text:
Next up at NextStep – Marteijn Mout who is the project managers of the 'New meter market model' at E.ON Benelux. E.ON is one of Europe's largest Energy providers with over 30 million customers in 30 countries. E.ON Benelux has around 270,000 customers and they have been a customer of OutSystems since 2004.
Describing the team's experience of using OutSystems Agile approach he told us that:
- They started with 5 iterations (sprints), with a period length of 2 weeks
- Sprint 6 was added to address new change requests
- Weekly progress reports with 2 demo's after every sprint and 2 week testing periods after demos
- Change Management requests were registered in OutSystems Agile Network (OAN), ECT and assumptions documented
Key challenges that the experienced:
- They were initially skeptical of the OutSystems size and scope estimate for the project – but happy that the results were delivered as expected.
- Intensive collaboration – Marteijn quipped that "I'm not sure how many emails I received from and sent to the OutSystems team – but there were a LOT. It wasn't bad – but was an indication of the degree of collaboration!"
- "We had some initial challenges on combining Waterfall and Agile methods – especially in planning – we were lucky that OutSystems' approach was flexible on that front, and we were able to combine planning into a single planning/test plan."
- The greatest issue was they found was the temptation to keep adding new requirements – and it was important to keep that reigned in where possible.
End results of the project were a success!
- The Customer Portal and underlying SMDR "engine" meet all requirements and more!
- OutSystems were a good sparring partner for architectural and functional matters
- OutSystems were flexible as promised (planning/adaptability)
- Communication lines efficient, effective and sound – even with a LOT of emails 😉
- Realistic planning/sizing and no budget overruns
- The Agile approach and technology is a big success factor for the project!
How are you coping with the amount of inter-team communications that's required by Agile – what tools are you using?


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Marine Geosciences
The Marine Geosciences Section supports research on processes that occur on and below the seafloor and at the water/sediment/rock interface. The section also supports facilities dedicated to such research.
The international Ocean Drilling Program (ODP) explores the Earth's crust beneath the ocean revealing the composition, structure, and history of the submerged portion of Earth's surface. The Marine Geology and Geophysics program supports research on all aspects of geology and geophysics of the ocean basins and margins, as well as the Great Lakes.

