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Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a "base composition signature" (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.
1. A method of determining the absence of a bioagent in a sample comprising:
a) contacting said sample suspected of containing nucleic acid encoding said bioagent with at least one pair of oligonucleotide primers which are capable of hybridizing to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of said bioagent;
b) treating said variable nucleic acid sequence under amplification conditions capable of producing an amplification product of said variable nucleic acid sequence;
c) performing spectroscopy to determine the molecular mass or base composition of all amplification products; and
d) comparing said molecular mass to one or more molecular masses of amplification products or said base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein the lack of a match indicates that said bioagent is absent from said sample.
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[0001] This invention was made with United States Government support under DARPA/SPO contract BAA00-09. The United States Government may have certain rights in the invention.
[0002] The present invention relates to methods for rapid detection and identification of bioagents from environmental, clinical or other samples. The methods provide for detection and characterization of a unique mass or unique base composition signature (BCS) from any bioagent, including bacteria and viruses. The unique mass or unique BCS is used to rapidly identify the bioagent(s).
[0003] Rapid and definitive microbial identification is desirable for a variety of industrial, medical, environmental, quality, and research reasons. Traditionally, the microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through direct examination and culture of specimens. Since the mid-1980s, researchers have repeatedly demonstrated the practical utility of molecular biology techniques, many of which form the basis of clinical diagnostic assays. Some of these techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). These procedures, in general, are time-consuming and tedious. Another option is the polymerase chain reaction (PCR) or other amplification procedure that amplifies a specific target DNA sequence based on the flanking primers used. Finally, detection and data analysis convert the hybridization event into an analytical result.
[0004] Other techniques for detection of bioagents include high-resolution mass spectrometry (MS), low-resolution MS, fluorescence, radioiodination, DNA chips and antibody techniques. None of these techniques is entirely satisfactory.
[0005] Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. However, high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents ("cluttered" background). Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
[0006] Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
[0007] Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem. 69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998; Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
[0008] U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences that comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
[0009] U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
[0010] WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
[0011] WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand, which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
[0012] PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by Ms.
[0013] U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
[0014] WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
[0015] WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
[0016] Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need.
[0017] One embodiment of the present invention is a method of determining the absence of a bioagent in a sample comprising a) contacting the sample suspected of containing nucleic acid encoding the bioagent with at least one pair of oligonucleotide primers which are capable of hybridizing to sequences of the nucleic acid, wherein said sequences flank a variable nucleic acid sequence of the bioagent; b) treating the variable nucleic acid sequence under amplification conditions capable of producing an amplification product of the variable nucleic acid sequence; c) performing spectroscopy to determine the molecular mass or base composition of all amplification products; and d) comparing the molecular mass to one or more molecular masses of amplification products or the base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein the lack of a match indicates that the bioagent is absent from the sample. In one aspect of the invention, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of the invention, the bioagent is a bacterium, virus, parasite, fungi, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps a)-d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps a)-c) on a different plurality of known organisms from those in step d). Preferably, the one or more molecular mass is contained in a database of molecular masses. In another aspect of the invention, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
[0018] FIGS. 1A-1H and FIG. 2 are consensus diagrams that show examples of conserved regions from 16S rRNA (FIGS. 1A, 1A-2, 1A-3, 1A-4, 1B, 1B-1, and 1B-2), 23S rRNA (3′-half, FIGS. 1C-1, 1C-2, and 1D; 5′-half, FIGS. 1E-F), 23S rRNA Domain I (FIG. 1G), 23S rRNA Domain IV (FIG. 1H) and 16S rRNA Domain II (FIG. 2) which are suitable for use in the present invention. Where there is overlap or redundancy between the figures, the overlap is simply provided as an orientation aid and no additional members of the sequence are implied thereby. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. Bases in capital letters are greater than 95% conserved; bases in lower case letters are 90-95% conserved, filled circles are 80-90% conserved; and open circles are less than 80% conserved. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. The nucleotide sequence of the 16S rRNA consensus sequence is SEQ ID NO:3 and the nucleotide sequence of the 23S rRNA consensus sequence is SEQ ID NO:4.
[0019]FIG. 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in FIG. 1C.
[0020]FIG. 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved.
[0021]FIG. 4 is a schematic diagram of base composition signature determination using nucleotide analog "tags" to determine base composition signatures.
[0022]FIG. 5 shows the deconvoluted mass spectra of a Bacillus anthracis region with and without the mass tag phosphorothioate A (A*). The two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
[0023]FIG. 6 shows base composition signature (BCS) spectra from PCR products from Staphylococcus aureus (S. aureus 16S—1337F) and Bacillus anthracus (B. anthr. 16S—1337F), amplified using the same primers. The two strands differ by only two (ATE CG) substitutions and are clearly distinguished on the basis of their BCS.
[0024]FIG. 7 shows that a single difference between two sequences (A14 in B. anthracis vs. A15 in B. cereus) can be easily detected using ESI-TOF mass spectrometry.
[0025]FIG. 8 is an ESI-TOF of Bacillus anthracis spore coat protein sspE 56mer plus calibrant. The signals unambiguously identify B. anthracis versus other Bacillus species.
[0026]FIG. 9 is an ESI-TOF of a B. anthracis synthetic 16S—1228 duplex (reverse and forward strands). The technique easily distinguishes between the forward and reverse strands.
[0027]FIG. 10 is an ESI-FTICR-MS of a synthetic B. anthracis 16S—1337 46 base pair duplex.
[0028]FIG. 11 is an ESI-TOF-MS of a 56mer oligonucleotide (3 scans) from the B. anthracis saspB gene with an internal mass standard. The internal mass standards are designated by asterisks.
[0029]FIG. 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFA buffer showing that charge stripping with tributylammonium trifluoroacetate reduces the most abundant charge state from [M-8H+]8− to [M-3H+]3−.
[0030] The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with PCR-based BCS or mass technology using "intelligent primers" which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The high-resolution MS technique is used to determine the molecular mass and/or base composition signature (BCS) of the amplified sequence region. This unique mass or unique "base composition signature" (BCS) is then input to a maximum-likelihood detection algorithm for matching against a database of masses or base composition signatures in the same amplified region. The present method combines PCR-based amplification technology (which provides specificity) and a molecular mass detection mode (which provides speed and does not require nucleic acid sequencing of the amplified target sequence) for bioagent detection and identification.
[0031] The present method allows extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure. The method leverages ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives. Thus, the methods are useful in a wide variety of fields, including, but not limited to, those fields discussed below.
