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A potent and highly specific insecticidal toxin comprising 45 residues and three intramolecular disulfide bonds, as well as a method for selecting potent inhibitors of insect calcium channels.
1. A polypeptide which binds calcium channels of invertebrate pests which is substantially non-toxic against vertebrates, comprising a polypeptide isolated from Hydronyche versuta having a molecular mass of approximately 4,400 to 4,500 Daltons, and comprising about 45 amino acids.
2. The polypeptide of
This application is a Divisional of Ser. No. 09/780,874, filed Feb. 9, 2001, now U.S. Pat. No. 6,583,264 which claims priority to U.S. provisional application 60/181,532, filed Feb. 10, 2000, the entire contents of which are incorporated by reference herein.
The present invention relates to biological compounds, and genes encoding biological compounds, for use as pesticides, as well as methods for obtaining such compounds.
Unfortunately, it has increasingly been seen over the past several decades that employment of conventional chemical insecticides often leads to undesirable environmental consequences. Such consequences include toxicity to non-target organisms such as birds and fish, and human health hazards. Furthermore, pesticide management in the United States and elsewhere in the world is becoming increasingly complicated due to the evolution of insect resistance to classical chemical pesticides. Despite over 10 billion dollars being spent each year to control phytophagus insects, global losses in the food supply due to insects is still estimated to be about 20 to 30 percent (See, Oerke, Estimated crop losses due to pathogens, animal pests and weeds, 72–78 in Crop, production and crop protection: Estimated losses in major food and cash crops (Elsevier, Amsterdam 1994)). There remains, therefore, an urgent need to develop or obtain substances that can be used safely in the fight against insect pests.
Over the past several years, there have been proposed a number of "environmentally friendly" strategies to combat highly resistant insect pests such as certain species of cotton bollworm (e.g., Helicoverpa zea).
One recently introduced approach to insect management is the production of transgenic crops that express insecticidal toxins, such as engineered potato, and cotton crops that express protein toxins from the soil bacterium Bacillus thuringiensis (Estruch, J. J. et al., Transgenic plants: An emerging approach to pest control, Nature Biotechnology 15, 137–141, 1997).
A variation of this strategy is the release of insect-specific viruses that have been genetically engineered to express insecticidal neurotoxins (Cory, J. S. et al., Field trial of a genetically improved baculovirus insecticide, Nature 370, 138–140, 1994). Baculoviruses, for example, are arthropod-specific viruses with no member of the baculovirus family known to infect either vertebrates or plants. The infectivity of some baculoviruses is restricted to a few closely related species within a single family of lepidopterous insects (moths and butterflies) (See, e.g., U.S. Pat. No. 5,639,454). Some baculoviruses, such as the beet armyworm nuclear polyhedrosis virus, target only a single species. As a result of their high degree of specificity, baculoviruses have long been envisaged as potential pest control agents and were first used as such in the 1970s. Their specificity means that baculoviral insecticides complement natural predators, rather than replacing them, as is the case with many chemical insecticides. However, to date, baculoviruses have met with only limited commercial success. Most naturally occurring baculoviruses take 4–7 days to kill their hosts, with some species taking considerably longer. During this time the insect continues to feed and cause crop damage, thus limiting the ability of baculoviral insecticides to compete with chemical agents.
This shortcoming has been addressed by engineering recombinant baculoviruses that express insect-specific neurotoxins. Expression of heterologous insect toxins not only reduces the time interval between virus application and insect death, but also reduces the mean feeding time (Prikhod'ko et al., Effects of simultaneous expression of two sodium channel toxin genes on the properties of baculoviruses as biopesticides, Biological Control 12, 66–78, 1998). Importantly, introduction of genes for insect-selective toxins does not alter the intrinsic infectivity of the baculovirus or its natural host range (Black et al., Commercialization of baculoviral insecticides, in The Baculoviruses (ed. Miller, L. K.) 341–387 (Plenum Press, New York, USA, 1997)).
New approaches to insect-pest management have stimulated interest in peptide toxins from the venoms of animals, particularly spiders and scorpions, that prey on insect species.
Zlotkin et al., An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site, Arch. Biochem. and Biophysics 240, 877–887, 1985), disclose two insect selective toxins from the venom of the scorpion Leiurus quinqestriatus, one of which induced fast excitatory contractive paralysis of fly larvae while the other induced slow depressant flaccid paralysis, with both affecting sodium conductance in the neurons. Likewise, Canadian patent 2,005,658 (issued: Jun. 19, 1990 to Zlotkin et al.) discloses an insecticidally effective protein referred to as "LqhP35" derived from the scorpion Leiurus quinquestriatus hebraeus.
A number of investigators have also recognized spider venoms as a possible source of insect-specific toxins for agricultural applications (See, Jackson et al., Ann. Rev. Neurosci. 12, 405–414 (1989)). For example, U.S. Pat. No. 4,855,405 (issued: Aug. 8, 1989 to Yoshioka et al.) and U.S. Pat. No. 4,918,107 (issued: Apr. 17, 1990 to Nakajima et al.) both disclose glutamate-receptor inhibitors obtained from the venom of spiders as possible insecticidal agents. In U.S. Pat. No. 5,457,178 (issued: Oct. 10, 1995), U.S. Pat. No. 5,695,959 (issued: Dec. 9, 1997), and U.S. Pat. No. 5,756,459 (issued: May 26, 1998), Jackson et al. disclose a family of insecticidally effective proteins isolated from the venom of the spiders Filistata hibernalis (a common house spider) and Phidippus audax (a "jumping spider").
A particular group of spiders which has generated considerable investigative interest are the funnel-web spiders. WO 89/07608 (published: Aug. 24, 1989, Cherksey et al.) discloses low molecular weight factors isolated from American funnel-web spider venoms which reversibly bind to calcium channels. Adams et al., Isolation and Biological Activity of Synaptic Toxins from the Venom of the Funnel Web Spider, Agelenopsis aperta, in Insect Neurochemistry and Neurophysiology, Borkovec and Gelman (eds.) (Humana Press, New Jersey, 1986) teaches that multiple peptide toxins which antagonize synaptic transmission in insects have been isolated from the spider Agelenopsis aperta. In WO 93/15108, a class of peptide toxins known as the ω-atracotoxins are disclosed as being isolated from the Australian funnel-web spiders (Araneae:Hexathelidae:Atracinae) by screening the venom for anti-Helicoverpa ("anti-cotton bollworm") activity. Such toxins are disclosed to have a molecular weight of approximately 4000 amu, to be of 36–37 amino acids in length, and capable of forming three intrachain disulfide bridges. One of these compounds, designated ω-ACTX-Hv1 has been shown to selectively inhibit insect, as opposed to mammalian, voltage-gated calcium channel currents (Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-atracotoxin-Hv1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559–566 (1997)). Homologues of ω-ACTX-Hv1 have been isolated from the Blue Mountain funnel-web spider Hadronyche versuta (See, Wang et al., Structure-function of ω-atrocotoxin, a potent antagonist of insect voltage-gated calcium channels, Eur. J. Biochem. 264, 488–494 (1999)).
While some insecticidal peptide toxins isolated so far from scorpions and spiders offer promise, there still remains a significant need for compounds which display a wide differential in toxicity between insects, and non-insects, and yet which have significant insecticidal activity and a quick action.
The present inventors have isolated, and structurally and functionally characterized, a novel insecticidal toxin, designated ω-atracotoxin-Hv2a, from the venom of the Australian funnel-web spider H. versuta. This toxin is a highly potent and specific antagonist of insect calcium channels. The toxin of the present invention shows no significant sequence similarity to any previously isolated insecticidal toxins, and it shows no sequence or structural homology with the omega-atracotoxin-Hv1 family of insecticidal toxins previously isolated from H. versuta (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559–566 (1997); Wang et al., Structure-function studies of ω-atracotoxin, a potent antagonist of insect voltage-gated calcium channels, Eur. J. Biochem. 264, 488–494 (1999)).
The present invention teaches the use of ω-atracotoxin-Hv2a, or the gene coding for the toxin, as a biopesticide, either alone or in combination with other insecticidal toxins or genes thereof. It further teaches the use of the toxin, or the gene coding for the toxin, as a screen for natural or non-natural compounds that specifically inhibit insect calcium channels. Furthermore, the present invention provides in the determination of the toxin's three-dimensional structure, a model for developing non-peptidic mimics of the toxin that could be used as foliar pesticide sprays.
In a first embodiment of the present invention, there is provided a polypeptide toxin that is toxic to adult and/or larval insects having a molecular mass of approximately 4,478 Daltons and a length of 45 amino acid residues. The polypeptide is capable of forming three intrachain disulfide bonds. Activity may be demonstrated by rapid paralysis of insects and/or potent inhibition of whole-cell calcium currents in isolated insect neurons. Phylogenetic specificity may typically be demonstrated by minimal activity in rat or chicken nerve-muscle preparations and/or minimal antagonism of calcium channel currents in isolated rat neurons.