subset: commoncrawl


text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 8, 1994
|Filing date
|Sep 4, 1991
|Priority date
|Sep 4, 1991
|Fee status
|Also published as
|Publication number
|Curt L. Milliman
|Original Assignee
|Gen-Probe Incorporated
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (6), Non-Patent Citations (70), Referenced by (40), Classifications (14), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The inventions described and claimed herein relate to the design and construction of nucleic acid probes to Staphylococcus aureus which are capable of detecting the organism in test samples of, e.g., sputum, urine, blood and tissue sections, food, soil and water.
Two single strands of deoxyribo- ("DNA") or ribo- ("RNA") nucleic acid, formed from nucleotides (including the bases adenine (A), cytosine (C), thymidine (T), guanine (G), uracil (U), or inosine (I)), may associate ("hybridize") to form a double stranded structure in which the two strands are held together by hydrogen bonds between pairs of complementary bases. Generally, A is hydrogen bonded to T or U, while G is hydrogen bonded to C. At any point along the chain, therefore, one may find the classical base pairs AT or AU, TA or UA, GC, or CG. One may also find AG, GU and other "wobble" or mismatched base pairs.
When a first single strand of nucleic acid contains sufficient contiguous complementary bases to a second, and those two strands are brought together under conditions which will promote their hybridization, double stranded nucleic acid will result. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may be formed.
A probe is generally a single stranded nucleic acid sequence which is complementary to some degree to a nucleic acid sequence sought to be detected ("target sequence"). It may be labelled with a detectable moiety such as a radioisotope, antigen or chemiluminescent moiety. A background description of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences is described by Kohne, U.S. Pat. No. 4,851,330, and Hogan et al., EPO Patent Application No. PCT/US87/03009, entitled "Nucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organisms."
Hogan et al., supra, also describes methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These methods require probes sufficiently complementary to hybridize to the ribosomal RNA (rRNA) of one or more non-viral organisms or groups of non-viral organisms. The mixture is then incubated under specified hybridization conditions, and assayed for hybridization of the probe and any test sample rRNA.
Hogan et al. also describes probes which detect only specifically targeted rRNA subunit subsequences in particular organisms or groups of organisms in a sample, even in the presence of many non-related organisms, or in the presence of the closest known phylogenetic neighbors. Specific examples of hybridization assay probes are provided for Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, the genus Mycobacterium, Mycoplasma pneumoniae, the genus Legionella, Chlamydia trachomatis, the genus Campylobacter, Enterococcus, the genus Pseudomonas group I, Enterobacter cloacae, Proteus mirabilis, the genus Salmonella, Escherichia coli, bacteria, fungi, and Neisseria gonorrhoea. Such probe sequences do not cross react with nucleic acids from the groups listed above, or any other bacterial species or infectious agent, under appropriate hybridization stringency conditions.
Staphylococcus aureus is the causative agent of a wide variety of infections in humans, including diseases of the skin and soft tissues. e.g., skin pustules, impetigo, bacteremia, osteomyelitis, renal abscess, pneumonia, meningitis, endocarditis, gastroenteritis and toxic shock syndrome (Kloos and Schleifer, Staphylococcus, in Bergey's Manual of Systematic Bacteriology, 1013-1019, 1986) Further, S. aureus is a prominent agent of nosocomial infections. Methicillin resistant strains (MRSA) have emerged as a major epidemiological problem in hospitals throughout the United States.
Within the genus Staphylococcus, S.aureus is the most clinically significant species due to the incidence and severity of the infections it can cause (Morse, Stapylococci, in Medical Microbiology and Infectious Diseases. Abraham Braude, editor, W. B. Saunders Company, Philadelphia, Pa., 1981).
Current methods used to identify Staphylococcus aureus include Gram Stain morphology, cell morphology, production of catalase, pigment production, susceptibility to lysostaphin and lysozyme and anaerobic production of acid from glucose (Kloos and Schleifer, Simplified Scheme for Routine Identification of Human Staphylococcus Species, 1 J. Clin. Microbiol. 82, 1975). In addition, there are several commercially available systems that allow strains to be biochemically characterized.
Among Staphylococcus species associated with human infections, S. aureus is unique in its ability to clot plasma (coagulase). It should be noted, however, that some Staphylococcus species found in animals, such as S. intermedius and S. hyicus may also share this property. Two different coagulase tests are commonly used to identify S. aureus. One is a tube test for free coagulase, and the other is a slide test for bound coagulase. The tube coagulase test is thought be be the more definitive of the two, however it can take several hours, or even an overnight incubation period to produce a result. Using the slide coagulase test, up to 10 to 15 percent of S. aureus stains may yield a negative result (Jorgeuen and Kloos, Staphylococcal Infections, Diagnostic Procedures for Bacterial Infections, 7th ed. B. B. Wentworth. American Public Health Association, Washington, D.C. 1987). Because of the existence of problem clinical isolates that may not be identified using current methods, and because of the coagulase-positive strains associated with animals, an alternative identification method is needed.
Thus, in a first aspect, the invention features a hybridization assay probe able to distinguish Staphylococcus aureus from other Staphylococcus species.
In preferred embodiments, the probe is complementary to rRNA or rDNA, e.g., a variable region of rRNA; at least 50% of the nucleotides in the oligonucleotide probe are able to hybridize to a contiguous series of bases in at least one variable region of ribosomal nucleic acid in Staphylococcus aureus; the probe is a nucleotide polymer able to hybridize to the rRNA of the species Staphylococcus aureus in the region corresponding to bases 338-367 of E. coli 23S rRNA, or a nucleotide polymer complementary thereto; and the oligonucleotide comprises, consists essentially of, or consists of the sequence (SEQ. ID. NO.: 1) CCACTCAAGAGAGACAACATTTTCGACTAC or oligonucleotides complementary thereto, with or without a helper probe, as described below.
By "consists essentially of" is meant that the probe is provided as a purified nucleic acid which hybridizes under stringent hybridizing conditions with the desired organism and not with other related organisms. Such a probe may be linked to other nucleic acids which do not affect such hybridization. Generally, it is preferred that the probe be of between 15 and 100 (most preferably between 20 and 50) bases in size. It may, however, be provided in a vector.
In related aspects, the invention features a nucleotide polymer able to hybridize to the above oligonucleotides, a nucleic acid hybrid formed with the above oligonucleotides, and a nucleic acid sequence substantially complementary thereto.
The probes of this invention offer a rapid, non-subjective method of identification and quantitation of a bacterial colony for the presence of specific rRNA sequences unique to all strains of Staphylococcus aureus.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
We have discovered DNA probes complementary to a particular rRNA sequence obtained from Staphylococcus aureus. Furthermore, we have successfully used those probes in a specific assay for the detection of Staphylococcus aureus, distinguishing S. aureus from its known and presumably most closely related taxonomic or phylogenetic neighbors.
With the exception of viruses, all prokaryotic organisms contain rRNA genes encoding 5S rRNA, 16S rRNA and a larger rRNA molecule known as 23S rRNA. Using methods known to those skilled in the art, variable regions of rRNA sequences from the 23S rRNA of Staphylococcus aureus were identified as described below. Other such sequences can be identified using equivalent techniques. These methods include partially or fully sequencing the rRNA of Staphylococcus aureus and closely related phylogenetic neighbors, aligning the sequences to reveal areas of maximum homology, and examining the alignment for regions with sequence variation. The examples provided below are thus not limiting in this invention.
With respect to sequencing, complementary oligonucleotide primers of about 10-100 bases in length were hybridized to conserved regions in purified rRNA that are specific to the 5S, 16S, or 23S subunits and extended with the enzyme reverse transcriptase. Chemical degradation or dideoxynucleotide-terminated sequencing reactions were used to determine the nucleotide, sequence of the extended product. Lane et al., 82 Proc. Natl. Acad. Sci. USA. 6955, 1985. In a less preferred method, genomic ribosomal RNA sequences may also be determined by standard procedure.
It is not always necessary to determine the entire nucleic acid sequence in order to obtain a probe sequence. Extension from any single oligonucleotide primer can yield up to 300-400 bases of sequence. When a single primer is used to partially sequence the rRNA of the target organism and organisms closely related to the target, an alignment can be made as outlined below. If a useful probe sequence is found, it is not necessary to continue rRNA sequencing using other primers. If, on the other hand, no useful probe sequence is obtained from sequencing with a first primer, or if higher sensitivity is desired, other primers can be used to obtain more sequences. In those cases where patterns of variation for a molecule are not well understood, more sequence data may be required prior to probe design.
After sequencing, the sequences are aligned to maximize homology. The rRNA molecule has a close relationship of secondary structure to function. This imposes restrictions on evolutionary changes in the primary sequence so that the secondary structure is maintained. For example, if a base is changed on one side of a helix, a compensating change is made on the other side to preserve the complementarity (this is referred to as co-variance). This allows two very different sequences to be aligned based on the conserved primary sequence and also on the conserved secondary structure elements. Once sequences are aligned it is possible to find the regions in which the primary sequence is variable.
We have identified variable regions by comparative analysis of rRNA sequences both published in the literature and sequences which we have determined. Computers and computer programs which may be used or adapted for the purposes herein disclosed are commercially available. Since the sequence evolution at each of the variable regions (for example, spanning a minimum of 10 nucleotides) is, for the most part, divergent, not convergent, we can confidently design probes based on a few rRNA sequences which differ between the target organism and its phylogenetically closest relatives. We have seen sufficient variation between the target organism and the closest phylogenetic relative found in the same sample to design the probe of interest.
We have identified the following useful guidelines for designing probes with desired characteristics. Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.