[0032] In some embodiments of the invention, the methods disclosed herein can be used for environmental testing. Detection and discrimination of pathogenic vs. non-pathogenic bacteria, viruses, parasites, fungi and the like, in samples of water, land, air, or other samples, can be carried out. Water samples can be obtained from, for example, lakes, rivers, oceans, streams, water treatment systems, rainwater, groundwater, water table, reservoirs, wells, bottled water, and the like. Air samples can be obtained from ventilation systems, airplane cabins, schools, hospitals, mass transit locations such as subways, train stations, airports, and the like. Conditions such as sick building syndrome can be detected. Land samples can be obtained from any location.
[0033] In other embodiments of the invention, the methods disclosed herein can be used to screen blood and other bodily fluids and tissues for pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like. Animal samples, including but not limited to, blood and other bodily fluid and tissue samples, can be obtained from living animals, who are either known or not known to or suspected of having a disease, infection, or condition. Alternately, animal samples such as blood and other bodily fluid and tissue samples can be obtained from deceased animals. Blood samples can be further separated into plasma or cellular fractions and further screened as desired. Bodily fluids and tissues can be obtained from any part of the animal or human body. Animal samples can be obtained from, for example, mammals and humans.
[0034] In other embodiments of the invention, the methods disclosed herein can be used for forensics. For example, medical examiners can use the present invention to determine the cause of death. In addition, epidemiologists, for example, can use the present methods to determine the geographic origin of a particular strain of bacteria or virus. For example, a particular strain of bacteria or virus may have a sequence difference that is associated with a particular area of a country or the world and identification of such a sequence difference can lead to the identification of the geographic origin and epidemiological tracking of the spread of the particular disease, disorder or condition associated with the detected virus or bacteria. In addition, carriers of particular DNA or diseases, such as mammals, non-mammals, birds, insects, and plants, can be tracked by screening SNPs, VNTRs, or polyA, for example. Diseases, such as malaria, can be tracked by screening commensals, such as mosquitos.
[0035] In other embodiments of the invention, the methods disclosed herein can be used for detecting the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like in samples of foodstuff or cosmetics. For example, food and wine can be examined for the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like. Particular types of foods susceptible to bioagent contamination, such as agricultural products, meat products and eggs, can be examined for pathogenic organisms such as E. coli and Salmonella species. Such examination procedures can be used by, for example, the wholesalers of foodstuffs and beverages, or by regulatory agencies such as the U.S. Department of Agriculture and the Food and Drug Administration. In addition, grapes and wines, for example, can be examined using the present methods to detect particular strains of bacteria or yeast that may indicate a particular time upon which to harvest the grapes or alter the wine-making process.
[0036] In other embodiments of the invention, the methods disclosed herein can be used for detecting the presence of bioagents in a container, such as a package, box, envelope, mail tube, railroad box car, and the like. For example, mail and package delivery entities and agencies, both domestic and abroad, as well investigative agencies such as the FBI and ATF can use the present methods to detect bioagents in containers.
[0037] In other embodiments of the invention, the methods disclosed herein can be used for detecting the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like in organ donors and/or in organs from donors. Such examination can result in the prevention of the transfer of, for example, viruses such as West Nile virus, hepatitis viruses, human immunodeficiency virus, and the like from a donor to a recipient via a transplanted organ. The methods disclosed herein can also be used for detection of host versus graft or graft versus host rejection issues related to organ donors by detecting the presence of particular antigens in either the graft or host known or suspected of causing such rejection. In particular, the bioagents in this regard are the antigens of the major histocompatibility complex, such as the HLA antigens.
[0038] In other embodiments of the invention, the methods disclosed herein can be used for detection and identification of livestock infections such as, for example, mad cow disease, hoof and mouth disease, and the like. Livestock includes, but is not limited to, cows, pigs, sheep, chickens, turkeys, goats, and other farm animals.
[0039] In other embodiments of the invention, the methods disclosed herein can be used for pharmacogenetic analysis and medical diagnosis including, but not limited to, cancer diagnosis based on mutations and polymorphisms, drug resistance and susceptibility testing, screening for and/or diagnosis of genetic diseases and conditions, and diagnosis of infectious diseases and conditions. The present methods can also be used to detect and track emerging infectious diseases, such as West Nile virus infection, mad cow disease, and HIV-related diseases.
[0040] The present methods can be used to detect and classify any biological agent, including bacteria, viruses, fungi and toxins. As one example, where the agent is a biological threat, the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes. In addition, the methods can be used to identify natural or deliberate engineering events including chromosome fragment swapping, molecular breeding (gene shuffling) and emerging infectious diseases.
[0041] Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species (Proc. Natl. Acad. Sci. U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins are DNA polymerase m beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G. Operons can also be targeted using the present method. One example of an operon is the bfp operon from enteropathogenic E. coli. Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential. The method can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
[0042] A theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor. The complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved. However, the present invention provides a straightforward strategy for obtaining information with the same practical value using base composition signatures (BCS). While the base composition of a gene fragment is not as information-rich as the sequence itself, there is no need to analyze the complete sequence of the gene if the short analyte sequence fragment is properly chosen. A database of reference sequences can be prepared in which each sequence is indexed to a unique base composition signature, so that the presence of the sequence can be inferred with accuracy from the presence of the signature. The advantage of base composition signatures is that they can be quantitatively measured in a massively parallel fashion using multiplex PCR(PCR in which two or more primer pairs amplify target sequences simultaneously) and mass spectrometry. These multiple primer amplified regions uniquely identify most threat and ubiquitous background bacteria and viruses. In addition, cluster-specific primer pairs distinguish important local clusters (e.g., anthracis group).
[0043] In the context of this invention, a "bioagent" is any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, parasites, fungi, toxin genes and bioregulating compounds. Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.
[0044] As used herein, a "base composition signature" (BCS) is the exact base composition from selected fragments of nucleic acid sequences that uniquely identifies the target gene and source organism. BCS can be thought of as unique indexes of specific genes.
[0045] As used herein, "intelligent primers" are primers which bind to sequence regions which flank an intervening variable region. In a preferred embodiment, these sequence regions which flank the variable region are highly conserved among different species of bioagent. For example, the sequence regions may be highly conserved among all Bacillus species. By the term "highly conserved", it is meant that the sequence regions exhibit between about 80-100%, more preferably between about 90-100% and most preferably between about 95-100% identity. Examples of intelligent primers which amplify regions of the 16S and 23S rRNA are shown in FIGS. 1A-1I. A typical primer amplified region in 16S rRNA is shown in FIG. 2. The arrows represent primers which bind to highly conserved regions which flank a variable region in 16S rRNA domain III. The amplified region is the stem-loop structure under "1100-1188."
[0046] One main advantage of the detection methods of the present invention is that the primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same primer pair can be used to identify any desired bacterium because it will bind to the conserved regions which flank a variable region specific to a single species, or common to several bacterial species, allowing nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition. For example, the 16S—971-1062, 16S—1228-1310 and 16S—1100-1188 regions are 98-99% conserved in about 900 species of bacteria (16S=16S rRNA, numbers indicate nucleotide position). In one embodiment of the present invention, primers used in the present method bind to one or more of these regions or portions thereof.
