips collagenned, foreheads botoxed, body hair lasered and even breasts enlarged in under an hour.


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Grid List
Double-zip Leather Shoulder BagITEM: 9651 Our ample shoulder bag has two outside zippered pockets, one large and one small, plus an open compartment. The double-zip top opens into two separate sections, one with a zipper pocket and cell phone pocket. Adjustable strap. 9 1⁄2"h x 12"w x 4"d. Imported. Leather clean.Learn More
Regular Price: $200.00
Special Price $80.00
Large Shoulder HandbagITEM: 0042 Large Shoulder Handbag. Soft supple leather, with three zip pockets, and a total of 5 compartments. Approx. 15"w x 13"h x 3"d Imported.Learn More
Regular Price: $98.00
Special Price $39.20
Leather Hobo HandbagITEM: 9009 The hottest hobo handbag. Our newest leather hobo includes a designer inspired woven shoulder strap, smart looking hardware, leopard print lining, full zip closure, and lots of pockets inside. You will love it! 14"w x 10"h x 3 1/4"d. Imported.Learn More
Regular Price: $110.00
Special Price $44.00
Leather Quilted Dome HandbagITEM: 9079 Urban sleek, and a quilted chic. Our quilted dome bag of soft lambskin leather has a polished style. Convenient double handle, full zipper top for easy access, inside zippered compartment and cell phone pocket. Wipe-clean lining. Approx 12" W x 10" H. Imported.Learn More
Regular Price: $70.00
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Leather Shoulder BagITEM: 9057 A shoulder bag or a clutch-your choice. It's large enough to hold your personal items, but small enough to work with any outfit. Crafted of beautiful, soft leather with a metal turn-key closure. Detachable shoulder strap. Convenient inside pockets and zippered compartment. 13"w x 6"h. Imported. Leather clean.Learn More
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Minnetonka Suede Fringe BagITEM: 9426 Expertly hand-crafted by Minnetonka,® this mini bag features layers of fringe and accents, and inside zipper pocket and organizer. Adjustable strap makes it a great shoulder or cross-body handbag. Inside snap keeps items safely secured. 5.5"w x 6"h x 1.5"d. Imported. Suede clean.Learn More
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Multi-textured Tote bagITEM: 9055 The fabulous multi-textured tote bag. This bag features a fashionable combination of faux croco, faux suede, printed lizard and smooth patent. Silver accents. Double handle for over-the-shoulder or hand carrying. Magnetic snap closure for extra security. Inside: zippered pocket plus two extra pockets. Wipe-clean lining. 12"h x 14"w x 8"d. Imported.Learn More
Regular Price: $70.00
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Suede Fringe HandbagITEM: 9025 Slouchy and suede with fantastic fringe. Our fun new hobo bag is totally chic and roomy. Crafted by Minnetonka® in soft suede leather, with a versatile, adjustable shoulder strap, inside zippered pocket, and other smart extras. 13"w x 8"h x 5"d. Imported. Leather clean.Learn More
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The Large Quilted ToteITEM: 9084 The quilted tote. For all-around practicality, you can't beat the handiness of a tote. Crafted of soft quilted fabric with a printed croc double handle and detachable key fob with silver-tone charms and tassel. Full zippered top closure. Inside zippered compartment with two additional pockets. Wipe-clean lining. Approx. 15" W x 14" H. Imported.Learn More
Regular Price: $48.75
Special Price $19.50


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Feed U.S. Kids with The BackPack Program
Roll over image to zoom in
Millions of low-income children in the United States rely on food support from the National School Lunch Program. But when school is not in session, children living in food-insecure households are at risk of going without the nutritious food they need.
Feeding America's BackPack Program was developed to address this need. Currently, there are nearly 5,000 BackPack Programs around the country, serving more than 230,000 children each year. Backpacks, filled with child-friendly, vitamin fortified and nonperishable food, are discreetly distributed to children to take home on weekends or before holiday vacations.
You can help! $25 helps Feeding America establish and strengthen BackPack Programs around the country, ensuring that children do not have to go to bed hungry or suffer the ill health created by poor nutrition.
Report from the Field
In 2012, Feeding America released a study showing that more than 16 million children nationwide now deal with the potential of hunger. Children are "food insecure" in every county in the United States, and, in more than 1,000 counties, more than one in four children are at risk of hunger.