First, the stability of the probe:target nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long A and T rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %G and %C result in a Tm about 2�-10� C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.
Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account in constructing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, dimethylsulfoxide (DMSO) and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5� C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form (i.e., those having at least about 14 out of 17 bases in a contiguous series of bases being complementary); hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid.
Second, probes should be positioned so as to minimize the stability of the probe:nontarget nucleic acid hybrid. This may be accomplished by minimizing the length of perfect complementarity to non-target organisms, avoiding G and C rich regions of homology to non-target sequences, and by positioning the probe to span as many destabilizing mismatches as possible. Whether a probe sequence is useful to detect only a specific type of organism depends largely on the thermal stability difference between probe:target hybrids and probe:nontarget hybrids. In designing probes, the differences in these Tm values should be as large as possible (e.g., at least 2� C. and preferably 5� C.).
The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly homologous base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, oligonucleotide probes preferred in this invention are between about 10 to 50 bases in length and are sufficiently homologous to the target nucleic acid.
Third, regions of the rRNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided.
As explained above, hybridization is the association of two single strands of complementary nucleic acid to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. In the case of rRNA, the molecule is known to form very stable intramolecular hybrids. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. If the target is the genomic sequence corresponding to the rRNA then it will naturally occur in a double stranded form, this is also the case with the product of the polymerase chain reaction (PCR). These double stranded targets are naturally inhibitory to hybridization with a probe. Finally, there can be intramolecular and intermolecular hybrids formed within a probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. Computer programs are available to search for this type of interaction.
Once a presumptive unique sequence has been identified, a complementary DNA oligonucleotide is produced. This single stranded oligonucleotide will serve as the probe in the hybridization reaction. Defined oligonucleotides may be produced by any of several well known methods, including automated solid-phase chemical synthesis using cyanoethylphosphoramidite precursors. Barone et al., 12 Nucleic Acids Research 4051, 1984. Other well-known methods for construction of synthetic oligonucleotides may, of course, be employed. 2 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning 11 (2d ed. 1989).
Once synthesized, selected oligonucleotide probes may also be labelled by any of several well known methods. 2 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning 11 (2d ed. 1989). Useful labels include radioisotopes as well as non-radioactive reporting groups. Isotopic labels include 3 H, 35 S, 32 P, 125 I, 37 CO and 14 C. Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcription. When using radio-labelled probes, hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radio-isotope used for labelling.
Non-isotopic materials can also be used for labelling, and may be introduced internally into the sequence or at the end of the sequence. Modified nucleotides may be incorporated enzymatically or chemically and chemical modifications of the probe may be performed during or after synthesis of the probe, for example, by the use of non-nucleotide linker groups. Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands. We currently prefer to use acridinium esters.
Following synthesis and purification of a particular oligonucleotide sequence, several procedures may be utilized to determine the acceptability of the final product. The first is polyacrylamide gel electrophoresis, which is used to determine size. 2 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, 11.51 (2d ed. 1989). Such procedures are known in the art. In addition to polyacrylamide gel electrophoresis, High Pressure Liquid Chromatography ("HPLC") procedures also may be used to determine the size and purity of the oligonucleotide product. These procedures are also known to those skilled in the art.
It will be appreciated by those skilled in the art that factors which affect the thermal stability can affect probe specificity and therefore, must be controlled. Thus, the melting profile, including the melting temperature (Tm) of the oligonucleotide/target hybrids should be determined. The preferred method is described in Arnold et al., Patent application Ser. No. 613,603 filed Nov. 8, 1990, entitled "Homogeneous Protection Assay assigned to Gen-Probe Incorporated, Mar. 6, 1992, Reel/Frame 6057/0433-34," hereby incorporated by reference herein, also published under European Patent Application Publication Number 0,309,230, Mar. 29, 1989.
For Tm measurement using a Hybridization Protection Assay (HPA) the following technique is used. A probe:target hybrid is formed in target excess in a lithium succinate buffered solution containing lithium lauryl sulfate. Aliquots of this hybrid are diluted in the hybridization buffer and incubated for five minutes at various temperatures starting below that of the anticipated Tm (typically 55� C.) and increasing in 2-5 degree increments. This solution is then diluted with a mildly alkaline borate buffer and incubated at a lower temperature (for example 50� C.) for ten minutes. Under these conditions the acridinium ester attached to a single stranded probe is hydrolyzed while that attached to hybridized probe is relatively protected from hydrolysis. The amount of chemiluminescence remaining is proportional to the amount of hybrid, and is measured in a luminometer by addition of hydrogen peroxide followed by alkali. The data is plotted as percent of maximum signal (usually from the lowest temperature) versus temperature. The Tm is defined as the point at which 50% of the maximum signal remains.
In addition to the above method, oligonucleotide/target hybrid melting temperature may also be determined by isotopic methods well known to those skilled in the art. It should be noted that the Tm for a given hybrid will vary depending on the hybridization solution being used because the thermal stability depends upon the concentration of different salts, detergents, and other solutes which effect relative hybrid stability during thermal denaturation. 2 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, 9.51 (2d ed. 1989).
Rate of hybridization may be measured by determining the C0 t1/2. The rate at which a probe hybridizes to its target is a measure of the thermal stability of the target secondary structure in the probe region. The standard measurement of hybridization rate is the C0 t1/2 which is measured as moles of nucleotide per liter times seconds. Thus, it is the concentration of probe times the time at which 50% of maximal hybridization occurs at that concentration. This value is determined by hybridizing various amounts of probe to a constant amount of target for a fixed time. For example, 0.05 pmol of target is incubated with 0.012, 0.025, 0.05, 0.1 and 0.2 pmol of probe for 30 minutes. The amount of hybrid after 30 minutes is measured by HPA as described above. The signal is then plotted as a log of the percent of maximum Relative Light Units (RLU) (from the highest probe concentration) versus probe concentration (moles of nucleotide per liter). RLU are a measurement of the quantity of photons emitted by the labelled-probe measured by the luminometer. The C0 t1/2 is found graphically from the concentration corresponding to 50% of maximum hybridization multiplied by the hybridization time in seconds. These values range from 9.0�10-6 to 9�10-5 with the preferred values being less than 3.5�10-5.
As described by Kohne and Kacian (U.S. Ser. No. 816,711, entitled "Accelerated Nucleic Acid Reassociation Method," filed Jan. 7, 1986 abandoned in favor of U.S. application No. 644,879, filed Jan. 23, 1991 issued Jul. 21, 1992 as U.S. Pat. No. 5,132,207, and is assigned to Gen-Probe Incorporated, Apr. 14, 1986, Reel/Frame 4538/0494, hereby incorporated by reference herein), also published under European Patent Application Publication Number 0,229,442, Jul 22, 1987, other methods of nucleic acid reassociation can be used.
The following example sets forth synthetic probes complementary to a unique rRNA sequence, or the corresponding gene, from a target organism, Staphylococcus aureus, and their use in a hybridization assay.
A probe specific for S. aureus was identified by sequencing with a primer complementary to the 23S rRNA. The phylogenetically near neighbors S. saprophyticus, S. epidermidis and S. avium and others were used as comparisons with the sequence of S. aureus.
This probe is 30 bases in length and hybridizes to the 23S rRNA of S. aureus corresponding to bases 338-367 of E. coli. To demonstrate the reactivity and specificity of the probe for S. aureus, it was used in a hybridization assay. The probe was first synthesized with a non-nucleotide linker, then labelled with a chemiluminescent acridinium ester as described in EPO Patent Application No. PCT/US88/03361, entitled "Acridinium Ester Labeling and Purification of Nucleotide Probes" filed Oct. 5, 1988. The acridinium ester attached to unhybridized probe is rendered non-chemiluminescent under mild alkaline conditions, while the acridinium ester attached to hybridized probe is relatively resistant. Thus, it is possible to assay for hybridization of acridinium ester-labelled probe by incubation with an alkaline buffer, followed by detection of chemiluminescence in a luminometer. Results are given in RLU, the quantity of photons emitted by the labelled-probe measured by the luminometer. The conditions of hybridization, hydrolysis and detection are described in Arnold et al., 35 Clin. Chem. 1588, 1989.
Nucleic acid hybridization was enhanced by the use of "Helper Probes" as disclosed in Hogan et al., U.S. Pat. No. 5,030,557, entitled "Means and Methods for Enhancing Nucleic Acid Hybridization", allowed Dec. 17, 1990, and hereby incorporated by reference herein. RNA was hybridized to the acridinium ester-labeled probe in the presence of unlabeled Helper Probes, oligonucleotide SEQ.ID. NO. 2 with the sequence of 5'GATGATTCGTCTAATGTCGACCTTTGTAACTCC 3' and oligonucleotide SEQ. ID. NO. 3 with the sequence of 5'CGGAATTTCACGTGCTCCGTCGTACTCAGGAT 3'.
In the following experiment, RNA released from one colony or >108 organisms was assayed. An example of such a method is provided by Murphy et al., U.S. Ser. No. 841,860, entitled "Method for Releasing RNA and DNA from Cells", filed Mar. 20, 1986, abandoned in favor of U.S. Ser. No. 298,765, filed Jan. 17, 1989, abandoned in favor of U.S. Ser. No. 711,114, filed Jun. 21, 1991, assigned to Gen-Probe Incorporated, May 23, 1986, Reel/Frame 4566/0901, hereby incorporated by reference herein, also published under European Patent Application Publication Number 0,288,618, Feb. 11, 1988. An RLU value greater than 30,000 RLU is a positive reaction; less than 30,000 is a negative reaction.