[0047] The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with nucleic acid amplification-based BCS technology using "intelligent primers" which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s). Although the use of PCR is preferred, other nucleic acid amplification techniques may also be used, including ligase chain reaction (LCR) and strand displacement amplification (SDA). The high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments. The resolved spectral lines are then translated to BCS which are input to a maximum-likelihood detection algorithm matched against spectra for one or more known BCS. Preferably, the bioagent BCS spectrum is matched against one or more databases of BCS from vast numbers of bioagents. Preferably, the matching is done using a maximum-likelihood detection algorithm.
[0048] In a preferred embodiment, base composition signatures are quantitatively measured in a massively parallel fashion using the polymerase chain reaction (PCR), preferably multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids must be present for detection of bioagents by MS. A wide variety of techniques for preparing large amounts of purified nucleic acids or fragments thereof are well known to those of skill in the art. PCR requires one or more pairs of oligonucleotide primers which bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer. The primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Taq polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis. The solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence.
[0049] The "intelligent primers" define the target sequence region to be amplified and analyzed. In one embodiment, the target sequence is a ribosomal RNA (rRNA) gene sequence. With the complete sequences of many of the smallest microbial genomes now available, it is possible to identify a set of genes that defines "minimal life" and identify composition signatures that uniquely identify each gene and organism. Genes that encode core life functions such as DNA replication, transcription, ribosome structure, translation, and transport are distributed broadly in the bacterial genome and are preferred regions for BCS analysis. Ribosomal RNA (rRNA) genes comprise regions that provide useful base composition signatures. Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes. For example, there are currently over 13,000 sequences in the ribosomal RNA database that has been created and maintained by Robin Gutell, University of Texas at Austin, and is publicly available on the Institute for Cellular and Molecular Biology web page (www.ma.icmb.utexas.edu/). There is also a publicly available rRNA database created and maintained by the University of Antwerp, Belgium at www.rrna.uia.ac.be.
[0050] These databases have been analyzed to determine regions that are useful as base composition signatures. The characteristics of such regions are: a) between about 80 and 100%, preferably >about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, preferably no more than about 50-250 nucleotides, and more preferably no more than about 60-100 nucleotides, between the conserved regions.
[0051] Due to their overall conservation, the flanking rRNA primer sequences serve as good "universal" primer binding sites to amplify the region of interest for most, if not all, bacterial species. The intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature.
[0052] It is advantageous to design the "intelligent primers" to be as universal as possible to minimize the number of primers which need to be synthesized, and to allow detection of multiple species using a single pair of primers. These primer pairs can be used to amplify variable regions in these species. Because any variation (due to codon wobble in the 3rd position) in these conserved regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal base". For example, under this "wobble" pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res. 24:3302-3306, 1996).
[0053] In another embodiment of the invention, to compensate for the somewhat weaker binding by the "wobble" base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs are 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G. Propynes are described in U.S. Pat. Nos. 5,645,985, 5,830.653 and 5,484,908, the entire contents of which are incorporated herein by reference. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, the entire contents of which are incorporated herein by reference. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, the entire contents of which are incorporated herein by reference.
[0054] In some embodiments of the invention, the methods described herein can be used for defense of germ warfare (allowing immediate identification of the bioagent and appropriate treatment). Bacterial biological warfare agents capable of being detected by the present methods include Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders), Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, and Vibrio cholerae.
[0055] Besides 16S and 23S rRNA, other target regions suitable for use in the present invention for detection of bacteria include 5S rRNA and RNase P (FIG. 3).
[0056] Biological warfare fungus biowarfare agents include coccidioides immitis (Coccidioidomycosis).
[0057] Biological warfare toxin genes capable of being detected by the methods of the present invention include botulism, T-2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin and saxitoxin.
[0058] Biological warfare viral threat agents are mostly RNA viruses (positive-strand and negative-strand), with the exception of smallpox. Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high. RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases.
[0059] Examples of (−)-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic feyer, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbillivirus).
[0060] Examples of (+)-strand RNA viruses include picomaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross River virus, Venezuelan equine encephalitis virus, Rubella virus, hepatitis E virus). The hepatitis C virus has a 5′-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3′-untranslated region of 240 nucleotides. The 5′-UTR and 3′-UTR are 99% conserved in hepatitis C viruses.
[0061] In one embodiment, the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a (−)-strand RNA virus. (+)-strand RNA viruses are double stranded RNA and replicate by RNA-directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template. Helicase unwinds the RNA duplex to allow replication of the single stranded RNA. These viruses include viruses from the family picomaviridae (e.g., poliovirus, coxsackievirus, echovirus), togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae (e.g., lymphocytic choriomeningitis virus, lassa fever virus), cononaviridae (e.g., human respiratory virus) and Hepatitis A virus. The genes encoding these proteins comprise variable and highly conserved regions which flank the variable regions.
[0062] In a preferred embodiment, the detection scheme for the PCR products generated from the bioagent(s) incorporates three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a BCS that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel.
[0063] In one embodiment, the method can be used to detect the presence of antibiotic resistance and/or toxin genes in a bacterial species. For example, Bacillus anthracis comprising a tetracycline resistance plasmid and plasmids encoding one or both anthracis toxins (px01 and/or px02) can be detected by using antibiotic resistance primer sets and toxin gene primer sets. If the B. anthracis is positive for tetracycline resistance, then a different antibiotic, for example quinalone, is used.
[0064] Mass spectrometry (MS)-based detection of PCR products provides all of these features with additional advantages. MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product with a unique base composition is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons. Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). For example, MALDI of nucleic acids, along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.).
[0065] In some embodiments, large DNAs and RNAs, or large amplification products therefrom, can be digested with restriction endonucleases prior to ionization. Thus, for example, an amplification product that was 10 kDa could be digested with a series of restriction endonucleases to produce a panel of, for example, 100 Da fragments. Restriction endonucleases and their sites of action are well known to the skilled artisan. In this manner, mass spectrometry can be performed for the purposes of restriction mapping.
[0066] Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
[0067] The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.
[0068] In general, the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS). In one embodiment of the invention, the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents). To achieve minimal latency, a continuous sample stream flows directly into the PGC-MS combustion chamber. When an increase in biomass is detected, a PCR process is automatically initiated. Bioagent presence produces elevated levels of large molecular fragments from 100-7,000 Da which are observed in the PGC-MS spectrum. The observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove(combining PCR and MS, preferably FT-ICR MS) is initiated. Optionally, alarms or other processes (halting ventilation flow, physical isolation) are also initiated by this detected.biomass level.