Feeding America provides low-income individuals and families with the fuel to survive and even thrive. As the nation's leading domestic hunger-relief charity, they serve the entire United States with more than 200 member food banks supporting 61,000 agencies that address hunger in all of its forms.
Free Standard Shipping in the U.S. on your entire cart when you donate $15.00 or more in our Gift That Gives More™ program. See Current Promotions for details.
Why a Gift That Gives More™ is a smart, effective donation.
100% of your donation will go to the program described above as a grant through GreaterGood.org. GreaterGood stores do not receive any profit from donations through this Gift That Gives More™; we bring it to you in the spirit of the greater good. We even pay the credit card transaction fee, so every cent of your donation goes to charity.
Please note: A free downloadable certificate is available to print for easy gift giving for any non-disaster related Gift That Gives More™. The "gift message" field during check-out does not apply to your Gift That Gives More™ certificate.
Your donation is 100% tax-deductible in the U.S.A.
You will receive a receipt from GreaterGood.org for your taxes. GreaterGood.org has ultimate authority and discretion with regard to the distribution of its funds. All expenditures made are consistent with the exempt purposes of GreaterGood.org.
Check with your employer: your company may match your donation.
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While I've "done" a lot of euthanasia procedures, JP was my very first pet that I had to euthanize on my own. I fully admit that I was a mess … and I know that it'll take time to heal. But what I personally learned a lot from it was this:
- If you have the opportunity to, euthanize your pet at home. It's comforting to your four-legged friend to not have to undergo a stressful car-ride or be subjected to a strange environment. While this isn't always feasible or possible, if you can make it happen, do so.
- Take the time to mourn. I took the whole weekend to spend every waking moment with JP before I euthanized him, spoiling him rotten in the process. I was fortunate enough to take time away from work afterwards too – I needed the mental health time to mourn my best friend.
- Ask your veterinarian for a high-fiber canned dog food for the last few weeks of your pet's life. I was feeding JP so much filet mignon and table food, he ended up with severe diarrhea, which made him (and his butt) miserable for a few days. By mixing in the high-fiber canned food (or probiotic) for the last few weeks, JP was able to eat to his heart's (and butt's) content while dining on delicious snacks.
- Never question what you want. Hours later, I frantically called the hospital where I dropped off JP's body for cremation, asking them to save me some fur. In my grief, I had forgotten to ask for it earlier. Someone then asked me if I really wanted his fur, and would it mean that much to me after he's gone (after all, it's still all around the house!). When mourning, any step helps… and yes, I did want his fur and all those clay paws!
Next Page: even more advice on losing a pet


text: > View Product Details Please select the size of the product. Please enter valid quantity. Zegna charcoal and light blue diagonal stripe silk-cotton tie overview details Retail: $195.00 Reduced: $146.99 20% Off : $117.59 color : We're sorry. This style is currently unavailable. Charcoal / Light Blue For a stylish and sophisticated finishing touch, look no further than a classic silk tie from Zegna. Color: Charcoal / Light Blue Lightweight diagonal striped silk-cotton blend woven Logo silk faille lining Measures approximately 58'' long x 3�'' at widest point 74% Silk / 26% Cotton; Dry Clean; Italy; style#320132801


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After a lot of hard work, long nights, and even longer days by our development team we are happy to announce that Carrara 8.5 is available for purchase in the DAZ 3D store. We would like to thank all of our loyal Carrara customers for their patience and support with this project.
Carrara 8.5 Pro is priced at $285.00 for a limited time. Carrara 8.5 Standard is priced at $149.95.
Current owners of Carrara will receive a 50% discount on either product for a limited time. Platinum Club members get an additional 40% off, which means it's never been a better time to join the Platinum Club.
Carrara 8.5 Pro
Carrara 8.5 Standard
Included in Carrara 8.5 Pro are the Michael 5 and Victoria 5 Pro Bundles and Carrara Shaders - over $250 worth of Genesis content.
Carrara 8.5 standard ships with the Michael 5 and Victoria 5 Starter Bundles and Carrara Shaders - over $140 worth of Genesis content.
