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1. A method for identifying or verifying members of the short chain dehydrogenase (SDR) family comprising the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family. 2. The method according to claim 1, further comprising a step (e) ranking of the positive SDR candidates obtained according to the number of amino acids matching with motifs MT1, MT2, MT3, MT4 and MT5. 3. The method according to claim 1, wherein in step (b) the target sequence is compared with core SDR motifs selected from (i) MT1:TGxxxGxG, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at position 90-110 relative to MT1, (iv) MT4:S(11-52:x)YxxxK and (v) MT5:PG, and wherein in step (c) positive SDR candidates are determined containing (i) at least the core SDR motifs MT1 and MT4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5. 4. The method according to claim 1, wherein in step (c) positive SDR candidates are determined containing (i) at least the core SDR motifs MV1, MV4 and one of MT2, MT3 and MT5 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5. 5. The method according to claim 1, wherein in step (c) positive SDR candidates are determined containing (i) the core SDR motifs MV1, MV4, MT2 and MT3 or MV1, MV4, MT2 and MT5 or MV1, MV4, MT2, MT3 and MT5. 6. The method according to claim 1, wherein positive SDR candidates are determined containing the core SDR motifs MV1, MV4, MT2, MT3 and MT5. 7. The method according to claim 1, wherein in step (c) positive candidates are determined containing at least 9 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4, and MT5. 8. The method according to claim 1, wherein MT2 is defined as NNAG. 9. The method according to claim 1, wherein MV4 is derived from the motif MT′4:S(11-52:x)YxASK by replacement of 0-2 amino acids. 10. The method according to claim 9, wherein in step (c) positive candidates are determined containing at least 9 of the 16 amino acids contained in the core motifs used. 11. The method according to claim 1, wherein MT2 and/or MT5 are extended for identifying or verifying FabG_SDRs, wherein MTy2:VxVNNAG, wherein V can be replaced and MTy5:PGFI, wherein F and/or I are used as search motif. 12. The method according to claim 1, further comprising one or more of the following further steps: (i) three-dimensional structure comparison and (ii) biological function analysis. 13. A member of the short-chain dehydrogenase (SDR) family identified with the method according to claim 1. 14. The SDR according to claim 13, wherein it is selected from the SDRs shown in Tables 1-5. 15. A method for providing modulators for members of the short chain dehydrogenase (SDR) family comprising the steps (a) providing one or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family and (b) providing modulators, which enhance or inhibit the activity of the members of the short chain dehydrogenase family. 16. The method according to claim 15, wherein step (a) comprises the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) Mv4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family. 17. The method according to claim 15, wherein in step (b) a protein sequence alignment with known SDR sequences is performed for pre-selecting possible modulators. 18. A method for evaluation of lead-candidates for possible modulators of a member of the SDR family comprising the steps (a) providing one or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family, (b) ranking the target sequences according to the number of amino acids matching with the core SDR motifs used and (c) deriving lead-candidates from metabolites of evolutionary related SDR enzymes. 19. The method according to claim 18, wherein step (a) comprises the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV 1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family. 20. A method for providing a pharmaceutical agent comprising the steps (a) providing tone or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family, (b) providing modulators, which enhance or inhibit the activity of the members of the short chain dehydrogenase family and (c) formulating said modulators as pharmaceutical agent. 21. The method according to claim 20, wherein step (a) comprises the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family. 22. The method according to claim 20, wherein step (b) comprises the steps (a) providing one or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family and (b) providing modulators, which enhance or inhibit the activity of the members of the short chain dehydrogenase family. 23. The method according to claim 20, wherein a modulator is provided, which enhances the activity of the members of the short chain dehydrogenase family. 24. The method according to claim 20, wherein a modulator is provided, which inhibits the activity of the members of the short chain dehydrogenase family. 