The following data show that the probe did not cross react with organisms from a wide phylogenetic cross section. These ATCC isolates were tested in duplicate, the RLU given is the average of the duplicates.
______________________________________ RESULTATCC # Organism RLU (+/-)______________________________________ 1 33604 Acinetobacter calcoaceticus 5417 - 2 15309 Acinetobacter lwoffii 5336 - 3 10049 Actinomyces israelii 3327 - 4 19411 Actinomyces pyogenes 3010 - 5 11563 Aerococcus viridans 2391 - 6 7966 Aeromonas hydrophilia 15644 - 7 27061 Alcaligenes denitrificans 1864 - 8 8750 Alcaligenes faecalis 3903 - 9 6051 Bacillus subtilis 2510 -10 23745 Bacteroides fragilis 6085 -11 10580 Bordatella bronchiseptica 3452 -12 25238 Branhamella catarrhalis 2790 -13 9172 Brevibacterium linens 3714 -14 33560 Campylobacter jejuni 4674 -15 18804 Candida albicans 2796 -16 27872 Capnocytophaga ochracea 3020 -17 29094 Chromobacterium violaceum 5819 -18 14501 Clostridium innocuum 2161 -19 13124 Clostridium perfringens 3393 -20 11913 Corynebacterium diphtheriae 3650 -21 9345 Corynebacterium haemolyticum 2944 -22 10700 C. pseudodiphtheriticum 2014 -23 19410 C. pseudotuberculosis 2048 -24 373 Corynebacterium xerosis 2070 -25 32045 Cryptococcus neoformans 3134 -26 35073 Deinococcus radiodurans 2991 -27 15994 Derxia gummosa 2522 -28 19433 Enterococcus faecalis 2520 -29 19414 Erysipelothrix rhusiopathiae 1732 -30 10798 Escherichia coli 4607 -31 13253 Flavobacterium 3941 - meningosepticum32 10379 Gemella haemolysans 2804 -33 19418 Haemophilus influenzae 3382 -34 23357 Klebsiella pneumoniae 5242 -35 33152 Legionella pneumophila 6149 -36 33313 Leuconostoc paramesenteroides 1912 -37 35152 Listeria monocytogenes 2501 -38 27570 Micrococcus kristinae 2059 -39 4698 Micrococcus luteus 3150 -40 27566 Micrococcus lylae 4628 -41 186 Micrococcus roseus 2312 -42 13077 Neisseria meningitidis 5834 -43 19247 Nocardia asteroides 2209 -44 33225 Oerskovia turbata 3278 -45 27402 Oerskovia xanthineolytica 3024 -46 17741 Paracoccus denitrificans 2032 -47 33314 Pediococcus acidilactici 2813 -48 14955 Peptococcus magnus 1420 -49 27337 Peptostreptococcus 3954 - anaerobius50 6919 Propionibacterium acnes 1944 -51 25933 Proteus mirabilis 6970 -52 25330 Pseudomonas aeruginosa 3075 -53 33071 Rahnella aquatilis 2494 -54 25592 Rhodococcus bronchialis 1516 -55 33591 Staphylococcus aureus 658694 +56 25904 Staphylococcus aureus 583913 +57 12600 Staphylococcus aureus 543927 +58 27660 Staphylococcus aureus 543617 +59 12598 Staphylococcus aureus 542628 +60 29213 Staphylococcus aureus 531156 +61 29247 Staphylococcus aureus 527566 +62 43866 Staphylococcus aureus 504506 +63 12598 Staphylococcus aureus 472820 +64 13301 Staphylococcus aureus 468886 +65 33589 Staphylococcus aureus 463814 +66 27661 Staphylococcus aureus 400398 +67 25923 Staphylococcus aureus 299651 +68 35662 Staphylococcus cohnii 3770 -69 29887 Staphylococcus epidermidis 3294 -70 14990 Staphylococcus epidermidis 2839 -71 35983 Staphylococcus epidermidis 2658 -72 12228 Staphylococcus epidermidis 2145 -73 29970 Staphylococcus haemolyticus 2155 -74 29885 Staphylococcus hominis 3716 -75 29663 Staphylococcus intermedius 3769 -76 43867 Staphylococcus saprophyticus 2959 -77 15305 Staphylococcus saprophyticus 2665 -78 11631 Staphylococcus simulans 10296 -79 27836 Staphylococcus warneri 6348 -80 13813 Streptococcus agalactiae 2509 -81 9811 Streptococcus mitis 2028 -82 6303 Streptococcus pneumoniae 2584 -83 19615 Streptococcus pyogenes 6451 -84 23345 Streptomyces griseus 4516 -85 17802 Vibrio parahaemolyticus 6487 -86 9610 Yersinia entercolitica 3211 -______________________________________
The above data confirm that the novel probes herein disclosed and claimed are capable of distinguishing Staphylococcus aureus from its known nearest phylogenetic neighbors.
Other embodiments are within the following claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 8(2) INFORMATION FOR SEQ ID NO: 1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1:CCACTCAAGAGAGACAACAT TTTCGACTAC30(2) INFORMATION FOR SEQ ID NO: 2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:GATGATTCGTCTAATGTCGACCTTTGTAACTCC33(2) INFORMATION FOR SEQ ID NO: 3:( i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:CGGAATTTCACGTGCTCCGTCGTACTCAGGAT32(2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:GTAGTCGAAAATGTTGTCTCTCTTGAGTGG30(2) INFORMATION FOR SEQ ID NO: 5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:GUAGUCGAAAAUGUUGUCUCUCUUGAGUGG30(2) INFORMATION FOR SEQ ID NO: 6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:CCACUCAAGAGAGACAACAUUUUCGAC UAC30(2) INFORMATION FOR SEQ ID NO: 7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:CTACTAAGCAGATTACAGCTGGAAACATTGAGG33(2) INFORMATION FOR SEQ ID NO: 8:( i) SEQUENCE CHARACTERISTICS:(A) LENGTH:32(B) TYPE:nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:GCCTTAAAGTGCACGAGGCAGCATGAGTCCTA32
|Cited Patent
|Filing date
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|VACCINE FOR $i(BRANHAMELLA CATARRHALIS)
|U.S. Classification
|536/24.32, 435/883, 536/23.7, 435/882, 536/24.3, 435/884, 435/6.15, 435/6.12
|International Classification
|Cooperative Classification
|European Classification
|Dec 13, 1991
Owner name: GEN-PROBE PRODUCTS COMPANY, CALIFORNIA
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Achcham Yenbadhu Madamaiyada Movie Review & Rating: Achcham Yenbadhu Madamaiyada is a Tamil action romantic film written and directed by Gautham Menon. It features Silambarasan and Manjima Mohan in lead roles. While Baba Sehgal, Daniel Balaji, Sathish Krishnan played the prominent supporting cast roles. It is simultaneously made in Telugu as Sahasam Swasaga Sagipo with Naga Chaitanya and Manjima Mohan in lead roles. After so many postponements, The film Achcham Yenbadhu Madamaiyada movie is going to be released on November 11, 2016. Check out Simbu's "Achcham Yenbadhu Madamaiyada" Movie complete review, rating, story line, plus points, minus points, verdict, and box office collection details from the below article.
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Achcham Yenbadhu Madamaiyada (AYM) Tamil Movie Review & Rating
Simbu's Achcham Yenbadhu Madamaiyada Movie Cast and Crew (Credits):
Release Date: November 11, 2016
Language: Tamil
Star Cast: Silambarasan, Manjima Mohan, Baba Sehgal, Daniel Balaji, Sathish Krishnan
Director: Gautham Menon
Producer: Gautham Menon
Editor: Anthony Gonsalves
Cinematographer: Dan Macarthur
Music Director: A. R. Rahman
Achcham Yenbadhu Madamaiyada Tamil Movie Theatrical Trailer
Achcham Yenbadhu Madamaiyada (AYM) Movie Story:
Simbu is a college going youth who falls in love with Manjima Mohan, his sister's best friend. As time passes by, he gets close to Leela and takes her for a long road trip.
The twist in the tale arises when the beautiful road trip changes upside down because of a horrible accident. Thereafter, things become even more difficult for the couple as a dreaded gang starts attacking them.
Who are these people ? What do they have to do with such a young couple ? and how will Naga Chaitanya save his girl friend from this chaos. That forms the rest of the story.
Achcham Yenbadhu Madamaiyada Movie Highlights:
- Simbu.
- Screenplay.
- Pre-Climax.
- Music & BGM.
- Cinematography.
Achcham Yenbadhu Madamaiyada Movie Drawbacks:
- Climax.
- Songs Placement.
- Slow Narration.
On the whole, Achcham Yenbadhu Madamaiyada is well-balanced love action drama which will surely be loved by our younger generation. On the flip side, the movie has some negative elements like the silly climax, slow narration.
Overall, Simbu's Achcham Yenbadhu Madamaiyada ended up as a watchable movie for the weekend.
Achcham Yenbadhu Madamaiyada (AYM) Movie Rating:
- Critics Rating: 2.75/5
- Audience Rating: 3/5
Achcham Yenbadhu Madamaiyada Movie Box Office Collections:
Achcham Yenbadhu Madamaiyada movie minted Rs 10.10 crore in first three days in the state.
That's it guys this is the perfect & genuine review and rating of Simbu's Achcham Yenbadhu Madamaiyada Tamil Movie and Box Office Collections. If you like this Achcham Yenbadhu Madamaiyada (AYM) Tamil Movie Genuine Review & Collections. Then please share it with your friends. But, Don't forget one thing i.e. please watch the movie in theatres. Kill Piracy and save the industry.
Stay tuned to this page for more updates regarding Simbu's AYM Tamil movie First 3 Days (First Weekend), First Week and Total Run Collections in Tamilnadu, Overseas, Worldwide.
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text:
Curt Robert Johann Schleusener
- Date of birth: Sep 29, 1926
|Let the memory of Curt be with us forever
This memorial website was created in memory of our loved one, Curt Schleusener, . Curt served in the Merchant Marines from 1945-1947 and then in the United States Army from 1950-1952. We honor him for his service to country and community. We will remember him forever.
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Artificial Coconut Palm
Coconut Palm Tree With Fruits
- Height 5ft 11" /180cms
- Same day dispatch via 24 hr next working day courrier service (unpotted items only and orders before 2pm)
- Fitted to small starter planter as standard
Height – 5ft 11" / 180cm
Starter Planter - 8inches
This stunning artificial Coconut palm tree stands at 180cm tall and comes fitted in an 8' starter pot seen in the first image. This artificial Coconut palm tree features a replica trunk and mid to dark green polyester fronds. A 100% botanically accurate replica from the detailed leaves to the palm fibres on the trunk. This palm comes with two replica coconut fruits attached. Too add the extra tropial touch this palm tree displays two hanging coconut fruits (one big, one small).
Our replica palm trees are premium quality, made using top quality silks, plastic and latex to ensure they look lush and healthy for many years.
Where can I use this Artificial Coconut palm tree?
You will be able to create an everlasting tropical look with this fake palm tree! Ranging from a tropical wedding to a themed room in your home or office. Ideal for conservatories where it will not wither or die like real palms do, or simply to fill an empty corner. Perfect addition to swimming pool areas or any office environment where some greenery is required.
Finishing touches
This artificial palm comes fitted to a small starter planter. Please select a planter from the drop down menu if you wish to have this tree potted into a decorative pot.
Interesting fact about Coconuts!
The scientific term of the coconut palm tree is cocos nucifera. The word coconut is most likely to come from the Spanish and Portuguese word coco, which has a meaning of "a grin", "a monkey face" due to the resemblance of the coconut fruit to a human face, or a monkey head, since there are three small indentations on the shell of the fruit. Nucifera, in its turn, comes from Latin, and means "nut-bearing", where fero means "I bear" and nux is "a nut"..
Delivery & Dispatch
Orders placed Monday - Friday will be dispatched within 24 hours (weekend will be next working day) providing they are not fully potted or handbuilt trees, these may take up to 5 working days before dispatch. Your goods will be delivered using either Royal Mail 1st class or 24 hour courier depending on size and weight of parcel. If you have a deadline either call or make a note on your order. We will e mail you on the day of dispatch so you know your parcel is on its way to you.
Returns & Refunds
If you are not completely satisfied with your product or your product is faulty or damaged, you can return the goods to us for a refund. Please see terms & conditions for more information. If your item is made to your own specifications and sizes it is classed as a bespoke non standard item.