The preferred toxin of the present invention is omega-atracotoxin-Hv2a (SEQ ID NO:1), abbreviated as omega-ACTX-Hv2a or ω-ACTX-Hv2a, as defined herein:
The toxins of the present invention may be isolated from spider venom or chemically synthesized and oxidized/folded using similar techniques to those described previously for production of synthetic omega-atracotoxin-Hv1a (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559–566 (1997), both of which are incorporated by reference in their entirety herein). The toxin could also be prepared from a synthetically constructed gene using recombinant DNA techniques as the authors have described previously for an unrelated protein (Riley et al., Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain, Eur. J. Biochem. 219, 877–886 (1994) which is incorporated in its entirety herein). A DNA probe coding for the amino sequence of the toxin may be used to isolate the gene coding for the protein or the corresponding preprotein or preproprotein using standard molecular biological techniques. The natural or synthetic gene(s) may be inserted into appropriate overexpression vectors for production of the toxin. In particular, the gene for the protein, preprotein, or preproprotein may be inserted into the genome of an appropriate insect vector, such as a baculovirus. Alternatively, transgenic plants may be constructed that express the toxin or the preprotein or preproprotein form of the toxin. Thus, the invention also provides insect viruses and plant species engineered to express the toxins of this invention.
In another embodiment of the present invention, there is provided variants of ω-ACTX-Hv2a, wherein a "variant" is defined as a polypeptide that corresponds to or comprises a portion of ω-ACTX-Hv2a, or is homologous to ω-ACTX-Hv2a. For the purposes of this invention, "homology" between two peptide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second.
In particular, a polypeptide is "homologous" to ω-ACTX-Hv2a if a comparison of their amino acid sequences reveals an identity greater than about 30% (which is usually sufficient to indicate structural homology). Such a sequence comparison can be performed via numerous computer algorithms in the public domain.
In yet another embodiment of the present invention, there is provided a method of screening for, or designing, an antagonist of insect calcium channels. This method involves selecting or designing a substance which inhibits the binding of ω-ACTX-Hv2a, or a variant thereof, to insect calcium channels and testing the ability of the substance to act as an antagonist of insect calcium channels. The term "insect calcium channel" refers to any insect calcium channel that is inhibited by ω-ACTX-Hv2a.
There is also provided by the present invention, a method of screening for substances for insecticidal potency and phylogenetic specificity, the method comprising: (a) measuring the ability of a substance to inhibit the binding of ω-ACTX-Hv2a, or a variant thereof, to insect calcium channels; (b) measuring the ability of the substance to antagonize insect calcium channels; and (c) determining whether the substance has minimal activity against vertebrate calcium channels. Preferably the substance isolated by use of such method has high phylogenetic specificity being defined herein as greater than 100-fold selectivity for insect over vertebrate calcium channels, and preferably greater than 1000-fold selectivity for insect over vertebrate calcium channels.
According to yet another embodiment of the present invention there is provided an insecticidal composition for delivering ω-ACTX-Hv2a, a variant thereof, or an inhibitor of insect calcium channels, discerned by the methods described above. For example, where the toxin, variant, or calcium channel antagonist can be expressed by an insect virus, the virus encoding the toxin, variant, or calcium channel antagonist can be applied to the crop to be protected. The virus may be engineered to express ω-ACTX-Hv2a, a ω-ACTX-Hv2a variant, or one of the calcium channel inhibitors either alone, in combination with one another, or in combination with other insecticidal polypeptide toxins that may result in synergistic insecticidal activity. The virus may be formulated in an agriculturally acceptable carrier, diluent and/or excipient. Suitable viruses include, but are not limited to, baculoviruses.
Alternatively, the crop itself may be engineered to express ω-ACTX-Hv2a, a ω-ACTX-Hv2a variant, or a calcium channel antagonist, discerned by the above described methods, either alone, in combination, or in combination with other insecticidal polypeptide toxins that may result in synergistic insecticidal activity. Crops for which this approach would be useful include cotton, tomato, green bean, sweet corn, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower, and field lupins.
Alternatively, the insecticidal agent may be delivered directly to the crop in an agriculturally acceptable carrier, diluent and/or excipient. Delivery could, for example, be in the form of a foliar spray. Insect infestation of crops may be controlled by treating the crops and/or insects with such compositions. The insects and/or their larvae may be treated with the composition, for example, by attracting the insects to the composition with an attractant.
There is disclosed an extremely potent and specific polypeptide-antagonist of insect calcium channels identified by SEQ ID NO:1 and referenced herein as "omega-ACTX-Hv2a" or "ω-ACTX-Hv2a". Such antagonist consists of forty-five amino acid residues, has a molecular mass of approximately 4,478 Daltons, and is capable of forming three intrachain disulfide bonds. Such antagonist has been found to be toxic to both adult and/or larval insects and to display more than 50,000-fold specificity for invertebrate over vertebrate voltage-gated calcium channels.
SEQ ID NO:1 may be derived by chemically synthesizing the same and oxidizing/folding the peptide using similar techniques to those described previously for production of synthetic omega-atracotoxin-Hv1a (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, ω- atracotoxin-HV1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559–566 (1997), both of which are incorporated by reference in their entirety herein). The polypeptide antagonist may also be derived by isolation from spider venom, in particular the venom of Hadronyche versuta and other Australian funnel-web spiders of the genera Hadronyche and Atrax.
The polypeptide antagonist SEQ ID NO: 1 may also be derived by constructing a synthetic gene coding for the polypeptide (e.g., based on computer-based back-translation), cloning the gene into an appropriate vector, transforming a cell line with the vector, causing the polypeptide to be expressed, and purifying the polypeptide. Expression systems may contain control sequences, such as promoters, enhancers and termination controls such as are known in the art for a variety of hosts (See, e.g, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Press (1989) which is incorporated herein in its entirety). The expression systems may also contain signal peptide and proprotein sequences that facilitate expression of the toxin gene and/or folding of the toxin.
The polypeptide toxins of the present invention may be prepared using recombinant DNA techniques such as described in Riley et al., Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain, Eur. J. Biochem 219, 817–886 (1994) (such reference being incorporated by reference in its entirely herein) which was authored by certain of the present inventors.
The polypeptide toxins of the present invention may be prepared in both prokaryotic and eukaryotic systems. Constructs may be made wherein the coding sequence for the polypeptide is preceded by an operable signal peptide which results in secretion of the protein. The particulars for construction of expression systems and purification of peptides, and cleavage from fusion peptides are well known to those of ordinary skill in the art. Technology for introduction of DNA into cells includes four general methods: (1) physical methods such as microinjection, electroporation and the gene gun (See, e.g., Johnston et al., Gene gun transfection of animal cells and genetic immunization, Methods Cell. Biol. 43(A), 353–365 (1994)); (2) viral vectors (See, e.g., Eglitis et al., Retroviral vectors for introduction of genes into mammalian cells, Biotechniques 6(7), 608–614 (1988)); (3) chemical methods (See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Vol. 1, Greene Publishing Associates/John Wiley & Sons (1993); Zatloukal et al., Transferrinfection: A highly efficient way to express gene constructs in eukaryotic cells, Ann. N.Y. Acad. Sci. 660, 136–153 (1992)), and (4) receptor-mediated mechanisms (See, e.g., Wagner et al., Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor mediated gene delivery and expression of transfected genes, Proc. Natl. Acad. Sci. USA 89(13), 6099–6103 (1992)). As would be understood by one of ordinary skill in the art, minor modification of the primary amino acid sequence of SEQ ID NO:1 may result in a polypeptide which has substantially equivalent or enhanced activity as compared to SEQ ID NO:1. By "modification" of the primary amino acid sequence it is meant to include "deletions" (that is, polypeptides in which one or more amino acid residues are absent), "additions" (that is, a polypeptide which has one or more additional amino acid residues as compared to the specified polypeptide), "substitutions" (that is, a polypeptide which results from the replacement of one or more amino acid residues), and "fragments" (that is, a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the specified polypeptide). By "modification" it is also meant to include polypeptides that are altered as a result of post-translational events which change, for example, the glycosylation, amidation, lipidation pattern, or the primary, secondary, or tertiary structure of the polypeptide.
Preferred "substitutions" are those that are conservative, ie., wherein the residue is replaced by another of the same general type. In making changes, the hydropathic index of amino acids may be considered (See, e.g., Kyte. et al., J. Mol. Biol. 157, 105–132 (1982), herein incorporated by reference in its entirety). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide having similar biological activity. In making such changes, the substitution of amino acids whose hydropathic indices are within �2 is preferred, those that are within �1 are more preferred, and those within �0.5 are even more preferred. Similarly, select amino acids may be substituted by other amino acids having a similar hydrophilicity, as set forth in U.S. Pat. No. 4,554,101 (herein incorporated by reference in its entirety). In making such changes, as with the hydropathic indices, the substitution of amino acids whose hydrophilicity indices are within �2 is preferred, those that are within �1 are more preferred, and those within �0.5 are even more preferred.
Amino acid changes may be achieved by changing the codons of the DNA sequence making use, for example, of known redundancy in the code:
Preferably any variant is homologous to ω-ACTX-Hv2a. Sequence comparison can be performed via numerous computer algorithms that are well known to the skilled artisan. An homologous peptide may be produced in accordance with the present invention, for example, by conventional site-directed mutagenesis (which is one avenue for routinely identifying residues of the molecule that are functionally important or not), by random mutation (by "mutation" it is meant an alteration in the primary structure of the polypeptide due to changes in the nucleotide sequence of the DNA which encodes it), by chemical synthesis, or by chemical or enzymatic cleavage of ω-ACTX-Hv2a, and other techniques known to those of ordinary skill in the art.