Here's a quick look at some of the new features:
Native DSON Importer means seamless integration of:
- TriAx™ Weight Mapped figures (Genesis, G2F, G2M)
- General Weight Mapped figures
- Legacy Parametric Figures (4th Generation and earlier)
- DSON presets
- Scenes
- Scene Subsets
- Wearable(s) Preset
- Character Preset
- Properties Preset
- Shaping Preset
- Pose Preset
- Hierarchical Material(s) Preset
- Material(s) Preset
- Shader Preset (preset must be for a Carrara shader)
- Camera(s) Preset
- Light(s) Preset
- Support for multiple UV sets (through Hierarchical Material(s) Presets and Material(s) Presets)
Native AutoFit functionality means:
- Conversion of legacy parametric clothing to TriAx™ weigh mapped figures
- Automatic Morph Projection quickly gives you the fit you need for your figure's shape.
- Automatic Weight Map Projection allows AutoFit clothing to bend and pose smoothly with the figure.
- Morphs built into clothing are carried through the AutoFit process.
Improved Sequencer, Keyframing and Animation Tools
- New keyboard shortcuts to:
- Play/Stop animation
- Advance the frame
- Preview a frame
- Key Frame Filters
- Sample – Creates a key at every frame of the animation.
- Smooth – Smooth out wild keys to get an even looking animation
- Reduce – Programatically determine which keys are unecessary and eliminate them.
- Graph Editor Box allows for fine tuning of animations
- Easily adjust timing
- Simple yet powerful manipulation of amplitude
- Scale, and translate key frames along the Graph Editor.
- Marquee selection of key frames for Graph Editor.
Smart Content and CMS Integration
- Carrara can now access the CMS database; PostgreSQL or Valentina.
- New smart content pane allows users to browse smart content by:
- File
- Category
- Product
- File view provides selection based filtering so that the user only sees items compatible with the current instance tray selection.
- Double click and drag and drop loading are both supported in Smart Content Tab.
Parameters Actions
- Zero, Memorize and Restore:
- Figure
- Figure Shape
- Figure Pose
- Selection
- Selection Shape
- Selection Pose
- Selection Tree
- Selection Tree Pose
- Selection Tree Shape
- Provides users with powerful tools to reset a pose, shape or both.
- Memorize and Restore allow user to take a snap shot of the model's current pose and/or shape and restore all or part of it.
Improved Fit to Functionality
- Automatic projection and following of morphs for TriAx™ Figures
- No more relying on built in clothing morphs to fit clothing over a figure. Carrara automatically projects any morphs for a TriAx™ figure onto items conformed to the figure.
Improved Viewport Avatars
- Spotlight, Sunlight, Moonlight, Tube Lights and Shape lights have all received a makeover and can easily be identified in the viewport.


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Red Hat Third Quarter Exceeds Expectations
Linux undertaking Red Hat has recorded a 24.3 million dollar profit in the third quarter of 2008, bettering last year's numbers.
Red Hat sales rose 22% to 165 million dollars compared to the third quarter of 2007. Software subscriptions for the U.S. company increased over the same period by 17% to 135.5 million dollars. The results exceeded analysts' expectations, despite economic times, the company's acquisition of Qumranet and undesirable foreign exchange rates.
Jim Whitehurst, president and CEO as of a few months ago, was pleased with the results and was hopeful about the future. He said that IT purchasing decisions will undergo some extreme rethinking considering the financial crisis and that open source companies such as his have a better chance at weathering the storm with their "low cost, high value" software. Linux Magazine Online had the opportunity to interview the Red Hat chief early December in Munich.
Issue 177/2015
Buy this issue as a PDF
NoScript Flaw
Mozilla's script blocker add-on could be putting malware sites on the whitelist.
RIP SSLv3
The Internet community officially banishes the notoriously unsafe Secure Sockets Layer protocol.
Mate 1.1 Arrives
Popular desktop environment continues the Gnome 2 legacy – with new support for the Gnome 3 toolkit.
US Government Requires HTTPS
The Obama White House has issued a memorandum telling all US government agencies they must use HTTPS for all websites and web communication.
Mozilla Signs Firefox Add-Ons
New program will dial up security for the Firefox browser.
Fedora 22 Arrives
Red Hat's community distro embraces the cloud.