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Interface Monitoring System to Promote Residual Limb Health
Co-Principal Investigators:Yonggang Huang and Stefania Fatone (Northwestern University); Todd Coleman (UCSD); and John Rogers (Northwestern University)
Co-Investigators: Lilly Tran, MS; Ryan Caldwell, CP
Funded by: Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) and National Institute of Biomedical Imaging and Bioengineering (NIBIB) 5R01EB019337
Status: In Progress
Project Overview
This proposal aims to develop a stretchable and flexible sensor technology capable of transforming healthcare from reactive and hospital-centered to preventive, proactive, evidence-based, and person-centered. The goal is to offer 'skin-like' properties, to enable intimate, complete non-invasive integration with the patient. The resulting 'epidermal' electronic devices (Kim, Lu et al. 2011) may allow clinicians to monitor their patients, and the general public to assess, continuously, their health and wellbeing. The proposed interface monitoring system, designed to promote residual limb health in persons who wear prostheses, in physical forms that are skin-like, may demonstrate key technological and scientific advancements towards evidenced-based and person-centered prosthetic care.
The work involves collaboration across three institutions to:
- Develop 'skin-like' pressure, strain and temperature sensors, with wireless operation, as well as hydration and blood flow sensors that operate inside a prosthetic socket.
- Develop computational modeling and algorithms for statistical signal processing of the sensor data and pattern recognition to create a user-friendly interface for clinicians and patients.
- Apply the proposed sensor technologies and data processing and pattern recognition techniques to prosthetic clinical practice. The continuous capture, storage and transmission of sensor data are critical to the design of lower limb prosthetics for improved health and healthcare.
The proposed work will improve health by leading development of new biomedical imaging devices for early detection and prevention of health problems and assessment of health status. In addition to prosthetic care, the proposal may address an unmet need for a model system for individualized healthcare, in which continuous sensing, monitoring and assessment are performed using wireless epidermal sensors instead of traditional lab-based instrumentation.
Progress Update
Focus Groups Used to Gather Stakeholder Input
As a first step in ensuring the clinical relevance and usefulness of the proposed development work, we convened two focus groups, one group consisting of Certified Prosthetists and one group of lower-limb prosthesis users, to gather input on system configuration and data presentation. The focus groups were held at NUPOC on January 21 and March 16 2015, respectively. The very fruitful discussions were recorded and currently are being transcribed for further analysis.
Kim, D. H., N. Lu, R. Ma, Y. S. Kim, R. H. Kim, S. Wang, J. Wu, S. M. Won, H. Tao, A. Islam, K. J. Yu, T. I. Kim, R. Chowdhury, M. Ying, L. Xu, M. Li, H. J. Chung, H. Keum, M. McCormick, P. Liu, Y. W. Zhang, F. G. Omenetto, Y. Huang, T. Coleman and J. A. Rogers (2011). "Epidermal electronics." Science333(6044): 838-843.
NUPOC would like to acknowledge the support of Ottobock in providing two residual limb models for use in this project.
NUPOC would like to thank the prosthetists and prosthesis users who participated in our focus groups for their candid and helpful comments. We also would like to acknowledge research extern, Matthew Quigley, for his invaluable help in preparing for the focus groups. Additionally, we thank Dr. R. J. Garrick for moderating, Piper Kruse for audio recordings, and Linda India for support with catering and administration.
Zhang Y, Webb RC, Luo H, Xue Y, Kurniawan J, Cho NH, Krishnan S, Li Y, Huang Y, and Rogers JA (2016) Theoretical and Experimental Studies of Epidermal Heat Flux Sensors for Measurements of Core Body Temperature. Adv. Healthcare Mater. 2016, 5, 119–127.
Fatone S, Huang Y, Rogers J, Coleman T (2017) Interface Monitoring System to Promote Residual Limb Health. Ignite Talk and poster at the 2017 NSF Smart and Connected Health (SCH) Principal Investigators Meeting, March 20-22, Boston, MA.
Tran L, Han S, Caldwell R, Rogers J, Coleman T, Fatone S (2017) Ability of Epidermal Sensors to Measure Lower-Limb Temperature during Activity with a Prosthesis Simulator. The 43rd Academy Annual Meeting & Scientific Symposium of the American Academy of Orthotists & Prosthetists (AAOP), March 1-4, Chicago, IL.
Fatone S, Caldwell R (2016) Socket-Related Research Collaborations at Northwestern University. Scheck Fair. April 8-9, Lombard, Illinois.
Tran L, Caldwell R, Quigley M, Fatone S (2016) Stakeholder Input on the Development of a Residual Limb Monitoring System. 12th Annual Lewis Landsberg Research Day, April 7, Northwestern University, Chicago, IL (poster).
Tran L, Caldwell R, Quigley M, Fatone S (2016) Stakeholder Input on a Residual Limb Monitoring System. 42nd American Academy of Orthotists and Prosthetists Annual Meeting & Scientific Symposium, March 9-11, Orlando, FL.
Tran L, Caldwell R, Quigley M, Fatone S (2016) Stakeholder Input on Development of a Residual Limb Health Monitoring System. NUPOC Biennial Symposium for the German-Speaking Travel Fellows of Initiative '93, February 25, Chicago, Illinois.
Tran L, Han S, Caldwell R, Rogers J, Coleman T, Fatone S (2016) Ability of epidermal sensors to measure lower limb temperature during activity with a prosthesis simulator. Midwest Chapter of the American Academy of Orthotists and Prosthetists, November 3-5, Rosemont, IL.
Tran L, Fatone S, Caldwell R (2015) Stakeholder Input on Development of a Residual Limb Health Monitoring System. Midwest Chapter, American Academy of Orthotists and Prosthetists Annual Meeting and Scientific Session, May 27-29, Rosemont, IL.
Fatone S, Huang Y, Rogers J, Coleman T, Tran L (2015) Interface Monitoring System to Promote Residual Limb Health. Ignite Talk at the 2015 NSF Smart and Connected Health (SCH) Principal Investigators Workshop: Amplifying Discovery through Shared Resources and Defining and Measuring Health Impact in SCH Research, June 30-July 1, 2015, Arlington, VA. [Access PDF, see Section #57, pages 115-120.]
Fatone S, Huang Y, Coleman T, Rogers J, Banks A, Tran L, Caldwell R, Brown D, Pharr M (2015) Interface Monitoring System to Promote Residual Limb Health (poster). 2015 NSF Smart and Connected Health (SCH) Principal Investigators Workshop: Amplifying Discovery through Shared Resources and Defining and Measuring Health Impact in SCH Research, June 30-July 1, 2015, Arlington, VA.
Leitner Tammy and Capitanini Lisa. Human Body Merges with Technology in 'Biohacking' Trend, NBC 5, Chicago, IL (Published Monday, Sept. 21, 2015).
Related Publications
Clark V. (March 2017) O&P Research Supports Evidence-based Care. The O&P Edge, 16(3):20-34. http://www.oandp.com/articles/2017-03_01.asp
Co-Principal Investigator, John Rogers, Joins Northwestern University
Co-Principal investigator, collaborator and innovator, John Rogers, joined Northwestern University in autumn 2016 as the Louis Simpson and Kimberly Querrey Professor of Materials Science and Engineering, Biomedical Engineering and Medicine.
Monitoring Limb Health
Signals from skin-like sensors can be wirelessly monitored