[0069] The accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance 13C and 15N isotopes, and assignment of the charge state for any ion. The cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow. The latter two problems are addressed by operating with a resolving power of >100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA. The resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [M-14H+]14− charge state of an 84mer PCR product is poorly characterized and assignment of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components are visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward. The [13C,15N]-depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al., Nucl. Acids Res. 20:4515-4523, 1992).
[0070] While mass measurements of intact nucleic acid regions are believed to be adequate to determine most bioagents, tandem mass spectrometry (MSn) techniques may provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3′ terminus and a 5′ phosphate following internal cleavage) and the a-Base series(having an intact 5′ terminus and a 3′ furan).
[0071] The second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample.
[0072] If there are two or more targets of similar base composition or mass, or if a single amplification reaction results in a product which has the same mass as two or more bioagent reference standards, they can be distinguished by using mass-modifying "tags." In this embodiment of the invention, a nucleotide analog or "tag" is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses. Such tags are described in, for example, PCT WO97/33000. This further limits the number of possible base compositions consistent with any mass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined.
[0073] In another amplification reaction, the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C. The combination of the A/T reaction and GIC reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in FIG. 4 and Table 1.
[0074] The mass tag phosphorothioate A (A*) was used to distinguish a Bacillus anthracis cluster. The B. anthracis (A14G9C14T9) had an average MW of 14072.26, and the B. anthracis (A1A*13G9C14T9) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS. The deconvoluted spectra are shown in FIG. 5.
[0075] In another example, assume the measured molecular masses of each strand are 30,000.115 Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand.
[0076] Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing. The detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes.
[0077] Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels. The program emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database (e.g. GenBank) is used to define the mass basecount matched filters. The database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted the maximum likelihood process is applied to this "cleaned up" data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.
[0078] In one embodiment, a strategy to "triangulate" each organism by measuring signals from multiple core genes is used to reduce false negative and false positive signals, and enable reconstruction of the origin or hybrid or otherwise engineered bioagents. After identification of multiple core genes, alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and potential primer binding sites flanking variable regions are identified. Next, amplification target regions for signature analysis are selected which distinguishes organisms based on specific genomic differences (i.e., base composition). For example, detection of signatures for the three part toxin genes typical of B. anthracis (Bowen, J. E. and C. P. Quinn, J. Appl. Microbiol 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
[0079] The present method can also be used to detect single nucleotide polymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly and accurately. A SNP is defined as a single base pair site in the genome that is different from one individual to another. The difference can be expressed either as a deletion, an insertion or a substitution, and is frequently linked to a disease state. Because they occur every 100-1000 base pairs, SNPs are the most frequently bound type of genetic marker in the human genome.
[0080] For example, sickle cell anemia results from an A-T transition, which encodes a valine rather than a glutamic acid residue. Oligonucleotide primers may be designed such that they bind to sequences that flank a SNP site, followed by nucleotide amplification and mass determination of the amplified product. Because the molecular masses of the resulting product from an individual who does not have sickle cell anemia is different from that of the product from an individual who has the disease, the method can be used to distinguish the two individuals. Thus, the method can be used to detect any known SNP in an individual and thus diagnose or determine increased susceptibility to a disease or condition.
[0081] In one embodiment, blood is drawn from an individual and peripheral blood mononuclear cells (PBMC) are isolated and simultaneously tested, preferably in a high-throughput screening method, for one or more SNPs using appropriate primers based on the known sequences which flank the SNP region. The National Center for Biotechnology Information maintains a publicly available database of SNPs (www.ncbi.nlm.nih.gov/SNP/).
[0082] The method of the present invention can also be used for blood typing. The gene encoding A, B or 0 blood type can differ by four single nueleotide polymorphisms. If the gene contains the sequence CGTGGTGACCCTT (SEQ ID NO:5), antigen A results. If the gene contains the sequence CGTCGTCACCGCTA (SEQ ID NO:6) antigen B results. If the gene contains the sequence CGTGGT-ACCCCTT (SEQ ID NO:7), blood group 0 results ("−" indicates a deletion). These sequences can be distinguished by designing a single primer pair that flanks these regions, followed by amplification and mass determination.
[0083] In other embodiments of the invention, all of the aformentioned methods can be used to determine or confirm the absence of a bioagent in a sample. For example, when the molecular mass or base composition is compared to one or more molecular masses or base compositions obtained from a plurality of known organisms and no match is obtained, the absence of a particular bioagent is determined or confirmed. In these methods of determining the absence of a bioagent, a positive control can be used to confirm the integrity of the system. Such positive controls include, but are not limited to addition of a known bioagent to the sample and confirming the presence of the known bioagent in the sample by obtaining a match from the comparison step. Alternately, residual primer signal can be detected, thus indicating that the system integrity is intact. Alternately, an internal probe can be used to indicate system integrity. In some embodiments, one or more primers or primer pairs can be used to carry out the aformentioned methods in, for example, a 96 well microtiter plate. In each of the wells in which the present methods are carrier out, thus, is an internal probe, residual primer, or known bioagent that can be used to determine the integrity of the system.
[0084] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.
[0085] In one embodiment, nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry. Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al.). The nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
[0086] FTICR Instrumentation: The FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber. The spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute. The Bruker data-acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments. For oligonucleotides in the 20-30-mer regime typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z.
[0087] Modified ESI Source: In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator. The ESI ion optics consists of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz. A lab-built electro-mechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the "open" position via a TTL pulse from the data station. When in the "closed" position, a stable electrospray plume is maintained between the ESI emitter and the face of the shutter. The back face of the shutter arm contains an elastomeric seal that can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position. When the shutter is triggered, a "time slice" of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir. The rapid response time of the ion shutter (<25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir.
[0088] Apparatus for Infrared Multiphoton Dissociation A 25 watt CW CO2 laser operating at 10.6 μm has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications. An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet. Using standard IR-compatible mirrors and kinematic mirror mounts, the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
[0089] Table 1 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms. The primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in FIGS. 1A-1C. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The primer pairs are >95% conserved in the bacterial sequence database (currently over 10,000 organisms). The intervening regions are variable in length and/or composition, thus providing the base composition "signature" (BCS) for each organism. Primer pairs were chosen so the total length of the amplified region is less than about 80-90 nucleotides. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
[0090] Included in the short bacterial database cross-section in Table 1 are many well known pathogens/biowarfare agents (shown in bold/red typeface) such as Bacillus anthracis or Yersinia pestis as well as some of the bacterial organisms found commonly in the natural environment such as Streptomyces. Even closely related organisms can be distinguished from each other by the appropriate choice of primers. For instance, two low G+C organisms, Bacillus anthracis and Staph aureus, can be distinguished from each other by using the primer pair defined by 16S—1337 or 23S—855 (M of 4 Da).