Code.org and College Board Team Reach Out for Talented High School Coders
New partnership will bring more and better CS training to US schools
A Peek Inside TeslaCrypt Ransomware
Criminals offer online help over Tor network
Mumblehard Still Spams
Sophisticated malware is still present on Joomla and WordPress sites around the world.
Launchpad Gets Git
Future versions of Ubuntu's code service will support the popular Git version control system used with Linux and other open source projects.


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Dear Unregistered,
We are proud to introduce to you our first FREE online game from
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**Forum Helpers & Forum Moderators - Recruiting**
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I have about 346 jar files I want to install on my nokia n78 and dont want to do them 1 by 1... is there any way to speed up this process?
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text:
Who We Are
ANGELA – Social Media
ROSWITHA – Co-Founder & CEO
MIHAI – Co-Founder & MK
ALEXANDRA – Editor
How To Be Happy
Maintain The Color Of Your Clothes While Saving The Planet
We all want beautiful clothes, that look like new after the years have passed. We also want to save our planet and be more environmentally friendly. How can we have both without any hassling or
5 Ways Yoga Can Help You Fall in Love with Your Body
Quick And Healthy Snacks For The Office
Bloggers MBA Review: Are You Ready To Go Pro?
Learn The Habits Of Successful Women
This is not "just another habits eBook"! Each habit included was tested by us and we do them on a daily basis!
Are you successful? If not, you might want to rethink your daily activities and habits and add some new ones that can really help you live the life you've always dreamed of!
PRICE: $7 or how much you'd pay for a salad.
How To Be Beautiful
Easy Does It – How To Look Flawless In 7 Easy Steps
Get Ready With Me & Target Beauty Box
This post is promoted by Target
How do you start your day? Are you putting self-love on the first place?
As women, we are very intriguing, we either spend too much time or money on our appearance
The Easiest Way To Remove Make-Up: Best Facial Cleansing Wipes
Easter Wishlist: Gifts For Him And Her
9 Inspirational Hairstyle Ideas To Go With Any Hat
Wearing a hat doesn't necessary mean that your hair is having a bad day. If you choose to wear a hat in order to complete your amazing outfit, here is how to do it in style!
We chose 9 Hairstyles that will make your life a whole lot easier because you will be able to wear them with any hat. (winter or summer) Roswitha's favorite is No. #3 – that's how she wears her hat 90% of the time. What is yours?
PRICE: Did I mention that we are giving it for FREE?
How To Be Stylish
10 Summer Prints You Don't Wanna Miss This Season
Autumn/Winter 2015 Ready-To-Wear: Alexander McQueen Women's Collection
Autumn/Winter 2015 Ready to Wear: Chanel Women's Collection
Autumn/Winter 2015 Ready-To-Wear: Etro Women's Collection
Autumn/Winter 2015 Ready-To-Wear: Prada Women's Collection
What To Wear To A Pool Party
How To Build A Capsule Wardrobe
If you want to be fashionable on a tight budget and you own a small closet space, learning how to build a capsule wardrobe will save you.
This ebook talks about: What does it really mean, Who needs it, How to build it: essential items, how to use the same type of item for winter and summer, Outfits (casual, elegant, party, outdoor, school, work, etc) and How to spend your budget: find out the secret formula – that Roswitha developed – to see how much you should spend on any item.
PRICE: Coming Soon!


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As I'm progressing farther along, I'm wondering why I'm putting myself through this mess again lol. When I look at my children, I see why. It seems like when I wasn't pregnant I sat around all day dreaming about new baby, and not "new pregnancy". It's like the joys of being a mom made me totally forget about the horrible things you experience while pregnant. Now I'm here and feeling like crap--blehhhh. My only wish is for a healthy pregnancy and healthy baby, but as sadly as it sounds I'm sooo ready to be 40 wks. PLEASE PLEASE PREGNANCY STOP MAKING ME FEEL SO TERRIBLE!
Do you have the same feeling? (Moms with other children)
Honestly, I hate the first trimester. I had those feelings then, but not anymore. I love being pregnant, but only the second and third trimester. I would be pregnant all of the time if it were like this.
ME(25) & DH(32), 1 angel baby, DS Justus James born September 30,2009
Paisley Jo EDD June 2, 2013!! But will be here the last week of MAY!!!