text:
by Kimberly Spencer
(Birmingham, AL)
My recipe for pig logs with smoking dipping sauce.
Click here to post comments
Return to Recipe Contest Entry Form.
I'm the cook behind this website. I have been passionate about cooking soul food for as long as I can remember. Nothing excites me more than cooking up a much appreciated home cooked meal for my family and friends. I get asked all the time why I started this Web site. Here's the short version....
My family and close friends have always loved the way I cook delicious home cooked meals. [Read More]
© 2006–2015 Soul Food and Southern Cooking. All rights reserved.


text:
A/N: Cooould have written more baby type fic but noooo, my brain's a dick. Post - TATM.
Comments and feedback always greatly appreciated.
"I'll tell her to write an afterword. For you. Maybe you'll listen to her." She pauses for a moment, waiting. For a word, a correction, a gesture.
He doesn't say anything, he doesn't disagree, he doesn't acknowledge.
He lets her go.
(A stitch loosens.)
She hears the door slam behind his exit.
She sets up in the library, pages strewn before her, a book to be that she hates she hates she hates flowing from her fingertips. A typewriter with ancient keys that keep catching. She doesn't care, she wants to hammer at something, hit and punch.
Here is where he finds her. Hours, days, minutes later. She doesn't know. It doesn't feel like it matters.
He won't come in, just stands in the doorway, his gaze away, far. Not with her.
(A thread unties.)
"Where are we?"
"The vortex."
"When. I meant…when." She digs the heels of her hands into her eyes. She feels as though she's fighting something back, she doesn't know what.
(She is tired.
And tired of…
It hurts.
Her hearts hurt.)
She is asking when they are. (She is asking where she sleeps. She is so tired.)
"We didn't check diaries earlier. I may have said some things I shouldn't." (One thing. Husband. Stupid stupid mistakes. Spoilers. She hopes she hasn't spoiled them.)
He looks at her, finally. "You didn't. Wife."
She exhales a breath from deep inside. She feels her lungs expand. Her husband is looking at his wife. It should matter more right now.
Nothing feels right now.
"I'm going to bed." The pages scatter as she stands. She lets them. Her parents' fate at her feet. She walks over them.
He lets her go.
(A ribbon unbinds.)
She tears off her (mourning) dress, the corset fasteners torn and shredded. It pools at her feet. (It will never be worn again.)
A shirt that might have once been his, or is hers now through custom, is buttoned messily, incorrectly, to cover her skin.
The lights dim, but do not extinguish. (The TARDIS never leaves her in the dark.)
She lies on her side of their bed, time drags across her body. The TARDIS tries to lull her to sleep, an ethereal hum to quiet her mind.
She stares at the ceiling.
A thousand years, a minute, a month later the bed dips with his weight. She doesn't know when he came in, when he removed his jacket, his bow tie.
A chasm between them.
"I'm not sad for me, I'm sad for you." (For now. For here. She doesn't know who she's trying to convince. The TARDIS murmurs.)
"They were your parents."
"They're still my parents. And they're together." (She is sad. And happy, in a way that hurts to feel it. They're together, going forward. She is jealous, too, a little. She stamps it down.)
She doesn't say like us. She doesn't feel it, not right now.
Not right now.
(Her left heart runs; pumps and races and flees. Her right heart loves; and it is maimed and shredded.)
Her patchwork heart is breaking, ripping along the seams she'd stitched together (where she'd sewn in Rory and Amy and him). Bleeding out with every beat. Her ribs feel sticky.
She feels like she's drowning inside herself. She coughs a laugh that startles him.
Rivers don't drown.
They swell and burst their banks. And destroy everything around them.
(She'll destroy.
Everything around her.
She'll destroy him.
She needs to run.)
"I told her to go. And I'd do it again. She had to take the risk, to be with him. Even if it didn't work, she had to try."
"She would have been safe if she stayed."
"No, my love. She wouldn't have been." (Safe, or Amy. Not if she hadn't tried. Not if she'd stayed.)
"You could have made her stay. You don't know that she would have gone if you'd…"
(She does she does she achingly does as she thinks of pyramids and universes that never were and ripping time apart just to yell love at the man beside her. She is her mother's daughter. Amy would have gone regardless. She will love her always for it.)
He's just talking now, making noises, rambling and saying things that he hasn't thought through, words and words and words. He pulls threads he doesn't realise have lost their tension. He undoes her by accident of misplaced attention.
"You could have gone with them." (He says could. She hears should. Her heart completes unravelling.)
She misses the crack in his voice. Her ears too full of misheard meanings. Her chest too full of jumbled pieces.
(He's saying could as his hearts break at the idea of it. He's already lost her once, back, at the start. What if she'd gone with them?
He's missing the point.
She always chooses him.
She would have jumped.)
She feels the adrenaline start to surge in her veins. She stands as he reaches for her, a foot away from the bed before he realises she is moving. (The TARDIS moans.)
"River," his voice is startled and afraid. "You're crying."
"No," she denies because she doesn't believe it is true. She doesn't cry (in front of him.) She doesn't cry. (How can she be crying when her loving heart is dead? She feels nothing but the urge to run.) She doesn't cry (…and yet. Her face is wet.)
"No." She backs away. He cannot he cannot he cannot see this. She won't permit it.
River runs.
He is fast and desperate but not enough to catch her.
He lets her go.
To beat her to console room.
A stalemate now. A standoff. He is almost blocking her way. Almost.
Her fingers twitch.
She darts as quickly as she can to reach the doors, the latch cold and secure beneath her fingers.
The TARDIS holds her safe and will not open the doors as she rattles at the handle, whispered supplications of please and open and please again, oh please. He turns her around, softly, gently. She tries to turn her face away, the tears still streaking. His forehead rests against her temple, his hands gripping her wrists.
"Let me go." (The TARDIS howls.)
"Never, River. Never," he wraps the words and his arms around her.
A decade, a second, a millennium passes. She stills and eases and acquiesces. Her mother is lost to her now, gone gone forever gone. Her father. (Rory who had grounded her on 17 occasions. Rory who kept her favourite tea by the kettle. Rory who, without question or comment, held her hand on days when she showed up silent and solemn.)
"They're gone." The reality of it empties her out, she sags against him (and he against her.)
"You're not." His voice is hot against her cheek. (You're here, with me. Don't leave.)
She feels his mind reaching for hers, she flinches and he stops. (He's so haphazard with words, too little too much too wrong when he's trying too hard to be right. But…she knows how he feels about this, about her, about them.)
He reaches for her again. She lets him in.
Her senses explode. The pain, the piercing in her (right) heart as he tries to stitch it back together. He intertwines their lives into the muscle, threads it with his love for her. With the grief too, that they're both suffering. She can feel him sewing her back up, stemming the bleeding, healing her (and them) as best he can.
She pushes him back softly, reclaiming herself and space. She smiles at him and it is real and true and trimmed with a sadness that will take time to pass. It is mirrored on the man smiling back.
"You're not wearing any shoes."
She glances down and laughs "Or trousers, dear." (The TARDIS croons.)
He crooks an eyebrow at her, half a shrug and guilty grin. She kisses him.
She doesn't say I love you (nor does he). It doesn't need saying, it is being felt too keenly.
The latch behind her unclicks. She can leave if she chooses.
She doesn't.