[0091]FIG. 6 shows the use of ESI-FT-ICR MS for measurement of exact mass. The spectra from 46mer PCR products originating at position 1337 of the 16S rRNA from S. aureus (upper) and B. anthracis (lower) are shown. These data are from the region of the spectrum containing signals from the [M-8H+]8− charge states of the respective 5′-3′ strands. The two strands differ by two (AT→CG) substitutions, and have measured masses of 14206.396 and 14208.373�0.010 Da, respectively. The possible base compositions derived from the masses of the forward and reverse strands for the B. anthracis products are listed in Table 3.
[0092] Among the 16 compositions for the forward strand and the 18 compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary, corresponding to the actual base compositions of the B. anthracis PCR products.
[0093] A conserved Bacillus region from B. anthracis (A14G9C14T9) and B. cereus (A15G9C13T9) having a C to A base change was synthesized and subjected to ESI-TOF MS. The results are shown in FIG. 7 in which the two regions are clearly distinguished using the method of the present invention (MW=14072.26 vs. 14096.29).
[0094] In other examples of the present invention, the pathogen Vibrio cholera can be distinguished from Vibrio parahemolyticus with ΔM>600 Da using one of three 16S primer sets shown in Table 2 (16S—971, 16S—1228 or 16S—1294) as shown in Table 4. The two mycoplasma species in the list (M. genitalium and M. pneumoniae) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced resolving power for closely related organisms. In cases such as Bacillus anthracis and Bacillus cereus that are virtually indistinguishable from each other based solely on mass differences, compositional analysis or fragmentation patterns are used to resolve the differences. The single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
[0095] Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background.
[0097] Table 4 shows the expected molecular weight and base composition of region 16S—1100-1188 in Mycobacterium avium and Streptomyces sp.
[0098] Table 5 shows base composition (single strand) results for 16S—1100-1188 primer amplification reactions different species of bacteria. Species which are repeated in the table (e.g., Clostridium botulinum) are different strains which have different base compositions in the 16S—1100-1188 region.
[0099] The same organism having different base compositions are different strains. Groups of organisms which are highlighted or in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example, Bacillus anthracis can be distinguished from Bacillus cereus and Bacillus thuringiensis using the primer 16S—971-1062 (Table 6). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g. anthracis cluster) are distinguished at high resolution with focused sets of primer pairs. The known biowarfare agents in Table 6 are Bacillus anthracis, Yersinia pestis, Francisella tularensis and Rickettsia prowazekii.
[0100] The sequence of B. anthracis and B. cereus in region 16S—971 is shown below. Shown in bold is the single base difference between the two species which can be detected using the methods of the present invention. B. anthracis has an ambiguous base at position 20.
[0101] The mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in FIG. 8. The mass standard was a 20-mer phosphorothioate oligonucleotide added to a solution containing a 56-mer PCR product from the B. anthracis spore coat protein sspE. The mass of the expected PCR product distinguishes B. anthracis from other species of Bacillus such as B. thuringiensis and B. cereus.
[0102] An ESI-TOF MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1228 region of the B. anthracis 16S rRNA gene. The results (FIG. 9) show that the molecular weights of the forward and reverse strands can be accurately determined and easily distinguish the two strands. The [M-21H+]21− and [M-20H+]20− charge states are shown.
[0103] An ESI-FTICR-MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1337 region of the B. anthracis 16S rRNA gene. The results (FIG. 10) show that the molecular weights of the strands can be distinguished by this method. The [M-16H+]16− through [M-10H+]10− charge states are shown. The insert highlights the resolution that can be realized on the FTICR-MS instrument, which allows the charge state of the ion to be determined from the mass difference between peaks differing by a single 13C substitution.
[0104] ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5 μM) from the saspB gene of B. anthracis containing an internal mass standard at an ESI of 1.7 μL/min as a function of sample consumption. The results (FIG. 11) show that the signal to noise is improved as more scans are summed, and that the standard and the product are visible after only 100 scans.
[0105] An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standard was obtained following addition of 5 mM TBA-TFA buffer to the solution. This buffer strips charge from the oligonucleotide and shifts the most abundant charge state from [M-8H+]8− to [M-3H+]3− (FIG. 12).
[0106] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.
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Editor's Pick: Transparency Best Practices for Cloud Providers
Cloud Security Alliance Launches Registry: Not a Moment Too Soon
The Cloud Security Alliance has taken an important step by launching a free, publicly available registry that documents the security controls for cloud offerings. The registry is intended to promote cloud provider transparency and help users evaluate the security provided by the cloud providers they use or are considering. The new initiative is called the CSA Security, Trust & Assurance Registry (STAR) and is open to all IaaS, SaaS, and PaaS providers and will be online in late 2011. The author also discusses the confusing landscape of cloud providers and the benefits to cloud providers who participate in the registry.
Read Cloud Security Alliance Launches Registry: Not a Moment Too Soon >
Joe McKendrick
August 12, 2011

subset: commoncrawl


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Tim Donaghy would be proud of this game's officiating.
Compared to the NHL, pro wrestling looks legit.
I wonder how much money Bettman bet on the Kings tonight.
This series is more rigged than a Russian election.
NHL = pro wrestling, except pro wrestling is far more popular
Los Angeles should have a ticker tape parade for the refs.
The NHL now has a championship team in Los Angeles. Mission accomplished. Well done, refs.
The NHL couldn't risk the Kings losing in a game 7, so the refs took care of business tonight.
The NHL rivals the NBA for the worst officiating in professional sports. Both leagues are a bleeping joke.
With the Devils on the verge of a historic comeback from 3 games to 0 down, America was just starting to pay attention to this series.
But thanks to the crooked officiating, this series is all but over and only the diehard fans are still watching.
The NHL just rained on its own parade. The league is its own worst enemy.
@ Lets Go Mets
If you had a life, you would have far better things to do than spend time & money supporting teams that don't even represent your home state.
If the Knicks had territorial rights in New Jersey, the Nets would've never played there to begin with.
The Knicks have NO TERRITORIAL RIGHTS IN NEW JERSEY.
Get a clue, kcruz1.
I agree with the commenter above who said the Nets will eventually leave Brooklyn for California, just like the Dodgers did 55 years ago.
Brooklyn is die-hard Knick country and will never support the Nets.
Why else do you think the Nets owners decided to make the Barclays Center the smallest arena in the entire NBA with only 18,000 seats?
Even they know they're going to have a tough time trying to sell tickets in Brooklyn.
But heck, they got a new arena all to themselves, 20 acres of prime real estate and enormous tax breaks, so they took the money and ran.
And they'll soon be running to California once the Brooklyn experiment falls flat on its face.
@ DrKenB
The Nets moved to Brooklyn because they received over $1 BILLION IN TAXPAYER SUBSIDIES(new arena, 20-acre land grab, tax breaks) to do so, not because of weak fan support in New Jersey.