Oh i'm right there with you. I absolutely hate pregnancy and I never feel right. I keep focusing on the end result, having my little one here. My first two pregnancies were absolutely terrible! In fact it took me a long time to decide on number 3 and here I am.. I keep telling myself this is temporary.. and what I'll have in June will be forever.. but I can't help wishing the time away until she/he is here. This is definitely my last one and i just want to get this over with! LOL
Ugh! I can't say I enjoy this remarkably either! AND, DH and I had trouble conceiving - so, you would think after waiting 18 months to conceive that I would just be happy to be pregnant. I am, I am happy and really, really thankful that it finally happened. But, I would also be really, really thankful to feel a little better - and maybe get to have a glass of wine!
I am enjoying this pregnancy. Maybe because I am pretty sure it will be our last. Sometimes I feel like shit, sure, but oh well!
I can't wait to meet the baby, but not so much for the reason of not being pregnant anymore. But I do look forward to having a Corona on the first nice day this summer!
It took me 5 years to be ready to be pregnant again, that should say something. Then it took another year of fertility treatments, so by then I was really ready. I still hate pregnancy though.
OUCH! Pain in your hip/pelvic area? Learn more about SPD here and follow my journey
I HATE pregnancy. I'm sorry but I am not one of those moms who dreams of the day when I feel little baby kicking and moving around. I love babies. I love being a mom...I hate the process. I don't like gaining weight, I don't like doctor appointments and blood work, etc. I'm generally a miserable pregnant person, even in the second and third trimester. Give me back my clothes that fit. If I didn't get pregnant so easily, we probably would have just adopted. I know that's horrible to say, so judge away... not every woman is happy being pregnant. But, I love the end result so it's worth it.
Married to my best friend since 06.24.06
Momma to 3 princesses & two handsome football players
Colby Richard born 05.23.13
I'm a terrible pregnant person. And I feel so guilty! I didn't have a great 1st pregnancy between my m/s and my pubis symphosis dysfunction. It took me 2.5 years to decide if I wanted to have another baby. Then it took us almost 4 years to conceive. So, on one hand, I'm so grateful to have another baby. On the other hand, I hate being pregnant. This pregnancy is a replay of my first....and I just want to whine about it all the time!
So looking forward to holding my sweet baby in June. This doesn't last forever. But this is my LAST pregnancy!
Mommy to IzzyB 4/10/06 and Baby Girl #2 due 6/15/13


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by Carimah Townes Posted on May 20, 2015
The sound of silence.
by Tara Culp-Ressler Posted on March 11, 2015
"This move by politicians is an underhanded attempt to harm women most in need."
by Ian Millhiser Posted on January 28, 2015
As it turns out, the American people don't much like the idea of Republican elected officials allowing the five Republican members of the Supreme Court to do their dirty work for them.
by Emily Atkin Posted on October 25, 2014
"The Republican party does not have a platform for Latinos, that is true," he said. "But we're voting for the man."
by Esther Yu-Hsi Lee Posted on July 17, 2014
The "Helping Unaccompanied Minors and Alleviating National Emergency" bill would speed up deportations.
by Josh Israel Posted on July 10, 2014
Several of those who object to any limits on political speech have themselves been long-time supporters of a constitutional amendment to do just that.
by Scott Keyes Posted on December 20, 2013
Rep. Steve Stockman tries to pull a fast one on Texas voters.
by Zack Beauchamp Posted on October 6, 2013
A top Republican senator struggles to defend the GOP's position on the shutdown.
by Esther Yu-Hsi Lee Posted on August 16, 2013
Maricopa County Sheriff Joe Arpaio and his deputies have placed white wooden crosses in the Arizona desert where border crossers have died, as a way to deter immigrants considering crossing the border at the Gila Bend.
by Alan Pyke Posted on June 23, 2013


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Browsing articles in humane
News: Guest Posts
One dog at a time
For Havana's dogs, it's not the best of times, but it's not the worst either. Some improvement is due to the efforts of the non-governmental Cuban Association for the Protection of Animals and Plants (ANIPLANT), an organization focused on improving the lives of dogs and other animals in Havana. Founded in 1988 by Cuban entertainer Maria Alveres Riso, and Cuba's first prima ballerina, Alisia...