Recombinant DNA technology can be used to produce a recombinant expression vector virus of the polypeptide antagonists of the present invention. For example, a baculovirus expression vector such as the type disclosed in U.S. Pat. No. 4,879,236 (which patent is incorporated by reference in its entirety herein) may be produced. Other publications describing a method for recombinant protein expression using baculovirus vectors include Tomalski, et al., Nature 352, 82–85 (1991), Stewart et al., Nature 352, 85–88 (1991) and McCutchen et al., Biotechnology 9, 848–851 (1991). The recombinant expression vector virus could be applied to the area where the insect is a pest. When the virus is ingested by the insect its replication will begin. During replication, the gene for the insecticidally effective protein is expressed, resulting in the disablement or death of the insect. The virus may express ω-ACTX-Hv2a, or variant thereof, or a calcium channel antagonist discovered by methods described herein. The virus could also be engineered to express ω-ACTX-Hv2a, a variant thereof, or such calcium channel antagonist in the various combinations possible with one another, and furthermore in combination with other insecticidal polypeptide toxins. Such combinations may result in synergistic insecticidal activity. Hybrid bacterial cells, comprising a plasmid with the gene coding for polypeptide antagonists of the present invention may likewise be used to control insects in conformity with the present invention.
Insect calcium channel antagonists, viral vectors, and hybrid bacterial cells of the present invention may be applied in the form of a foliar spray comprising an agriculturally acceptable carrier. Crops for which this approach would be useful are numerous, and include, without limitation, cotton, tomato, green bean, sweet corn, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower, and field lupins. Such agents may also be applied to insects directly.
As would be understood by one of ordinary skill in the art, plants may be produced that express the polypeptide antagonists of the present invention. By "transgenic plant" it is meant any plant, or progeny thereof, derived from a "transformed plant" cell or protoplast, wherein the plant DNA (nuclear or chloroplast) contains an introduced exogenous DNA molecule not originally present in a native, non-transgenic plant of the same strain. Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers et al., Methods in Enzymol. 153, 253–277 (1987)), and pCaMVCN transfer control vector (available from Pharmacia, Piscataway, N.J.). Of course, as would be understood by one of ordinary skill in the art, other means of gene introduction into the cell may also be employed, such as electroporation (Fromm et al., Expression of genes transferred into monocot and dicot plant cells by electroporation, Proc. Natl. Acad. Sci. USA 82(17),5824–5828 (1985)), polyethyleneglycol-mediated transformation of protoplasts (Ominrulleh et al., Plant Molecular Biology 21, 415–428 (1993)), desiccation/inhibition-mediated DNA uptake, agitation with silicon carbide fibers, by acceleration of DNA coated particles, injection into reproductive organs, and injection into immature embryos.
If an expression vector of the present invention is used to transform a plant, it is preferred that a promoter be selected that has the ability to drive expression in the plant. Promoters that function in plants are well known in the art. Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., Proc. Natl. Acad. Sci. USA 87, 4144–4148 (1990)), cauliflower mosaic virus (CaMV 35S) promoter, S-E9 small subunit RuBP carboxylase promoter, and corn heat shock protein (Odell et al., Nature 335, 810 (1985)). The choice of which expression vector, and ultimately to which promoter a polypeptide coding region is operatively linked, depends directly on the functional properties desired, for example, the location and timing of protein expression and the host cell to be transformed. In a preferred embodiment, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell. Transformation vectors used to transform plants and methods of making those vectors are described, for example, in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are incorporated in their entirety herein by reference.
The present invention also encompasses DNA sequences encoding for SEQ ID NO:1 and variants thereof. The DNA sequences encoding for such active polypeptide sequences allow for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to such gene sequences. The short nucleic acid sequences may be used as probes for detecting the presence of complementary sequences in a given sample, or may be used as primers to detect, amplify or mutate a defined segment of the DNA sequences encoding for SEQ ID NO:1, and variants thereof. A preferred nucleic acid sequence employed for hybridization studies is at least 14 nucleotides in length to ensure that the fragment is of sufficient length to form a stable and selective duplex molecule. Such fragments may be prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology (described in U.S. Pat. Nos. 4,683,195 and 4,683,202, herein incorporated in their entirety by reference), or by excising selected nucleic acid fragments form recombinant plasmids containing appropriate inserts and suitable restriction sites.
Improved methods for screening for and/or designing antagonists of insect calcium channels are also provided. Given the large difference between the binding constants of ω-ACTX-Hv2a with respect to invertebrate versus vertebrate calcium channels, in particular those calcium channels associated with the insect neuronal system, ω-ACTX-Hv2a may be effectively used in screening procedures to identify new antagonists of insect calcium channels. Using conventional structure-activity analysis of identified insect calcium channel antagonists, routes of chemical design of other more potent antagonists may be pursued based on the identified antagonists.
One method for isolating compounds having insect calcium channel antagonist activity comprises the steps of: preparing an invertebrate cellular preparation having a substantial number of unbound calcium channels; adding an amount of ω-ACTX-Hv2a effective to substantially bind all of the calcium channels of the invertebrate cellular preparation; selecting a test compound; adding the test compound to the invertebrate cellular preparation bound with ω-ACTX-Hv2a; measuring the amount of ω-ACTX-Hv2a released by the addition of the test compound.
Another method for isolating compounds having insect calcium channel antagonist activity comprises the steps of: preparing an invertebrate cellular preparation having a substantial number of unbound calcium channels; selecting a test compound; adding the test compound to the invertebrate cellular preparation at a set concentration; allowing the test compound to incubate for a period of time sufficient to allow the test compound to bind with the calcium channels of the preparation; washing the invertebrate cellular preparation which was incubated with the test compound so as to remove any unbound test compound; adding an amount of ω-ACTX-Hv2a sufficient to bind all of the calcium channels of the untreated invertebrate cellular preparation; measuring the amount of test compound displaced by the addition of the ω-ACTX-Hv2a.
Preferably, any antagonist identified by the screening procedure will bind strongly to insect calcium channel(s) such that the dissociation constant (Kd) for its interaction with the calcium channel(s) is less than 10−7 M and more preferably less than 10−9 M. Preferably, the activity of the test compound against vertebrate calcium channels is then determined so as to discern the relative selectivity of the compound. In a preferred embodiment, phylogenetic specificity is greater than 100-fold for insect over vertebrate calcium channels, and more preferably greater than 1000-fold. As would be recognized by one of ordinary skill in the art, other types of competitive assays and pharmacological activity screening procedures known in the art may be adapted to utilize ω-ACTX-Hv2a to provide for improved screening of compounds for invertebrate calcium channel antagonism.
Test compounds may comprise a compound from an archive of natural compounds or from combinatorial libraries of peptidic and non-peptidic compounds.
Libraries of mutated toxins for the purposes of screening may be obtained by in vitro evolution of a gene for ω-ACTX-Hv2a as described previously for unrelated proteins (Stemmer, DNA shuffling by random fragmentation and re-assembly; in vitro recombination for molecular evolution, Proc. Natl. Acad. Sci. 91, 10747–10751 (1994); Stemmer, Rapid evolution of a protein in vitro by DNA shuffling, Nature 370, 389–391 (1994); Zhang et al., Directed evolution of a fucosidase from a glactosidase by DNA shuffling and screening, Proc. Natl. Acad. Sci. USA 94, 4504–4509 (1997), all of which are incorporated by reference in their entirety herein). This could be done using error-prone PCR of the entire ω-ACTX-Hv2a gene or digestion of the ω-ACTX-Hv2a gene with an appropriate enzyme followed by error-prone PCR reconstruction of the entire gene sequence. These error-prone PCR procedures could also be applied to the complete preproprotein gene sequence for ω-ACTX-Hv2a. The library of mutant ω-ACTX-Hv2a gene sequences could then be used to generate a series of ω-ACTX-Hv2a variant antagonists. These antagonists may then be screened for their ability to inhibit the binding of ω-ACTX-Hv2a, or a variant thereof, to insect calcium channels, or directly for their ability to inhibit insect calcium channels. Screening may be performed, for example, by phage display of a mutant gene library followed by selection of phage particles that bind tightly to insect calcium channels, or phage particles that inhibit the binding of ω-ACTX-Hv2a, or a variant thereof, to insect calcium channels. As would be understood by one of ordinary skill in the art, a mutant gene library could also be constructed by other standard molecular biological methods such as oligonucleotide cassette mutagenesis or construction of synthetic genes with certain nucleotide positions randomised.
The three-dimensional structure of ω-ACTX-Hv2a, which has been elucidated by the present inventors as set forth below, may also be used to search structure libraries for (or to design) either peptidic or non-peptidic compounds that resemble the key structural elements of ω-ACTX-Hv2a, particularly those regions found to be critical for activity by mutagenesis/truncation/modification experiments. These compounds could then be tested for their ability to inhibit the binding of ω-ACTX-Hv2a, or a variant thereof, to insect calcium channels.
The ω-ACTX-Hv2a (or variant thereof) used in a competitive assay may be radioactive or fluorescently labeled, all of which fall within the scope of the present invention. Screening of test compounds may be performed using either native or recombinantly produced calcium channels, or structurally-modified calcium channels.