Attendance at Izod would've been far stronger had it not been the ONLY ARENA IN THE ENTIRE NBA NOT ACCESSIBLE BY BUSES OR TRAINS.
By the time the Nets moved to the highly accessible Prudential Center in 2010, the franchise had already turned its back on the Garden State by committing to the Brooklyn move and removing "New Jersey" from its uniforms, merchandise and court.
Not to mention the fact that the team stunk to high heaven during its final 2 years in New Jersey.
Next time, DrKenB, try doing a little research on the subject instead of simply repeating the talking points of the Jersey haters from New York.
@ stshskt
During their 35 years in New Jersey, the Nets had 12 winning seasons, 22 losing seasons, and 1 season at .500(41-41).
If that's not a perennial loser, I don't know what is.
As for the Izod/CAA/Brendan Byrne Arena, it was obsolete by the late-1980s due to major advancements in the architecture & design of NBA arenas, and was never renovated & modernized like Madison Square Garden and other league venues.
Izod was also the only arena in the league without mass transit access, with one of the most poorly chosen locations in the league as well - The Swamp.
Considering the circumstances, it's amazing that any fans at all showed up to support the Nets during their tenure in the Garden State.
Not withstanding their numerous marketing & PR gimmicks, Nets ownership & management couldn't have created a less fan-friendly franchise if they tried.
Back in February, Howard gave up his right to opt out of his contract this summer, in return for the Magic's promise not to trade him.
So even if ESPN's story is true - and I seriously doubt that it is - Howard has no choice but to remain with the Magic next season.
This gives the Magic ample opportunity to shed contracts, make trades and sign free agents in order to improve enough to convince Howard to re-sign in 2013.
As for the Nets, they have very little of value to offer the Magic for Howard, since Brook Lopez is damaged goods with foot problems that could very well turn into a Yao Ming/Bill Walton-type chronic condition.
And BTW, New Jerseyans aren't the only ones not following the Nets to Brooklyn next season. Deron Williams ain't following them to Brooklyn either. But don't worry, Brooklynites, you'll be able to see Williams once a year when the Dallas Mavericks come to town.
New Jersey basketball fans gave the Nets far more support than they deserved during their years in the Garden State.
Lousy arena, lousy location and lousy mass transit in East Rutherford.
Lousy teams that rarely made the playoffs.
Yup, no doubt about it, New Jersey basketball fans gave the Nets far more support than they deserved.
@ jerzeyjolt
The Knicks, Sixers & Brooklyn Nets do NOT have territorial rights in New Jersey.
Deputy and future NBA commissioner Adam Silver has publicly stated that Newark would be an excellent home for an NBA franchise.
Governor Christie & Mayor Booker have publicly stated that they would seek & encourage another NBA team to replace the Nets in Newark.
@ stshskt
According to Forbes Magazine's annual review of NBA team finances, the Nets ranked among the NBA's TOP 5 TEAMS IN TICKET REVENUES during their 2001-03 glory years.
During their pre-Ratner NJ years -1977-2004 - the Nets consistently ranked in the TOP HALF OF THE LEAGUE IN TICKET REVENUES.
Despite having one of the league's worst teams(the Nets were perennial losers in NJ), playing in one of the league's worst arenas(IZOD), in one of the league's worst locations(The Swamp), with some of the league's worst mass transit access(none), New Jerseyans supported the Nets where it mattered most - at the box office.
Truth be told, New Jerseyans gave the Nets far more support than they deserved.
Next time, stshskt, do some research before shooting your mouth off.


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April Fools: Museum hosts "Rational Comedy for an Irrational Planet"
Calling All Science Enthusiasts and Jokers — Museum hosts "Rational Comedy for an Irrational Planet"
RALEIGH — Are you a science enthusiast that loves a geeky science joke? Then prepare to laugh a lot and even learn a little at the North Carolina Museum of Natural Sciences on Tuesday, April 1 at 7pm when science funny guy Brian Malow presents "Rational Comedy for an Irrational Planet." Drawing from physics, astronomy, biology and chemistry, Earth's (self-proclaimed) Premier Science Comedian is guaranteed to tickle your funny bone. And you don't have to be a scientist to enjoy the show — Malow's comedy is suitable for general audiences, particularly teens and adults. This show is part of the NC Science Festival.
Though Malow has been a comedian for nearly two decades, his love of science is much older than his love of comedy. Malow grew up reading science books and science fiction — even pursuing an undergraduate degree in pre-med and considering a career in science. After discovering comedy, he slowly transformed his enthusiasm for science into a perfect comedic niche.
"It was a very natural evolution, and not a calculated move," Malow says.
Since then, Malow has performed for the National Science Foundation, the American Chemical Society, the National Association of Science Writers, Apple, Dell, Microsoft, and many more. He has produced science videos for TIME magazine's website, co-hosted on the Weather Channel and contributed to Neil deGrasse Tyson's radio show. Other appearances include The Late Late Show with Craig Ferguson (CBS), Tech TV, Discovery and A&E. Malow will soon perform at the USA Science & Engineering Festival in Washington, DC.
But Malow is not just a science comedian. He is also an expert science communicator, and frequently gives talks to scientists to teach them to become better public speakers. At the Museum, he is the Curator of the SECU Daily Planet Theater and hosts Thursday night Science Cafes. Although his shows usually incorporate comedy, his goal is to ask interesting science questions and communicate his passion and interest in science to a wider audience.
"My work is all about public engagement with science — making things interesting and easy to understand. The humor is a part of that but I don't have to make everything funny, as long as it's interesting," says Malow. "Of course, for the April Fool's show, the humor will be front and center."
Admission is free. For more information, visit naturalsciences.org.


text: I am trying to build openbsd on my Sun 4/3XX. I did a cvs get src to get all the source. Now I cd to /sys/arch/sparc/conf and copied SUN4 to my hostname 'JUBILEE'. When I try to config JUBILEE it always returns: jubilee# config JUBILEE maxusers not specified; 8 assumed config: symlink(../compile/JUBILEE/machine -> ../../include): No such file or direc tory config: symlink(../compile/JUBILEE/sparc -> machine): No such file or directory *** Stop. However if I go to ../compile ther is a Jubille directory with a ln machine to ../../include and spare -> machine Because of this, I cant build a new kernel. Any ideas? I am new to the *BSD family. ALSO, is there any where to get some documentation for *BSD? I looked all over the OpenBSD and that "other"BSD home page but couldnt find anything really informative. I bought a bsd book but surely there are some specifics unique to each project Mikey


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Scott Hiley
Scott Hiley received his PhD in December 2007 from Yale University, where he was a Lopez Fellow in Medieval Studies, a Beinecke Summer Research Fellow, and Assistant Editor of Yale French Studies. He arrives at Northwestern from Reed College, where he taught French literature from the Middle Ages to the nineteenth century as well as French language.