News: Guest Posts
Innovative Approaches
This weekend I'll be the keynote speaker at the 5th International Symposium on Non-Surgical Contraceptive Methods of Pet Population Control. The conference title is a bit of a mouthful, but the basic idea is this: Can scientists develop a drug that will permanently sterilize dogs and cats? Or, put even more simply, can we make "the pill" for pets? Now a lot of you may be asking, "Don't we already...
News: Shirley Zindler
Dog to the rescue
June 21 is "Take Your Dog to Work Day." For those of us who treasure our dogs company, being able to have our companions with us on the job is such a bonus. I've been blessed as an animal control officer to be able to bring my girl Breeze, a rescued Doberman, with me to work. On a rough day in the field, just being able to reach over and stroke Breeze's silky coat can make the day bearable. I...
Dog's Life: Humane
A partnership aims to save animals
Raise taxes to save animals? In this economy? When a grassroots coalition of animal lovers, sick at heart about Miami-Dade County's perennially high shelter death rate, proposed asking voters to do just that, veteran rescuers responded with a resounding: "Yeah, right." But on election day last November, a solid majority of voters—65 percent— did exactly that via straw ballot, setting the county...
Culture: DogPatch
Find a soul mate is never easy, but Brooklyn based Sarah Oren Brasky, aka the "Dog Matchmaker," is an expert at improving the odds. Brasky's passion for dogs began in childhood and has continued into her adult life, where, in addition to her work with dogs—she also founded and edits the rescue blog Foster Dogs NYC —she teaches elementary school. Motivated by the conviction that adopting dogs...
Culture: Stories & Lit
Standing up for a dog in Haiti
It was a strange place to cry, but I had no other place to go. In Haiti, a 10-by-12-foot classroom in a small schoolhouse served as a makeshift hotel for the evening. I sat down on the end of the bed—a green army cot beside a laminated poster of the human eyeball—and sobbed. My tears lacked the grace of my 35 years; childlike, inconsolable, they were tears of shame. I felt that I'd done something...
Culture: Reviews
Published by CreateSpace
Dogs, cats, ducks, horses, goats, roosters, deer, snakes—animal control officer Shirley Zindler has seen (and helped) them all. As she makes clear in this collection detailing her experiences, working with the public and making a positive difference for animals can be a challenge, but it's one that she's embraced wholeheartedly. After reading this book, you're likely to look at your beleaguered...
Dog's Life: Humane
Bark n' Bitches: LA's First Humane Pet Shop
Lush red walls and vintage furnishings are the first clues that this is no ordinary pet store. When a dozen dogs scramble to the door for introductions, it's clear you've entered a retail hybrid: a hip, humane petshelter boutique. At Bark n' Bitches in the Fairfax District of Los Angeles, the retail store supports a rescue shelter, and the rescues snooze amid high-end merchandise and artsy black...
News: Guest Posts
Really need you
I've always had a soft spot for old dogs. The gray muzzles and cloudy eyes get to me every time. One of my own dogs, Rocky, a rescued Pug/Chihuahua mix, is quite elderly at around 14 years of age. He recently had a couple of major seizures and became completely paralyzed from the neck down. A day of intensive care at the vet gave a poor prognosis. He did not seem to be in pain so I made the sad...
Dog's Life: Humane
People Who Matter: Emelinda Narvaez, founder of NYC's Earth Angels
Emelinda Narvaez, an animal rescue advocate working in both Manhattan and the Bronx, has become a familiar face to people visiting the city's pet stores. She calls her rescues "earth angels," and fittingly, that is also the name of her animal-rescue group. Narvaez Emelinda, who says she's rescued over more than 10,000 animals over a span of 45 + years, was a nurse in a Bronx hospital. Until she...
Copyright © 1997-2015 The Bark, Inc. Dog Is My Co-Pilot® is a registered trademark of The Bark, Inc


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Real Housewives Playlist For Summertime Drama
As we head into the thick of a Bravo-filled summer, listen to these lovely ladies belt their hearts out about love. And parties. And then I rate them with gifs of themselves.
1. New Jersey:
2. I give NJ an…
…for making us listen to that.
3. Atlanta:
5. I give ATL a…
…because Kandi is a Grammy award-winning musician and Kim is, um, not.
6. New York:
8. I give NY a…
…because no one is more fabulous than Countess LuAnn.
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