The present inventors have found that acutely isolated cockroach and bee brain neurons are particularly suitable to provide the basis for a sensitive electrophysiological assay for assaying substances that interfere with the ability of ω-ACTX-Hv2a to inhibit insect calcium channels, while a variety of mouse sensory ganglion neurons are suitable to provide the basis for a sensitive electrophysiological assay for testing the ability of compounds to inhibit vertebrate calcium channels. It will be appreciated, however, that other insect and vertebrate cells or cell lines would also be suitable for use in this aspect of the present invention. For example, transient expression of cloned insect calcium channels in suitable cell lines or oocytes could form the basis of a suitable assay system.
Now turning to several examples that illustrate particular compositions and methods within the scope of the present invention. Such examples, and the figures associated therewith, are presented in order to make certain aspects of the present invention more clearly understood, and are not intended to limit the scope of the invention as described herein in any manner.
Lyophilized crude venom (1.25 mg) was dissolved in 50 μl distilled water, loaded onto a Vydac C18 analytical reverse-phase HPLC column (4.6�250 mm, 5 μm pore size), and eluted at a flow rate of 1 ml min− using a gradient of 5–25% Buffer B (0.1% TFA in acetonitrile) over 22 min, followed by a gradient of 25–50% Buffer B over 48 min. Buffer A was 0.1% TFA in water.
Isolated ω-ACTX-Hv2a (50 μg) was reduced and pyridylethylated, then digested with Staphylococcus aureus strain V8 type XVII-B protease [EC 184.108.40.206] for 6 hours at a toxin:protease ratio of 100:1 and a temperature of 37� C. The reaction was carried out in 50 mM Tris buffer, pH 7.8. The resulting peptide fragments (labeled V1–V4) were applied to a Vydac C18 analytical reverse-phase HPLC column (4.6�250 mm, 5 μm pore size), and eluted at a flow rate of 1 ml min−1 using a gradient of 5–60% Buffer B (0.1% TFA in acetonitrile) over 40 min. Buffer A was 0.1% TFA in water.
The primary structure of ω-ACTX-Hv2a was reconstructed from N-terminal and C-terminal sequencing of the complete toxin as well as sequencing of various fragments obtained from digestion with V8 protease. The primary structure is shown in
The three-dimensional structure of ω-ACTX-Hv2a was determined using standard two-dimensional homonuclear nuclear magnetic resonance (NMR) spectroscopy techniques familiar to those skilled in the art (see, Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-ACTX-Hv1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559–566, 1997; W�thrich, NMR of Proteins and Nucleic Acids (John Wiley & Sons, Inc., New York, 1986), both of which are incorporated in their entirety herein).
Neurons were isolated from the brains of adult European honey bees, Apis melliferaa, as the authors have described previously (see, Wang et al., Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bond, Nature Struct. Biol. 7, 505–513, which is incorporated in its entirety herein). Standard whole cell voltage clamp recordings were made of bee brain calcium channel (ICa), sodium channel (INa) and potassium channel (IK) currents at ambient temperature (22–24� C.). For bee neurons, recordings were made with fire polished borosilicate pipets of ˜6 M resistance when filled with intracellular solution of either of the following compositions (mM): CsCl 120, NaCl 5, MgATP 5, Na2GTP 0.3, EGTA 10, CaCl2 2 and HEPES 10, pH 7.3 (for INa and ICa) or KF 130, EGTA 10, CaCl2 2 and HEPES 10, pH 7.3 for recording IK. For recordings of ICa and INa the external solution consisted of NaCl 135, tetraethylammonium chloride (TEACl) 20, CsCl 5, BaCl2 5, HEPES 10, glucose 10, BSA 0.05%, pH 7.3. For IK recording the external solution consisted of (mM) NaCl 130, KCl 20, CaCl2 2.5, MgCl2 1.5, HEPES 10, glucose 10, BSA 0.05%, pH 7.3.
Neurons were voltage clamped at −90 mV and currents evoked by stepping the membrane potential from −60 to +60 mV. Toxin effects on ICa and INa were tested at the potential with largest inward current, usually −10 or 0 mV. In bee neurons the peak inward currents were usually abolished by 100 μM Cd2+, suggesting that the current was largely carried by Ca2+ channels. In a few bee neurons there was a rapidly activating, transient and Cd2+-insensitive current which was blocked completely by tetrodotoxin (TTX, 1 μM).
Bee brain neurons were isolated as described in Example 4 above. Mouse trigeminal ganglion neurons were isolated by gentle trituration of minced ganglia following a 20-min treatment at 34 C with papain (20 units ml−1) in a HEPES buffered saline (HBS) solution of composition (in mM): NaCl 140, KCl 2.5, CaCl2 2.5, MgCl2 1.5, HEPES 10, glucose 10, pH 7.3. Standard whole cell voltage clamp recordings were made of bee brain calcium channel (ICa), sodium channel (INa) and potassium channel (IK) currents and mouse sensory neuron ICa and INa at ambient temperature (22–24 C). The same internal solution as described for the bee brain recordings in Example 4 was used for recordings of mouse sensory neuron ICa and INa; the electrodes had a resistance of 1–2 M. The ICa external solution for the mouse neuron recordings contained (mM): TEACl 140, CaCl2 2.5, CsCl 2.5, HEPES 10, glucose 10, BSA 0.05%, pH 7.3, while INa, were recorded in HBS.
Neurons were voltage clamped at −90 mV and currents evoked by stepping the membrane potential from −60 to +60 mV. In mouse sensory neurons, the peak inward currents evoked in the presence of potassium and sodium channel blockers were abolished by 30 μM Cd2+. The inward currents recorded in HBS consisted of both TTX-sensitive and TTX-resistant components. Toxin effects on bee brain IK were determined over a range of membrane potentials (from −40 to +60 mV). Data were collected and analysed as described previously (see, Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-ACTX-Hv1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559–566, 1997).
In striking contrast, superfusion of high concentrations of ω-ACTX-Hv2a (1 μM, n 10) for 5 min had virtually no effect on ICa in mouse sensory neurons (see unfilled circles in
It may be concluded that ω-ACTX-Hv2a is a potent and extremely specific antagonist of insect voltage-gated calcium channels. The toxin has no effect on potassium and sodium currents in either bee brain or mouse trigeminal neurons. Based on the data presented in
While the invention has been described with respect to certain embodiments, those skilled in the art will readily appreciate that various changes and/or modifications can be made to the invention without departing from the scope of the invention, and such changes and/or modifications are to be included within the spirit and purview of this application and the scope of the appended claims.
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About the program
After-school kids clubs are designed to focus on specific areas of learning. Youth leave the program with a set of new skills and knowledge, and with a desire to learn even more.
How do "clubs" work?
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Manufacturer:SOCOM Gear Airsoft
- Velocity: 310 fps (0.2 g BB) / Range 100-110 feet
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- Magazine Capacity: 26 Rounds
- Green Gas Model
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Socom Gear M9 Airsoft GBB Gas Pistol Manual
The SOCOM Gear Full Metal M9A1 SOF is an excellent new pistol that was just added to the SOCOM Gear line up. The slide is solidly constructed of metal with white etching on the side for a realistic action and look. The lower frame is also metal for a realistic feel and weight; and the frame features a weaver rail to mount lasers and flashlights. A 14mm CCW threaded adapter is included so you can attach the included barrel extension on for an even more special operations look; the adapter can be removed in the event that you prefer a classic military look. The grips are textured panels for comfortable feel with or without gloves.
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by News Editor
getAbstract provides executives worldwide with the best in business knowledge. The subscription service now offers summaries of TED Talks; each summary incorporates hyperlinks to the exact section of the video where the speaker discusses the linked information.
by News Editor
Multimedia localization is more complex than other translation projects, and requires experience to craft a translation that fits the timing of the original production. SpokenHere has teamed up with HelpFlix to create easy-to-understand product-assembly demonstration videos for non-English-speaking viewers.
by David Kelly
Since it was introduced last year, Articulate Storyline has become one of the most popular eLearning rapid- development tools on the market. The learning curve for Storyline is less steep than for some tools, but there's still plenty to learn. Stephanie Harnett's new book intends to fill that gap. Here's an expert's review of this new guide.
by Joe Ganci
In August, Joe discussed browser compatibility with HTML5 and identified the most commonly used development tools you can use to publish eLearning lessons to HTML5. This month completes the review with a list of more tools that publish to HTML5, and others that are very useful in the eLearning development process.
by Stephen Haskin
If your organization includes video in its eLearning media mix, managing that content involves some different issues compared to other media. Here is an overview of the issues, and some specifics for dealing with them.
by News Editor
With VideoSync, users can now show video and slides side-by-side to more effectively highlight subject matter experts and increase audience engagement.
by Stephen Haskin
Video is a great resource for delivering content for learning, but it requires a lot of storage, and producing video that doesn't have huge bandwidth requirements is another challenge. The combinations of ways to render video can be daunting. This article covers what you need to know about video compression, whether you are already using video or are just getting started!
by News Editor
John Wiley & Sons, publisher of a number of books for professionals in the learning field, has announced a series of strategic branding changes. Among these changes is the discontinuation of the Pfeiffer brand. Read the details here!
by News Editor
From small-group presentations to stadium-filled sales seminars, Citrix connects people with a full range of video-enabled collaboration tools.
by Julie Dirksen
As an instructional designer, how do you handle situations where there's not a knowledge problem? Where people know what to do, but just don't do it? Here's a review of a recent research study on the influence of visceral experience on behavior. It will suggest a way that you may want to try when dealing with these cases.