Scott's primary research and teaching interests are in medieval French literature, and more especially in discursive formations linking literature, law, and theology. His current book project, tentatively entitled Reading between the Loans: Usury and the Economies of Literature in Medieval France, argues that vernacular French literature and legal and theological writings on usury in the twelfth and thirteenth centuries were part of a distinct discursive formation that elaborated new kinds of economic subjects and new strategies of reading and interpretation. For his next project, he plans to investigate the narrative structures of thirteenth-century prose romance through the lens of medieval texual culture. This year, he will offer courses on allegory in Old French Grail legends and on the stakes of narrative practice in the French Middle Ages.
Scott has lectured and presented nationally and internationally on topics ranging from medieval literature and economic culutre to seventeenth-century medievalism, theories of orality and texutality, and Jules Verne's technologies of exploration. His hobbies include backpacking, canoeing, skiing, and reading things that seem to have nothing to do with the Middle Ages.


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Aerobics and Conditioning
Aerobics and Conditioning
The following classes combine aerobic conditioning and strength training at varying intensity levels.
All Around Fitness (Free!)
Good for anyone who wants a low-impact workout, this moderately paced class focuses on improving aerobic endruance, strength and flexibility. Meets Monday, Wednesday and Friday; only one registration required for all three days.
Belly Dance Fitness
This class utilizes belly dance isolations to strengthen core, upper, and lower body muscle groups. All Fitness levels will benefit from this fun, unconventional workout.
Body Core (Free!)
This class is a bootcamp-style, intense total body workout that includes interval training, track work, cardio-kickboxing, weight and core work. If you want a fun and challenging workout which burns calories and develops endurance and agility, check out Body Core!
Boot Camp (Free!)
This class incorporates drills designed to enhance agility, speed, power, strength, and quickness. Check "lazy" at the door when you step into this workout. Boot Camp works you head to toe so you will be ready for anything life brings.
Cardio Karate (Free!)
This high energy class emphasizes martial arts moves in a vigorous choreographed workout.
Complete Conditioning (Free!)
Complete conditioning combines an all over resistance workout with a non-stop cardio workout. With the use of medicine balls, jump ropes, free weights and various bike and track cardio drills, participants can expect to reach a new level of physical fitness.
Conditioning for Life (Free!)
All levels of fitness and bantering are welcome in this class that will stretch and exercise your mind and body and will provide general conditioning exercises, and the relentless pursuit of trivial knowledge. Let a master of repartee take you down this time-tested road to fitness. Meets M/W/F. Only one registration required for all three days.
Core Essentials (Free!)
The Core Essentials class is a great way to improve posture and strengthen the core body area. Please arrive warmed up and ready to exercise. Free to members.
This elite-level fitness class will challenge you to attain personal growth through individual and team-building workouts in a competitive environment. The class will draw on the benefits of competition to increase muscle building via a combination of individual effort and participant collaboration. Through timed drills, core building, speed and endurance, cutting-edge strength moves, and dynamic full-body exercises you can expect a comprehensive fitness challenge which sets a new bar each week. This class is a full 60 minutes including warm up, circuit, team competition, and cool down. *NOTE: This advanced exercise class will be extremely rigorous, and is not appropriate for beginning exercisers or those with injuries. There will be a fitness test on the first day with eight foundational exercises to ensure that it is the right fit for each participant.*
EnhanceFitness (Free!)
A safe, effecitve and fun fitness program for older adults, EnhanceFitness is proven to increase cardiovascular health, muscular strength, flexibility and balance. Participants can either sit or stand during the class. Only one registration required for all three days. For information about off-site locations please contact Jen Nicodemus at jnicodemus[@]annarborymca.org.
Exercise for New Moms (Free!)
Come exercise and play with your baby at the same time-no babysitters needed! This is a great opportunity to meet other local moms and babies and to learn exercises you and your baby can do at home. The class will focus on restoring general strength, with some effort to elevate the heart rate. This class is ideal for moms with babies less than 12 months old. Please check with your healthcare provider, but generally this class is great for moms from four weeks after delivery (or six weeks after a C-section). Please bring a big blanket, a sense of humor and your little one!
Flex & Firm (Free!)
This class helps you lengthen and strengthen your body, using a fusion of Pilates, Yoga, Tai Chi and other techniques. While toning all the muscles in the body, there is special emphasis on the core. Workouts also focus on increasing flexibility to maximize the muscle building process and avoid injury. It is recommended that participants bring a yoga mat to class.
Great Arms
Firm and tone your arms and shoulders in this 30 minute workout. Using your body weight, dumbbells, and tubing you'll get the arms of your dreams and everyone will be saying "you have great arms!"
Group Strength Training
Start your day with an invigorating strength training workout. Each week we'll complete a different workout targeting all muscle groups, using dumbbells, medicine balls, stability balls and more. This class is geared toward those who want a guided strength training experience, with less emphasis on a cardiovascular workout.
Gyrokinesis® is an exercise system that increases the mobility of the joints and creates balance and equilibrium. Work your entire body through a series of movements performed seated and on mats on the floor using many key principles from pilates, dance, gymnastics and Tai-chi.
Power Lunch (Free!)
Make the most of your lunch hour with this 45 minute conditioning class. Strengthen and sculpt your muscles using resistance bands, balls, and free weights.
Step Survival (Free!)
A high intensity, very high energy, interval aerobic workout which emphasizes endurance and muscular conditioning. If you survive this class, you will feel great all day!
Strength Training for Runners
This class is geared toward runners of any experience level that are interested in adding a strength training component to their exercise regimen. We will focus on strengthening major muscles of the upper body, lower body, and core that are used most often during running workouts to improve postural alignment, prevent common running pains and injuries, and help increase speed.
Stroller Circuit (Free!)
Incorporate cardio, resistance training and core work while spending time with your little one. Bring your stroller and meet other parents on the track for a full body workout. Best for moms 6 weeks post-partum, but newborns are welcome.
Teen Strength & Conditioning
In this class, participants will engage in dynamic warm up routines, plyometric & agility drills, and weight lifting exercises while working in a fun and friendly atmosphere. Instructors will prescribe an exercise progression that will ensure improvements in strength, speed, and agility while keeping safety a priority.
Triathlon Training Class
*NOTE: THIS IS A TWO PART CLASS WITH BIKING/RUNNING ON THURSDAYS (BRICK WORKOUTS) AND SWIMMING SKILLS ON FRIDAYS.* This class is geared for anyone looking to learn how to train for a triathlon OR to simply learn a new and fun way to cross train. Each class includes Swim, Bike and Run instruction/workouts. Topics covered include Gear & Training, Nutrition, Flexibility, Strength Training & Injury Prevention. Prerequisite: participants should be able to comfortably swim 50 yards, Bike 10 minutes and walk/run 10 minutes. Culminates with a class "mini-tri" Saturday, December 21!