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Sir Winston Churchill's Marlborough: His Life and Times
- Alexis de Tocqueville's Democracy in America
Tocqueville's magnum opus, Democracy in America, made me a conservative. His profound analysis of liberty and equality in the American polity profoundly affected my views of liberty and equality. Likewise, his detailed discussion as to how history forms a nation's politics drifted my thinking away from abstract theories to more real historical realities. Truly a book of political science that all conservatives should be well acquainted with.
- Edmund Burke's Reflections on the Revolution in France
The book that begun a movement! And what prose! Such profound analysis of the effects of ideas! Never has ever such an elegant writer ever used his pen to such effect to denounce idealism and tyranny. Burke poses timeless conservative themes which, as history has proven time and time again, are as true as gravity. Burke does away with all social contracts or any other innate redundancies. Instead, Burke believes that we are the inheritors of a grand order, one of which if we deviate from brings chaos, ruin, and catastrophe. Sadly for some, he's right, as most of the time he is.
- Thomas Hobbes Leviathan
- Lord Macaulay's History of England


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Bank Of The Bahamas Limited
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Saturday, December 12 2015
Bay Ridge Quotes
John Morrison, winning trainer of Saythreehailmary's (No. 4): "She was coming into this race well and I knew she was going to run well and everything worked out. Jose gave her a perfect trip and I'm just happy about the results. We'll see how she comes out of this before we decide where to go next but we were looking at the Winter Showcase Day in February as a possibility. She's honest, she gives you everything she's got every time."
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EE MUSIC: European Energy Use Benchmarks for the Music Event Industry
EE MUSIC Greenhouse Gas Emissions
Another round-up of energy-saving top tips and tricks from EE MUSIC\'s travels across Europe.
News from Powerful Thinking
In his report EE MUSIC Ambassador Neven Prišuta gives an overview about the status quo in the Croatian music scene.
Introducing Richard Fletcher of De Montfort University, talking about festival audience engagement project Face Your Elephant\'s work with young engineers, and sustainable energy management at festivals.
Guest Blog: Introducing Elin Trogen of FIFTI (Fight It From the Inside), EE MUSIC Ambassador for Sweden, on sustainability in the Swedish events industry.
Energy efficient equipment - from lights to fridges.
The events implemented in the context of EE MUSIC have been a great success. About 1.000 visitors have been reached via Stakeholder Mobilisation Events such as EE MUSIC Launches and EE MUSIC Workshops.
Diana Simpson Hernandez, RCA product design graduate, talks about generating clean energy through music and design.
Sharing Tricks of the Trade: Top Tips to Save Energy from across Europe
Reporting from the PLASA Show 2014
EE MUSIC analysed the data submitted to the EE MUSIC IG Tools by users from across Europe to come up with some new energy use benchmarks for the European music event industry.
Average electricity use per m2 per year: 124 kWh/m2/year
Average gas use per m2 per year: 78 kWh/m2/year for gas
Only complete and robust data that included floor area and energy use over a 12 month period was included in the analysis.
The EE MUSIC benchmarks are in contrast to the previously existing Julie's Bicycle 'Performing Arts' benchmark based on data from UK performing arts venues – where gas consumption is higher than electricity. This benchmark is based on 3 years of data from around 300 theatres and concert halls in the UK.
Possible explanations for electricity use being higher than gas use for so many of the building groups in the EE MUSIC data set are:
We recommend that venues and clubs continue monitoring and reporting on their energy use so benchmarks can be further refined and country-specific benchmarks calculated for individual countries and circumstances.
Energy use per audience day: 0.47 L per audience day*
Average biodiesel consumption: 17%
This benchmark is calculated on the basis of 'audience days' (per person per day on site), a measure based on ticket sales. For example, for a two-day festival, the number of audience days would be: (number of one-day tickets sold for Day 1) + (number of one-day tickets sold for Day 2) + ((number of 2-Day tickets sold) x 2 ). Only complete and robust data that included audience day figures and energy use from festivals that were either completely or predominantly powered by diesel-powered generators were included in the analysis. The benchmark is based on data from festivals across a variety of sizes and includes all on-site diesel use (including concessions/traders where figures are available).
Average diesel consumption is lower than the existing Julie's Bicycle benchmark for outdoor events, which is based predominantly on UK festivals. This may be due to:
The share of biodiesel is also slightly higher among the EE MUSIC cohort. As with the UK benchmark however, this should not be extrapolated to the whole industry as events engaging with EE MUSIC are likely to have a higher share of biodiesel than the industry norm.
Lastly, it was possible to obtain preliminary diesel consumption benchmarks for the NL/DE country cluster and the UK/IE country cluster, which support the theory that overall fuel efficiency and/or mains grid use is more widespread outside the UK:
Netherlands & Germany: 0.46 L / audience day
UK & Ireland: 0.56 L / audience day
Aggregating the festival data sets in a meaningful way for benchmarking has been extremely challenging, as some festivals are powered almost entirely by diesel supplemented by only a little mains grid electricity, while others are powered predominantly by mains electricity supplemented with only a little diesel generator power. Their respective consumption of mains electricity or diesel is therefore not directly comparable, as the energy infrastructure is significantly different. For future development, we would recommend consulting with the industry to come up with a methodology under which festivals monitor their energy use in kWh whatever the energy source – however, there are numerous difficulties associated with this, not least the fact that generator efficiency varies widely so that converting diesel usage into kWh relies on numerous assumptions. More in-built data monitoring functionality and services will help resolve this issue in coming years, as more direct data on load and power draw will be available at source. However, the industry and its service providers will need to develop the capacity to process and analyse this data in a meaningful way. Companies such as Watt-Now in the Netherlands are starting to address this, providing a package of off-grid monitoring and management.


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Published on Amazon? If you have a book, e-book, or audiobook available on Amazon.com, we'll promote it on WritingForums.org for free. Simply add your book to our Member Publications section. Add your book here or read the full announcement.Dismiss Notice
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Odd Feelings
Published by Speedy in the blog Speedy's Blog Central. Views: 161
Well tomorrow or the day after im going to the doctors to tell them about how i have been feelings. Im not sure how long it takes, but i'm going to Anti-depressantway (though previously no a fan of the idea until lately).
Im kind of scared. I mean doctors come across this daily but i actualy feel relly sick deep inside and an *sigh* depressed.... but i just dont know how to say it to the doctor. i mean have the signs, trying to throwaway my girlfriend of 10 years, telling my family to get lost and sitting in the bath cutting my arms, punching walls, breaking mobile phones and everything else thats mine but as much as talking doesnt bother me, its just the first sentence when the doctor asks, so why are you here that bothers me. I just fear saying to him or her "Im dperessed" without thinking what they are thinking (I mean i dont care but id rather stay in my room all weekend than be judge "Oh he thinks his depressed" for that awkward period
The gitlfriend wants to come with me, i would like that but i've told her to stay at home.
If i take this route i wonder how i will feel in a week. I wonder if unlike now i will feel anything or if i will still feel nothing, but i can cope a little either.
I feel excited, if i felt something.
I hate doctors and getting on my knees, but F#@! it.
Updates later.
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"Lost My Name,a series of personalized children's books that spark imagination and creativity for the whole family.Using technology and original artwork, Lost My Name allows you to create one-of-a-kind stories, based on a child's name and location that incorporates personalized illustrations and text. No two stories are alike, making this a truly unique gift for kids… especially for those children who have more unique names!"
The Little Boy or Girl Who Lost Their Name
We all have that friend or family member that can NEVER find their name on anything. My granddaughter is one of those unfortunate kids. JayLynn – yes the L is capital and that is what throws everything off. She was so happy when she got this book and saw HER name in it.
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Dedication – And they will let you add a personal message in the front of the book ( at no extra charge ). This lets you mark a special event, such as Christmas or birthday.
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Characters – Well the characters in her book were her three brothers! And she ( and they ) loved that part. So she was able to have an adventure with her brothers!