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If there's one good thing that has come out of dabbling with the NeatDesk scanner, it's that my desk is now—or at least was for a moment—neat. That's not because my desk was overflowing with paperwork, receipts, and business cards that are now in digital form, so much as it is that I really like to clean my desk off before I start writing. I use a "box of crap" technique, in which I slop everything off my desk into a box. Works great. The NeatDesk, though? Not so great. Its first problem is that it's simply a scanner, and a sheet-fed one at that, with slots for "Documents" (8.5 x 11 inch paper), "Receipts", and "Cards". Useful when it works, but you won't be putting a stack of anything on the NearDesk for unattended scanning; the plastic slots are too thin. Then there's the software, the so-called "Neat Library". It's not free, for one, although you do get a license code for a copy when you buy the NeatDesk. (Presumably Neat is trying to prevent you from using the software with just any old scanner.) And the latest version, which also supports OS X (the platform on which I tested the NeatDesk), coughed when it came time to calibrate, but seemed to work when I fired up the program itself. Neat Library is terribly overwrought, though, for something with so little magic. It starts with false sample data in its database, which is confusing, instead of walking you through a scan of your own. (Who would buy a NeatDesk without business cards and receipts of their own ready to be scanned?) The OCR is spotty, and seems to have very little intelligence. I tried scanning Cory's new cards, which look like the back of a postcard, but are set in a clean Courier font. Neat Library decided his company was "Puce", misinterpreting "Place", while it registered his email domain as "craphound.cor" (emphasis mine). Is it too much to expect a business card scanner to know all valid top-level domains? (Another card from an Intel employee changed the @ symbol to an "ED".) All this haphazard scanning means you'll be spending a lot of time massaging the data, cutting and pasting items into the appropriate fields and doublechecking the card. It would be easier, and less time consuming, to simply punch the cards in by hand. I only scanned a grand total of seven cards, but every single one of them required some manual fixing. NeatDesk is a good idea, but they need to circle back and think about the problems they're trying to solve and the workflow that they're requiring of their users. (Look at the interface to Neat Library above (with personal information occluded); it looks like something Microsoft would have tossed off a decade ago, and it's about as enjoyable to use.) Business cards can come in a variety of looks and formats, but it wasn't like I was trying very hard to stump it. For $450 ($350 street) I expect scan-and-forget—and nothing less.
A couple of hours with the NeatDesk scanner (Verdict: Not worth the time or trouble)
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8 Responses to "A couple of hours with the NeatDesk scanner (Verdict: Not worth the time or trouble)"
I have the their mobile scanner, and I kind of like it. Their software is totally crap like you mentioned above. But the scanner I have does a decent enough job, and its super small and doesn't need an extra power cord to work.
As an FYI, I was looking at this one, but heard similar things about the OCR and other aspects of the Mac software — plus, it's pricer than its major competitor, the Fujitsu Scansnap series. (I've had my eye on the new model, the S1500M, for awhile…darn recession.)
* How to make your own network scanning rig
As MEF said, just get a Fujitsu ScanSnap.
I'm an employee of The Neat Company. I'm sorry you had a poor experience with NeatDesk.
I wanted to clarify that NeatDesk is more than simply a scanner. It's an auto-document feed scanner that comes bundled with NeatWorks software. The software enables users to digitally organize and manage their receipts, business cards and documents. Users can easily create expense reports, send contact information to Address Book and export data to other applications, such as Quicken. Additionally, every item is searchable through the application or Spotlight and everything is stored as a PDF.
Intelligent text recognition technology reads data off of receipts and business cards and parses fields accordingly. As you mentioned in your review, the accuracy is not 100%, however, I wanted to inform you that if something is incorrect or misread, the data can be dragged from the image to the field.
In regards to the hardware, you mentioned that you were unable to scan a stack of papers because the plastic slots are too thin. Could you please email me at email@example.com and provide more information on why you were unable to batch scan? I'd like to make sure that you did not have a defective hardware. The paper tray is designed to allow users to insert up to 10 receipts, 10 business cards and 10 documents, and scan them all in a single batch (or the paper tray can be removed to scan up to 50 pages at once).
I purchased a refurbished travel neatco scanner that's one year older than the current model. I love it. I keep everything…. I have receipts dating back almost 5 years and I have been placing them into this software. I have had very little issues. The scanning ocr isn't 100% accurate, but no scanner inserting data is. Most have common mistakes such as placing a "0" instead of an "O" or an "8" instead of an "S" due to the similarities of the letters and numbers. Personally, I love this product and the software. It isn't fun scanning one page at a time of all the data, but I couldn't afford a NeatDesk, but I'm definitely saving up for it.
I find all the comments thought-provoking, and the comments by Joel from Neat clarified some of the other statements. But I would like to hear more from Joel regarding the Fujitsu Scansnap, as my first thought when I encountered the Neat products, esp. NeatDesk, was how the two compared, and why I should be willing to spend more for NeatDesk. The claims for both seem about the same…
FYI, I have the mobile NeatWorks (Neat Receipts) scanner and it scans black and white 8.5×11 pages at 4ppm. There is also a load delay and a delay after the scan button is pushed… which brings the realistic scan rate to 3ppm.
Color is twice as slow… about 2ppm.
I have both scanners. I think they are both great and reasonably quick. Also I like the software and am a fan. It doesn't take too long to figure it out. I don't really care about the character recognition so much, so that isn't something I feel either way about. It's just nice to have clean desk and clean pockets. It's nice to be able to email documents off to people. The mobile scanner is nice to be able to take anywhere and get work done. It's nice to look at my neat .pdf's on my phone if I want to.


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Pre-Release: Simon Berz, Cut Out
5. 06. 2015,
Record Release & Performance: Simon Berz - Cut Out
Coming Soon: Simon Berz - Cut Out
It's been a little while since our last physical release - all the
more we're very happy to announce the next release on Hula Honeys
Simon Berz
- half of insanity driven duo
will be
presenting a solo release in early June 2015.
who has seen Simon Berz perform knows that he is not your usual drummer
from the block. He has been experimenting with his drum kit -
modulating it with microphones, speakers and effects. The drum kit has
grown into the so-called "Rocking Desk" - and is nothing less than an
extended and hybrid instrument that can stir up the dirt.
With his Rocking Desk, Simon Berz crosses the border between analogue and electronic music. He is still the drummer, but he can not only play snare drums, rim shots or rides, but also make them scream and squeak in tone through the contact microphones placed on the drums.
The forthcoming release "Cut Out" comes on a double 7" record with a recording of a solo drum song by Simon Berz, a remix by Toktek and 2 x 25 loops that invite DJs and musicians to explore the sounds of the Rocking Desk further.
Artist Profile Simon Berz
Bit-Tuner nominated for Swiss Music Prize
The Swiss Federal Department of Home Affairs announced the nominees for the Swiss Music Prize 2015.
is among the 15 candidates, along with Joke Lanz, Bruno Spoerri, Oy and Nik Bärtsch, among others.
The seven jury members selected 15 nominees
representing a wide scope of the Swiss music scene out of a
pre-selection of approximately 50 musicians that were presented by an
independant expert group. The nominees come from the Jazz, Classical,
Folk Music, Experimental or Electronic scenes. In the words of the Jury,
they "influence the Swiss music scene from the edges and intersections
of genres with their dedication for an open-minded approach".
Sold out: Feldermelder - 3 Yards of Warmth
This one went really fast! The postcard of Feldermelder 's "3 Yards of Warmth" is sold out at our bandcamp store , there might be one or two copies left at Zurich's oOR record store . The webshop isn't ready yet - so you'll have to call them to find out: + 41 44 542 32 80
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Free Download: ASIC - Pentaprism EP
The third release on Hula Bytes comes from
. The
[hub03] Pentaprism EP
explores the in-betweens of Electronica, Techno, Glo-Fi and Poststep
with an architectural approach. The digital EP comes as
free download
(licensed under Creative Commons, BY-NC-SA) and as "Laser Cuttable".
Coming from his year-long
performing routine, ASIC has recorded two EPs that are being released
via Hula Honeys and sub_perversion simultaneously. While the TAPA EP on
explores the deeper Poststep influences, the
[hub03] Pentaprism EP
on Hula
Honeys opens the musical spectrum towards Glitch-Hop, Techno and Bass.
The tracks of the TAPA EP and the PENTAPRISM EP are rooted in live sessions. ASIC moves through soft, harsh or wild city scapes that are ever evolving, temptingly rough and delicate at the same time. A night time walk with this city's soundtrack is a journey full of surprises and twists. While skyline is of mere architectural beauty, the surfaces are full of mechanical decay and organic growth. Moody sounds can be lurking behind every corner.
Both releases are available as digital downloads. They are accompanied by PDF-Files that can be laser cutted on arbitrary material, similar to a vinyl record. The necessary script was developed and written by Amanda Ghassaei . Both the music and the script are licensed under Creative Commons (BY-NC-SA).
The release is available as free download on the
release page
Moods Artist in Residence: Simon Berz
The drummer and sound artist Simon Berz – half of
has been working on a very unique sonic world for years. He is
interested in the in-betweens: nature, people, media and machines are
his working material, his focus lies on instable states.