HIIT: High Intensity Interval Training
A total body workout consisting of high intensity interval training. Cardio, strength and ab intervals will be thrown in and mixed up each week to keep your body on its toes. Each muscle group is worked to fatigue using a circuit format that incorporates cardiovascular segments to keep the heart rate elevated. Weight can be adjusted for various fitness levels and modifications will be shown for cardiovascular segments.
TRX is a total body functional fat-burning workout that builds power, strength, flexibility, balance and mobility all at the intensity you choose. Our instructors will guide you through this revolutionary method, which utilizes a system of ropes and straps. You will quickly learn to work multiple muscles and joints simultaneously.
World Dance Workout
World Dance Workout is a fusion of dance moves from around the world, strength training, toning, stretching and much more! It is a total body workout designed to inspire, uplift, and motivate people of all ages adn fitness levels.
This class fuses latin rhythms and easy to follow moves to create a dynamic fitness program. You will achieve long term benefits while experiencing this one hour of calorie burning, body energizing, and awe inspiring movements meant to engage and captivate you.The routines feature interval training, using fast and slow rhythms to tone your body while burning fat. Add some Latin flavor and international zest into the mix and you've got Zumba. Who knew working out could be this much fun!
Zumba gold takes the Latin and international dance rhythms, found in the original Zumba, and modifies them for beginner participants and active older adults. It is created to emphasize the basic steps of Merengue, Salsa, Cha Cha, Belly Dance, Flamenco and Tango. Zumba Gold is a fun, easy, and an effective total body workout. It's great for the mind, body and soul.
Zumba Toning
Zumba Toning combines targeted body-sculpting exercises and high-energy cardio work with Latin-infused Zumba moves to creast a calorie-torching, strength-training dance fitness party. Students learn how to use light weights to enhance rhythm and tone all their target zones, including arms, abs, glutes, and thighs. Zumba Tonins is the perfect way for enthusiasts to sculpt their bodies while having a total blast.


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Hello Beautiful!
My dear and talented friend Amber Doe has been invited by the Arteles Creative Centre to be part of its March residency program! 2013 is definitely going to be her year to shine (or at least everyone else to see her bright star). I have known Amber for most of my New York life, meeting her just over four years ago. Amber has been an integral part in forming the way I see the world and how I react towards it. She's an incredibly talented artist who explores many issues I believe important to help evolve human thought: "gender, religion, identity, home, mysticism and the natural world."
Please check out her Indiegogo video and campaign, and have a listen to the Spotify playlist I made for her. Expect exciting things :)


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KERNERSVILLE, N.C. —Kernersville mother Celena Adams said she is now concerned more than ever for the safety of her child at the bus stop since 11-year-old Hasani Wesley's death.
Adams said the appeal process for a different route is "non-existent" and took months before getting a response from the Winston-Salem/Forsyth County school transportation department.
"It was told to me that it was on a first come first serve basis and that those would not be even looked at until September 10th which is after the school year started which to me Is unacceptable," Adams said.
Adams requested a safer bus stop for her 3rd grader in February.
The stop was just declared unsafe after 10 months.
School system representative Theo Helm said several factors are considered when declaring whether a bus stop is safe.
"From the traffic on the road, whether or not there are two lanes or four lanes or beyond that whether there are sidewalks on the road." Helm said.
Helm said currently the department does not calculate the requests based on danger or any other categories but are compiled together and are handled on a case by case basis.
Requests can be made through the particular school a child attends.
Adams plans on starting a petition or safer school by routes throughout the county.
You can find out more about appealing your child's bus route here.


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The Elite RealAxiom CT is a trainer for the riders that want to push themselves to their peak. This Trainer Includes a full suite of training software, from the Real video courses or the internet racing. With a completely…
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In Stock – The item is in stock at our warehouse. UK next day delivery available if ordered before 3pm or try our Click & Collect service (FREE).
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The Elite RealAxiom CT is a trainer for the riders that want to push themselves to their peak. This Trainer Includes a full suite of training software, from the Real video courses or the internet racing. With a completely wireless ANT+ head unit, there are no wires needed to connect to the drive unit or computer and is a good way to keep your bike looking neat and tidy. Like the Real Power model this has the RITMO feet which simulate the natural movement you make when you apply a little more pressure. The Elastogel roller has improved tyre grip, reduces tyre wear and vibrations which accumulates to be a 20% noise reduction. The high performance magnetic resistance unit is really responsive and has a built in power supply regulator. All of the features on this trainer make indoor training fun and suitable for the days where the weather is not on your side.
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Magnetic Powder
- Frame:
Folding frame for easy storage.
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Includes spare Quick-Release for those using Mavic or non-compatible skewers and the 2 real life classic race course are included, with additional courses as upgrades and add ones. Also the training software is provided, from real video courses to fully customised workouts.
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A buying guide that offers general advice on turbo trainers
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We offer a range of delivery services for our UK and International customers. UK Customers can select our FREE standard delivery option on most non bike orders over £20. Or if you're in a hurry you can upgrade to our Next Day, Saturday or Sunday delivery services. You can also use our free Click & Collect service, where you can collect your purchases from a choice of over 50 Evans Cycles stores. International customers can benefit from FREE delivery on most orders and save UK VAT if we are shipping outside of the EU.
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UK bike delivery charge includes assembly by our fully trained mechanics, pre-delivery inspection and safety check, packing in our custom bike box, multi-tool and delivery from only £9.99 per bike.Click here to see how we deliver your bike
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Funny interview of my friend Josh (done by me)
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Join Date: Oct 2010
Location: United Kingdom
Posts: 727
Funny interview of my friend Josh (done by me)
Thought this would be funny to upload, this was my friend Josh (who plays snare drum)'s interview of an acoustic gig he played this week. There are video's of him too!
P.S. Please excuse our accent, we're Scottish haha!
Here are english translations
How was your gig tonight Josh? It was alright
Were you nervous?- I was ******** it!
Really?- Yeah! Check my pants!
What made you chose those songs?- Why not other ones! - Cause I can't play any other songs
In time? -Nah, couldn't be bothered
Why didn't you pick Caught Up by Fall Of Troy - Cause it's ****?
So Josh, who's your favourite band at the moment? - I have alot of favourite bands
whats the best live band? - Stalwart Pandora1
Do you like Delgado (my other band) - (Rock on)
You coming to see Delgado on Sunday Night? £5 a ticket? - Maybe!
Thanks Josh - This is getting gay now! Bye
Hahaha, pretty funny. We call him Milkybar Kid, he looks like the kid from the Milkybar advert and his guitar is also called Milkybar Kid (Don't ask why haha).
Mapex, Sabian, Vic Firth.
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