text: 0 Coil Master Kbag $8.54 Posted on December 5, 2016 by cheapvapingdeals Total Views Today: 1 Total Views: 1,094 Tweet Email Tweet Email From Coil Master comes the Kbag. This carrying case can easily fit your favorite mod, back up mod, atomizers and juice for the day without being bulky and cumbersome. $8.54 Use code YEAHBABY at checkout You Will Receive: 1x – PU/EVA ZIP Coil Master Kbag (13"x 8.3"x 1.9") Related


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Thread: Is the search feature broken?
06-02-04, 15:36 #1Registered User
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Unanswered: Is the search feature broken?
It seems the search forum freature is not working. I every search I do comes up with nothing even when I see the word in the first few posts. It would be a great help if this worked. Also it seems that sometimes I make a post and it dissappears. At first I thought I was loosing my mind and well I still do.
06-02-04, 15:40 #2Registered User
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yes it is broken. It sucks because searching is a very useful feature. Hopefully someone will fix it soon.


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WA8 8BS, Mersey Valley Golf and Country Club: environmental permit application advertisement
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The consultation period for this application has ended. You can view current licences on the Public Registers.
View the application submitted by Mersey Valley Golf and Country Club for St Michaels Golf Course, Widnes.


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- Weight capacity 250 lbs
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Safed Candles Orb Candle with Blue, Red and Pink Stripes
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Safed Candles Orb Candle with Blue, Red and Pink Stripes
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This item is a great decorative piece that will brighten your home!


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Wokingham, United Kingdom (PressExposure) September 03, 2014 -- Park House School, a Computer Science Centre of Excellence has just signed a three-year contract with local ICT provider Ntegra. Ntegra will provide the school with a fully managed ICT service to meet the school's increasing needs for enterprise quality ICT.
Park House School felt it was important to work with an ICT partner that would embrace the school's ethos of innovation and creativity that led to them becoming the Centre of Excellence for Computer Science in the South of England and are excited to have found that in locally based Ntegra.
Head Teacher Derek Peaple said "ICT is playing an increasingly important role in both curriculum delivery and the operation of the school. It was vital that we selected a partner who understands that role and that is willing to develop the ICT services in partnership with the school".
David Marson, Chair of Governors at Park House School said, "Ntegra is a forward thinking local company. This three year contract will enable the school to upgrade its ICT provision in cost effective manner, and to continue to innovate in a way that enhances the learning experience for students for example by enabling effective and managed use of students own devices."
Andy Davies, Ntegra's CEO said, "We are delighted to be working in partnership with Park House School and the team. Education is one of our core markets and we are really excited about providing the school with a high quality ICT solution that will make a real difference to students, teachers and administration staff."
Ntegra have plans to expand their school IT support and other services, and are seeking opportunities to extend their services to both local and secondary schools. Andy Davies goes on to say, 'We are progressing discussion with a number of secondary and primary schools in West Berkshire and Hampshire. Our service is built on the quality and expertise that we offer to our private sector clients in a flexible and affordable manner.'
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We went on our usual run at the park late this afternoon despite the headache I had suffered since after lunch. On our second round, we noticed this adorable white dog with brown patches tied to a bench. She looked like a cross between a spitz and a corgi. A girl in rollerblades was playing with her for a bit and and then left her alone. We figured the owner was just jogging nearby.
My headache turned into a dull throbbing that I had to sit down. I secretly also wanted to be near the dog. She was so well-behaved, just sitting next to the bench and looking at passers-by. It was hard not to notice her.
My husband and I took turns having her get to know us by smelling our hands and then we sat at the bench across from where she was so that we can keep an eye on her. A woman approached us and said that the dog had been tied to that bench for about 30 minutes already. She had witnessed how its human had put her there and left in a hurry. She was concerned that the dog was deliberately left in the park for good so she alerted the park security.
About 10 minutes after we sat across from her, she started howling in a low tone and giving out short barks. She was starting to feel lonely. My chest kept tightening by the second and my eyes were starting to well up. I walked over to her and she immediately started to lick my hands and the exposed parts of my legs. She had me right there and I was ready to bring her home.
Of course the husband had to bring me back to earth and remind me that we had two other dogs, a male lab and a young, female beagle, who might not welcome a new addition to the family. Our territorial persian cat was another thing. Still, he too couldn't help coming with a name for our new friend.
As we were discussing her fate, a young woman who had also been running approached and asked about the dog. She was willing to take her in despite the fact that she was only renting a room nearby. It was clear she fell in love with our new furry friend. We were just waiting for the park security to come by and take note of the girl's contact number in case the owner came back looking for the dog. The girl was beginning to get very excited, and so was her husband who was training nearby for the national muay thai meet. About an hour after we discovered the dog, a middle-aged woman and someone who was clearly her helper came running towards us. The woman was muttering, explaining to no one in particular that she was buying fish and lost track of time. They took the dog and hurriedly walked away from us.
My husband had a few conclusions as to what happened. He thought he heard the woman scolding the helper. I was a little put off that the owner did not even acknowledge us, nor thank us for keeping her dog company. The concerned woman, whose name we later found out was Astrid, was a little ticked off as well.
As we were walking home, I realized that my headache was gone and in its place was a hint of a heartache. I wish we had our phones with us so we could have taken her photo. We were going to name her Dani, from Daenarys of Game of Thrones. 🙂


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It will challenge and change you in all the best ways, and I am super honored to have her here today, sharing a bit of her lovely words about identity and living out loud who you really, really are! (Because hey... who among us doesn't forget THAT sometimes, too? We will dive in to Identity a little later this month, but today, I am beside-myself excited to introduce you to The Life Artist, Erika Morrison:
(So good, right? You will fall in love with her, too! I just know it!)
One of the most powerful Truths that I think we all so often forget is the importance of knowing - truly knowing - who we are... and then being her (or him) out loud and with joy!
We know the truth of how we were intricately created and fashioned by God... we want to know and believe the truth that we are special...
...It's just that Sometimes We Forget!
As if Erika's words alone were not beautiful enough... there is this breathtaking Book Trailer to enjoy too!
Bandersnatch (Full Length Trailer) from Melissa Kane on Vimeo.
Today is Day SIX of 31 Days of Truths We Know...! If you missed the kick-off and official 'Home page' for this series, you can go HERE and catch up! You will find links for each days' postings, added daily throughout the month. If you'd prefer to have them delivered right to you, I invite you to enter your email address to the right ('Subscribe Here') at the top of this page and subscribe to the blog. That way, once a week, each post is delivered to your inbox in a single email over the weekend and you'll be sure not to miss a thing!


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Refrigerant level transducer probes for use in industrial and commercial refrigeration systems
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French Fields: Complete Series 3 DVD
Starring Anton Rodgers, Julia McKenzie, Pamela Salem, Philip Bird, Karen Ascoe, Zulema Dene, Philip McGough, Eric Dodson, Nicholas Courtney, Frank Lazarus
All six episodes from the third series of the ITV sitcom starring Anton Rodgers and Julia McKenzie.
After having followed their son to live in France, William and Hester Fields discover it's not as easy as they expected adapting to the changes, especially when they have an elderly housekeeper they can't understand.
Episodes comprise: 'French with Tears', 'The Merry, Merry Pipes', 'Make for the Hills', 'Surprise, Surprise', 'Darling Daughters' and 'Hail and Farewell'.
- Format: DVD
- Region: 2
- Certification: (PG)
- Catalogue number: 7953579
- Studio: Network
- Release date: 19/09/2011
- Genre: Comedy
- Colour: Colour
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