Simon Berz looks out for the beauty in any given moment and the value of the unimpressive. In the past years, he's developed manyfold approaches to sounding material. With his self-developed Lithophon, he uses an array of stones to create fragile rhythms and noises through analogue electronics and feedbacks. This approach is also his – so to say – standard when he baits unheard sounds out his drum set.
The renowned Jazz club Moods invited Simon Berz to their last residency
of the
year. He is curating three nights under the theme of "instabile moments"
in December, featuring like minded artists:
, Simona Bosco,
Flo Stoffner, Moose Jackson & Rob Cambre, Vincent Membrez, Linda
Vogel, Xu Fengxia, Bettina Boller and
, of course.
Read the full article
"Lisboa / Skagaströnd" by Jen Reimer + Max Stein out now:
The 16th release on Hula Honeys has been cooking up for quite a
while. Finally, we're happy to release
Jen Reimer + Max Stein
"Lisboa / Skagaströnd" on tape and as digital download. The tape edition
is limited to 100 copies and can be ordered via our
Bandcamp store
Reimer (CAN) and Max Stein (USA) share a fascination with reverberant
spaces. Drawing inspiration from the hidden harmonies and secret rhythms
present in urban sound enviroments, they create immersive multichannel
sound works for processed horn and field recordings: conceived,
composed, performed and recorded in the spaces themselves.
This release features two performances in resonant, yet very different locations. "Lisboa" was recorded in the former cistern Mãe D'Áqua, an architectural cathedral for water and it's sounds. In "Skagaströnd", Jen Reimer + Max Stein take their listeners into the inside of an old chimney in Iceland, and slowly reveal it's glistening, fulminating wavy sound. Both works make the entanglement of sound and space tangible for those who let themselves glide into the recordings.
The tape version comes with a riso-printed poster by Hraïr Hratchian and a download code.
FELL and the Incubate Kidsorchestra
out now!
The 2nd EP of FELL is out now via our
Bandcamp store
. The release is available as CD or Digital Download.
"FELL and the Incubate Kidsorchestra" documents a creative journey FELL undertook at
Incubate Festival
in late 2013 together with the Kidsorchestra. FELL collaborated with the kids in several workshops.
Starting out from scratch, they used any sounding material they could find, such as personal belongings, found objects and trash to build simple instruments and to record rhythmic patterns and sounds. The elements found on this EP were designed, tried out and modified collaboratively. FELL created four pieces of music out of the material by adding beats and melodies to the recordings of the Kidsorchestra and winding the recorded patterns, frazzled voices and athmospheric glimpses through a digital microcosm and the guts of selfmade electronics. The EP is being released in collaboration with
Incubate Festival
. A first track is now up on our
Bit-Tuner: The Japan Syndrome Release
On March 26th 2014, Bit-Tuner's second live album «The Japan Syndrome» will be released as 12" vinyl. After a successful digital release in late 2013, Hula Honeys is teaming up with
Berenice Recordings
to bring you this special edition live album.
Recorded at Zurich's Helsinki Klub, «The Japan Syndrome» is a fragmentary snap shot, a rampant audio(visual) trip through Japan's largest cities, that
visited in spring 2013. He captures these cities via field recordings to then transform the found sound into his characteristic dark beats and minimalistic sounds.
«The Japan Syndrome - Vinyl Edition» is a coproduction between Hula Honeys and Berenice Recordings . The record will be available via our Bandcamp store , and in the shop your choice.


text:
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You can't go wrong with Kool Vue parts! From restoration to performance products, Kool Vue has got you covered! Kool Vue is a tried and tested brand trusted by thousands of DIY people and body shops alike; it provides only the best quality products for its customers, and best of all it comes with a 2-year Kool Vue limited warranty!
Fits the following 2008 Subaru Outback Submodels
When restoring the A/C Condenser of your heating or air conditioning system, Spectra carries the most suitable Original Equipment (OE) quality auto component for your car. To guarantee best fit and ideal performance, Spectra A/C Condenser was created to meet or surpass OEM parts standards. It comes with the exact same structure of the original A/C Condenser that came with your motor vehicle so that you're assured of its performance. Furthermore, given that Spectra A/C Condenser is an Original Equipment quality replacement Automotive Part, it is relatively quick to mount.
Fixing or customizing your vehicle usually requires replacing components. Because out of date automotive parts perform a lot less proficiently, it is crucial to switch to fresher ones to lessen the possibility of endangering additional auto or truck parts. In addition, replacing automotive parts which are in inferior form helps you ride with confidence and permit your motor vehicle to operate with maximum effectiveness.
Fits the following 2008 Subaru Outback Submodels
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Replacing parts is a key task in repairing or modifying your vehicle. It's valuable to replace outdated and worn-out automotive parts mainly because they function inadequately and they risk damaging other car or truck parts. The major rewards of changing automotive parts which are in terrible shape can include driving with a peace of mind and getting the motor vehicle running at highest possible effectiveness.
Fits the following 2008 Subaru Outback Submodels
Using Denso A/C Condenser definitely helps make the whole task of repairing your heating and air conditioning system a lot easier. Due to the fact these replacement A/C condensers fulfill or surpass the requirements of their original equipment counterparts, they are convenient in preventing poor performance and abrupt break down. Having the benefits of OEM quality design, high quality materials, and enhanced manufacturing methods, Denso A/C Condenser presents reliable performance and durability.
One of the most effective procedures to fix an automobile's heating and air conditioning system straight into its prime form is thru changing the impared OE auto components. Whenever auto parts are allowed to continue in their bad or damaged state,they only risk ruining other adjacent car or truck parts. For more comfortable trips and worry-free repairs, change parts immediately after they indicate evidence of failure.
Fits the following 2008 Subaru Outback Submodels
For dependable A/C Condensers, always rely on GPD. These A/C Condensers are designed and manufactured from high quality components to meet or surpass OE requirements and specifications. With its high standard facilities, these AC parts assure quality and functionality like no others.
Fits the following 2008 Subaru Outback Submodels
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As its name clearly suggests, Free YouTube Video Downloader is a software tool that you can use to download videos from
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YouTube to ALLPlayer is an application that will allow you to download video files from the most popular video-sharing website
Super Free Youtube Video Downloader 3.0 is a powerful application that enables you to download videos from the
YouTube HD Downloader is a program that downloads YouTube videos to hard drives, enabling users to play video and audio content offline on
Quick YouTube Downloader is a free program that downloads videos from YouTube and saves them to multiple formats and qualities.
A YouTube Downloader & YouTube Converter which supports unlimited downloading tasks to download videos from YouTube.com to your local
YouTube MP4 to MP3 Converter is a simple file converter. Instead of converting one format of video into
All Free YouTube to MP4 Converter is a multi-functional software program not only can play, accelerate and
Agood YouTube to Mp4 3GP Converter allows you to download videos from YouTube.com and convert them to MP4 or 3GP video formats. It provides
Agrin Youtube Video to AVI MP4 WMV Converter is a useful video conversion tool that provides you with the function of downloading and converting
Agood Rip DVD to Youtube FLV MP4 AVI WMV Ripper is the excellent ripping DVD software that can help you rip DVD to


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Home Page - Illinois Public Media
Today is the last day to support WILL in our current fiscal year. Every gift makes a huge difference!
The latest content from Illinois Public Media
(Photo: Brian Mackey/WUIS)
News Local/State
(Aikman Wildlife Adventure)
News Local/State
Aikman Drops Proposed Site For Wildlife Park Near Arthur, Will Seek New Location
(Jim Meadows/Illinois Public Media)
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As He Retires from WILL, Dave Dickey Cites "Serving The Public" As Career Highlight
(Photo: Darrell Hoemann)
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June Is The Fourth Wettest Month On Record
News Local/State


text:
Dr. Glen T. Stuhring, MD14048 Juanita Dr NE
Kirkland, WA 98034 (7.2 miles away)
Dr. Daniel J. Riegel, MD19125 N Creek Pkwy Ste 204
Bothell, WA 98011 (6.4 miles away)
Dr. Steven W. Rittenberg, MD1909 214th St SE Ste 110
Bothell, WA 98021 (4.2 miles away)
Dr. Timothy J. Halm, MD19401 40th Ave W Ste 230
Lynnwood, WA 98036 (less than 1 mile away)
Dr. Katherine D. Bumstead, MD4112 Harbour Pointe Blvd SW # 1
Mukilteo, WA 98275 (4.4 miles away)
Dr. Timothy S. Anderson, DO18208 66th Ave NE Ste 200
Kenmore, WA 98028 (4.8 miles away)
Dr. Christopher J. Telge, DO18208 66th Ave NE Ste 200
Kenmore, WA 98028 (4.8 miles away)
Dr. Theresa A. Platz, MD10025 NE 186th St
Bothell, WA 98011 (5.9 miles away)
Dr. Lee R. Argue, MD12800 Bothell Everett Hwy Ste 120
Everett, WA 98208 (5.4 miles away)
Dr. Robert K. Kelley, MD11800 NE 128th St Ste 560
Kirkland, WA 98034 (9.1 miles away)
Dr. William W. Bacon, MD1025 153rd St SE Ste 200
Mill Creek, WA 98012 (4.2 miles away)
Dr. Laurel E. Morrison, MD11800 NE 128th St Ste 300
Kirkland, WA 98034 (9.1 miles away)
Dr. Shawn H. West, MD7315 212th St SW
Edmonds, WA 98026 (2.2 miles away)
Dr. Russel W. Goldman, MD11821 NE 128th St Ste D
Kirkland, WA 98034 (9.2 miles away)
Dr. Catherine J. Dhanki, MD16708 Bothell Everett Hwy Ste 201
Mill Creek, WA 98012 (3.8 miles away)
Dr. Mitchell J. Peterson, MD11011 Meridian Ave N Ste 200
Seattle, WA 98133 (8.3 miles away)
Dr. Shirley T. Chen, MD12910 Totem Lake Blvd NE Ste 101
Kirkland, WA 98034 (9.1 miles away)
Dr. Jeffrey B. Cantor, MD19401 40th Ave W Ste 230
Lynnwood, WA 98036 (less than 1 mile away)
Dr. Rachna Gadhok, MD12333 NE 130th Ln Ste 310
Kirkland, WA 98034 (9.3 miles away)
Dr. James I. Kim, MD1818 121st St SE
Everett, WA 98208 (5.8 miles away) – More Locations
* Please verify when scheduling an appointment. If you are this provider and would like to confirm what you practice, please update your free profile.
Appointment Request
Procedures Commonly Performed By Family Practitioners
- Pelvic Exam & Pap Smear
- Immunizations (Vaccinations)
- X-Ray
- Hormone Replacement Therapy
- Joint Injections


text:
I am writing an Jersey Response filter. I am using Jersey 1.17. I want to get access to some attributes of the httpServletRequest in the filter API. The way what i am doing right now is as below. Is it safe to inject the servletRequest like in the snippet below or will this cause some kind of concurrency issues? If there are multiple requests coming in conncurrently, will the servletRequest in different requests overwrite each other? Thanks for your hlep.
public class LoggingFilter implements ContainerResponseFilter {
@Context private HttpServletRequest servletRequest;
public ContainerResponse filter(final ContainerRequest req, final ContainerResponse resp) {
String s = this.servletRequest.getAttribute("xxx");


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We are loading you into the search listing pages...{{/if}} {{/if}} {{/if}} {{/if}}
{{Current}} just saw you!{{/if}} {{#if LastTopSpotPurchaseIsMulti }} {{#if HasAnyTopSpot}}
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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
35 year old woman | Colusa, CA, USA | Seeking men 30-38 within 100 miles
We always fulfill our own expectations....
Short version. In the beginning there was...California. Spent a few years in SC (Accent: yes), a winter in NE (Nebraska, not New England... and one -winter- was enough). California is home. So.. Me: Quirky, educated, generous and I have a big, soft heart. I DO have an opinion and am NOT afraid to express it. I have a 4 y/o son with ADHD. He is my shining star. I'm fairly fit, but hate exercise. I'd rather ride. Now there's a core workout. Screw P90X. I'm of the 'Dance with the one that brought you' school of life. Nerd x country girl combo with a thing for WW comics. And if you know what that is, plus! I am a lifelong horsewoman. I've been riding since I could walk. I don't much like cats. A cat has to be pretty special. In 34 years, there's only been one. I am a fantastic cook, I love it, it's a passion, my calling in life, after my son. I throw killer super bowl and world series parties, and really, any kind of party or celebration. Hate cut roses, diamonds, boxed chocolate and other overdone, thoughtless cliches. Cool exotic pet: Seahorses. Someday I will have a 6ft tall reef tank with a dozen seahorses. Color: the pacific ocean right off Monterey Bay? Where the otters play in the kelp beds? That rich, vibrant, gray/blue/teal/green? Yeah. That color. I am not rude. I'm honest. ;) Stand up to, for and with me, please. Pushovers need not apply. You? All I ask is to please: Be looking for something/someone real. I'm no Cindy Crawford, nor do I wish to be. Be reasonably fit. Don't have to be a modern Adonis, but please don't be the local hospital's leading heart attack candidate. Like or at least don't hate on the Giants or the Niners. And, ok I'm not sorry, but Dodgers, Yankees, Raiders and Cowboys fans probably shouldn't apply at all. You'd hate me soon enough. Make me laugh. Laugh at yourself. Laugh at me. Laugh with me. I think you get the notion. Be trustworthy, responsible... but don't let that make you stuffy, boring, or monotonous. Be open to the possibility of including my son in your life, if/when the time is right. We are a package deal. Be honest with yourself, as well as with me. Be emotionally and financially stable. (can you manage your own life? cuz if not, I don't want you near mine. i 'ain't yo mama'). Its my turn to be a priority. If you're just looking for options, keep on truckin, my friend. Vivere la vita. Ciao!
Book club , Camping , Cooking , Dining out , Fishing/Hunting , Gardening/Landscaping , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Travel/Sightseeing , Watching sports , Wine tasting
Baseball, Billiards / Pool, Bowling, Cycling, Other types of exercise, Swimming, Walking / Hiking
I exercise 1-2 times per week.
I like Birds, Dogs, Exotic Pets, Fish, Horses, Other
I've had'em all at different times in my life. Reptiles, horses, pigs, chickens, dogs, cats, fish, rodents. Currently 'fur-baby'-less.
Any sort of water, beaches. Amusement parks, DISNEYLAND, irish pubs, music festivals, open-air markets. Travel, art, theater. Fisherman's Wharf, Santa Cruz, SF, ATT Park, Monterey, Charleston, Tuscany, French Riviera, wine country and, and, and..
California State University, Chico, Chico, CA
FOOD (real mex, texmex, ital are favs). Sunshine. Giants. Niners. 'Good' music. Laughter. Old corvettes. Kitchen stuff. Comic books. Sea air. BandB's. Thunderstorms. Flip flops. Hammocks. Pearls. Gardens. Books. Wine. Artisan beer. Horses...
Ballgames. Boating, floating, fishing, horses, outdoors. Reading, painting, playing with my son. Pool, darts, bowling. Laying on the beach, or by the pool. Exploring. Cooking is a passion. Thus the herb/veggie garden hitch.
I am an avid reader. Just finished The Voice in the Wind. Good stuff. Now on The Thorn Birds. 'Course there's the prerequisite Dr. Suess and Shel Silverstein, per my kiddo.
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|What she is looking for
|5'4" (162cms)
|5'8" (172cms) to 6'2" (187cms)
|Body type:
|About average
|Slender, About average, Athletic and toned, A few extra pounds
|Black, Blue, Brown, Grey, Green, Hazel
|Auburn / Red
|Black, Light brown, Dark brown, Blonde, Salt and pepper
|Yes, but trying to quit
|No Way, Occasionally, Cigar aficionado, Yes, but trying to quit
|Social Drinker
|Social Drinker, Moderately
|Retail / Food services
|No preference
I love food. I've been in the industry forever, one way or another. Serving. Bartending. Manager. Cook. I love it all. Even spent a year as a butcher.
|Less Than $25,000
|Less Than $25,000, $25,001 to $35,000, $35,001 to $50,000, $50,001 to $75,000, $75,001 to $100,000, $100,001 to $150,000, $150,001+
|Never Married, Widow / Widower, Divorced
|Have kids:
|Yes, and they live at home (1)
|Yes, and they sometimes live at home, No, Yes, and they live away from home, Yes, and they live at home
|Wants kids:
|Not sure
|Someday, Not sure, No, but it's OK if my partner has kids
|White / Caucasian
|Latino / Hispanic, Native American, Pacific Islander, White / Caucasian
I am half Viking. Literally. The other half is your typical American mutt. lol. british, german, scott, irish and italian are the identifiable ones, i believe. But every ounce of me is 100% American, and fiercely so.
|Christian / Other
|Christian / Catholic, Christian / Protestant, Spiritual but not religious, Other, Christian / Other
I love, believe and trust in God. No church. If your views differ, cool. In my fam, we have agnostics, atheists, Buddhists, Lutherans, Episcopalians... and they went and sent me to Catholic school. You can imagine how that went. Can we say rebel?
|Some college
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral
Went to school for Architecture and fell in love with food instead. Chico didn't have a culinary division. 'nuff said. Now it would be pretty pointless. And a waste of $$, after a decade or so in the industry. :)


text:
By Marlene Y. Satter |
April 23, 2015
Singles and people who died relatively young were the most likely to die with no assets, according to an EBRI study.
By Ellen Uzelac |
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For three decades, Jim Poterba has studied how taxation affects household and business decision-making. The big takeaway for financial advisors: one size does not fit all.
By John Sullivan |
May 23, 2012
A recent study from the Employee Benefit Research Institute found that, despite falling during the first part of the last decade, the number of poverty-stricken seniors is once again growing.
By Danielle Andrus |
December 22, 2011
While the majority of older Americans say they will retire at a later age than they previously thought, the percentage of people near retirement who say they will probably never retire is going down, the Employee Benefit Research Institute found.
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text:
Foothill High School Sacramento, CA Alumni
Foothill Sacramento, CA Alumni Photos
What is this site?
- Search for FHS Lost Friends
- Send messages to alumni
- View FHS Alumni Profiles
- Share your memories
- Share photos with others
- Discuss past activities
Click on the Yearbooks below to view a copy Online at Classmates.com®. If you don't see your class's yearbook, check out our yearbook page.
Famous Alumni
There are currently no Foothill Sacramento, CA notable alumni listed. Be the first to add an FHS famous alumni.
If you know someone who graduated from FHS in Sacramento, CA that has passed away, you can create a memorial for them.
There are currently no memorials for Foothill Sacramento, CA alumni.
- Class of 1999 Brian Laxo
- Denise Luttrell
- Class of 2016 Gabrielle Romero
- Class of 2015 Gabriell Romero
- Tanya Karpuk
- Class of 1982 Diana Shuburt
- Class of 1973 David Ellinworth
- Class of 1995 Kellye Boyd
- Class of 1995 Bradley Boehle
- Class of 1985 Tony Hays
- Class of 1976 Henry Peele
- Class of 1974 Leanne Hopkins
- Class of 1991 Lynette Schleeter
- Class of 1986 Angie Perowitz
- Class of 1989 Carolyn Anderson
- Class of 1977 Frank Espegren
- Class of 2001 Surina Green
- Class of 1988 Gwen Agee
- Class of 1982 Kimberlie Loftus
- Class of 1991 Laurie McCullough
July Birthdays
|No Birthday's this month
Foothill Sacramento, CA Alumni Merchandise
The Foothill High alumni site is made so that students and teachers from the Sacramento high school can reconnect with those that they met while attending high school. You can share memories of those activities that you were involved with at Foothill HS and you can view other alumni and read what other former students have posted about the time they spent at Foothill. Foothill High School alumni can relive their experiences from high school by sharing memories and pictures, reconnecting with those alumni listed, and more.
Latest Updates from Foothill High School Alumni
Brian Laxo has someone searching for them.
Denise Luttrell has someone searching for them.
Gabrielle Romero has someone searching for them.
Gabriell Romero has someone searching for them.
Plus many more
Most recent: 8 days ago
|Dianna Jackson requested a FHS yearbook from 1980. April
|Someone visited Sandra Lewis's profile. April
|Someone visited Roberta Isham's profile. April


text:
Edmunds.com features a complete listing of Toyota dealerships in Burlington County, making it simple to the find new or used Toyota vehicle of your dreams. Compare local dealer prices in Burlington County with instant online quotes for Toyota car, truck, wagon, SUV, and minivan models. At Edmunds.com car buyers can search a comprehensive network of trusted Toyota car dealerships in their region, allowing them to pinpoint the lowest prices at the most convenient location.
Non Display Review Name "I purchased my Corolla about 2 years ago and I have been extremely satisfied with the vehicle, and have had not problems with the car to this date. Th"... Read more Review by: Cad123illac
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Non Display Review Name "I just want to start out and let you all know after the 2 previous Certified Pre-Owns I have bought in the past 1 year and 3 months that this dealersh"... Read more Review by: cabbit75
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Non Display Review Name "Bought a 2011 camry se. Got a good deal and good deal on trade. Service after the sale has been great so far. Hope this continues! " Read more Review by: camryse11
Non Display Review Name "Bought a Toyota corolla last week and couldn't be happier. It was a great experience! Tim W was who helped me, he was really friendly and patient. He "... Read more Review by: ninjja15


text:
Surgical Specialist, Orthopaedic Surgeon
36 years of experience
Video profile
Accepting new patients
36 years of experience
Video profile
Accepting new patients
10650 Park Rd
Ste 120
Charlotte, NC 28210 Phone number & directions
Ste 120
Charlotte, NC 28210 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Thomas Buter:
View the full profile to get information on:
- Qualifications
- What's important to you
View full profile
Patient Reviews
Patient comments (0)
The overall average patient rating of Dr. Thomas Buter is Poor.
Specialties & Research
Dr. Thomas Buter, MD is board certified in Orthopaedic Surgery.
- He has special expertise in 3 areas:
- Hip Osteoarthritis
- Knee Osteoarthritis
- Osteoarthritis (Hand and Wrist)
Education & Awards
Dr. Buter graduated from University of Michigan Medical School, Ann Arbor.
Dr. Buter has 10 hospital affiliations:
- Mercy Hospital
- Presb Orthopaedic Hospital
- Spanish
Home > Find a Surgeon > NC > Charlotte Surgeons > Dr. Thomas Buter, MD
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- Additional Information
- Dr. Thomas Henry Buter MD is a male Orthopaedic Surgeon, has 36 years of experience and practices in Orthopaedic Surgery.
- Where does Dr. Thomas H. Buter practice?
- Dr. Thomas Buter has 2 office locations.
Dr. Thomas Buter practices Orthopaedic Surgery near Charlotte, NC.
See Charlotte Surgeons.


text:
Family Practitioner, Primary Care Doctor
32 years of experience
32 years of experience
Valley Hi - North Laguna
6600 Bruceville Rd
Sacramento, CA 95823 Phone number & directions
6600 Bruceville Rd
Sacramento, CA 95823 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Ralph Libet:
View the full profile to get information on:
- Qualifications
- What's important to you
View full profile
Patient Reviews
Have you visited Dr. Libet? Share your experience.
Write a Review
Write a Review
Education & Awards
Dr. Libet graduated from University of California at San Francisco.
- Hebrew
Home > Find a Family Doctor > CA > Sacramento Family Physicians > Dr. Ralph Libet, MD
Similar doctors nearby
Dr. Archie James
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- Additional Information
- Dr. Ralph Arnold Libet MD is a male Family Practitioner, has 32 years of experience and practices in Family Medicine.
- Where does Dr. Ralph A. Libet practice?
- Dr. Ralph Libet has 1 office location.
Dr. Ralph Libet practices Family Medicine near Sacramento, CA.
See Sacramento Family Physicians.


text:
volatile organic compounds Companies in Gabon
Keyword: volatile organic compounds ×
Industry: University / Academia / Research ×
Location: Africa ×
Location: Gabon ×
Ionicon Analytik Ges.m.b.H.
based in Innsbruck, AUSTRIA
IONICON is the world's leading producer of trace gas analyzers with market-leading, real-time, single-digit pptv-level detection limits for VOCs using the unique Proton Transfer Reaction – Mass Spectrometry (PTR-MS) and Selective Reagent Ionization – Mass Spectrometry (SRI-MS) technology. IONICON also produces trace calibration devices for analytical instruments and ...
Environnement S.A
based in Poissy, FRANCE
Environnement S.A is the leading company in the field of instrumentation for the environment. Ever since its creation in 1978, Environnement S.A has been dedicated to the study, development, manufacture and supply of advanced on-line gas, particulate and water analyzers, using the most recent technologies. Environnement S.A proposes a comprehensive range of continuous ...
Syft Technologies Ltd
based in Christchurch, NEW ZEALAND
Syft Technologies develop, manufacture and market VOC analysis solutions. We work closely with our customers to understand their VOC analysis needs, then develop efficient solutions that deliver real operational and financial benefits. Our success is built on identifying breakthrough analysis techniques and developing them into robust, market-focused solutions. We began ...
based in München, GERMANY
Clariant is an internationally active specialty chemical company, based in Muttenz near Basel. The group owns over 100 companies worldwide and employed 21 202 employees at the end of 2012. In the financial year 2012, Clariant produced a turnover of CHF 6 billion. Clariant is divided into seven business units: Additives; Catalysts; Function Minerals, Industrial & Consumer ...
Sigma-Aldrich Co., LLC
based in Bellefonte, PENNSYLVANIA (USA)
Sigma-Aldrich is a leading Life Science and High Technology company whose biochemical, organic chemical products, kits and services are used in scientific research, including genomic and proteomic research, biotechnology, pharmaceutical development, the diagnosis of disease and as key components in pharmaceutical, diagnostics and high technology manufacturing. ...
Ashtead Technology Ltd
based in Letchworth, UNITED KINGDOM
Ashtead Technology was formed in 1985 when Ashtead Group plc acquired SubSpek Limited, which at the time provided underwater positioning, survey and inspection equipment rental from a base in Aberdeen. In 1994 the Singapore base was opened, focussing on the offshore oil and gas industry and the subsea telecommunications cable market in Asia Pacific. The first US ...
Pollution Srl
based in Budrio, ITALY
POLLUTION is an experienced designer and manufacturer of analytical instruments for the on-site chemical analysis of volatile compounds. Since 1991 POLLUTION has been a leader in micro GC and emissions FID technologies. Markets served: Environmental, Energy & Process (natural gas, petrochemical) , Health & Safety. POLLUTION's young and highly motivated team, on top of ...
Sitelab Corporation
based in West Newbury, MASSACHUSETTS (USA)
Sitelab Corporation manufactures portable laboratory instruments and test kits used to measure concentrations of petroleum hydrocarbons in soil, sediment or water on-site. Popular applications include field screening samples for TPH, BTEX, PAHs, GRO and DRO. Sitelab`s Ultraviolet Fluorescnece (UVF) technology is very sensitive to gasoline, jet fuel, diesel fuel, coal ...
Spill Doctor (SA)
based in Cape Town, SOUTH AFRICA
The Jesbra Group, now trading as Spill Doctor in Africa, represents some of the worlds industry leading providers of environmental, safety and health products. With over a decade of international experience and 25 years operating in Africa, Spill Doctor has consolidated a range of internationaly recognised products that both our regional and international clients regard ...
Aurora Biomed Inc.
based in Vancouver, BRITISH COLUMBIA (CANADA)
Aurora Biomed is a worldwide leader in the design and development of lab automation solutions for life science, environmental science, drug discovery/safety and chemical analysis research. We provide technologies and services which facilitate a higher sample throughput while improving quality, accuracy and precision. Aurora`s product range includes automated ...
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