25. The method according to claim 20, wherein the validation of a modulator or a function of a SDR enzyme found with an algorithm using core SDR motifs is performed with biochemical methods. 26. The method according to claim 20, wherein expressed sequence tags and gene sequence comparison are used to provide a function of the member of the short chain dehydrogenase family, which has been identified or verified with an algorithm using core SDR motifs. 27. The method according to claim 20, wherein a modulator or a function of an SDR enzyme found with an algorithm using core SDR motifs is validated high throughput function screening for function identification, UHTS for lead compounds, molecular homology modelling, substrate docking simulations, tissue expression, cDNA arrays or analysis of disease in animal or in vitro model systems. 28. The method according to claim 20, wherein a human SDR enzyme is provided and the pharmaceutical agent is applied for therapeutic or diagnostic purposes. 29. The method according to claim 28, wherein the human SDR enzyme is selected from the human SDRs shown in Table 1 or 2. 30. The method according to claim 20, wherein an SDR from a pathogen and/or a fungi is provided to obtain a high specific pharmaceutical agent. 31. The method according to claim 30, wherein the SDR is selected from the SDRs shown in Table 3, 4 or 5. 32. The method according to claim 20, wherein an SDR enzyme with high homology is provided, which constitutes an essential enzyme. 33. The method according to claim 20, wherein an SDR enzyme with low homology or high divergence between different species is provided, which allows for a species specific modulation. 34. A pharmaceutical agent obtainable by a method according to claim 20. 35. The pharmaceutical agent according to claim 34 for the prophylaxis, treatment and/or diagnosis of diseases. 36. The pharmaceutical agent according to claim 34, which is a fungicide or antibiotic. 37. A method for detection of clinically relevant polymorphisms or single nucleotide polymorphisms comprising the steps (a) providing one or more target sequences or members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family, (b) ranking the members of the short chain dehydrogenase family according to the number of amino acids matching with the core SDR motifs applied, and (c) comparing evolutionary patterns within the SDR enzymes. 38. The method according to claim 37, wherein disease mechanisms are characterised; 39. The method according to claim 37, wherein metabolisms of xenobiotics are characterised. 40. The method according to claim 37, wherein structure-function relationships are identified and/or substrates of SDR members with unknown function are identified. 41. The method according to claim 20, wherein a pharmaceutical agent for affecting immune regulation is provided by developing a modulator for 17β HSD type 3, 17β HSD type 7, 17β HSD type 8, 17β HSD type 10, 11β HSD-1, CR1, UDP glucose epimerase, SDR_SRL, AF067174, AF151840, AF151844, AF0078850, Fvt-1, HEP-27, DKFZ_ORF, WWOX_ORF, or CR3. 42. The method according to claim 20, wherein a pharmaceutical agent for affecting autoimmunity is provided by developing a modulator for 17β HSD-3, 17β HSD-8, 11β HSD-1, AF057034, U89717, CR1, AF0078850, HEP-27, or CR-3. 43. The method according to claim 20, wherein a pharmaceutical agent for wound healing or partial recovery is provided by developing a modulator for 17β HSD-3, 17β HSD-8, 11β HSD-1, U89717, CR1, AF0078850, HEP-27, or CR-3. 44. The method according to claim 20, wherein a pharmaceutical agent for treatment of leukemia is provided by developing modulators for 17-β HSD-10 or Fvt-1. 45. The method according to claim 20, wherein a pharmaceutical agent for apoptosis regulation is provided by developing a modulator for 17β HSD-10, U89717, SDR_SRL; or for providing a pharmaceutical agent for affecting immune response by providing a modulator for AF016509, or providing a pharmaceutical agent for the treatment of cancer by providing modulators for AF016509, or providing a pharmaceutical agent for affecting cell growth by providing a modulator for U89717, or providing a pharmaceutical agent for the treatment of lung carcinoma by providing a modulator for SDR_SRL, or providing a pharmaceutical agent for the regulation of inflammation or vasculitis by providing a modulator for DKFZ_ORF.

<SOH> TECHNICAL BACKGROUND <EOH>The short chain dehydrogenase/reductase (SDR) protein family (H. Jörnvall et al., Biochemistry 34 (1995), 6003 - 6013 ) is an old conserved protein family, the members of which show a residue identity level of only 20-30%. However, it has been found that the three-dimensional structure of members of the SDR family are highly similar, determining their functions and affiliation to the SDR family (U. Oppermann et al., Enzymology and Molecular Biology of Carbonyl Metabolism 6, Weiner et al. eds., Plenum Press, New York (1996), p. 403-415). While initially only two structures of SDR enzymes restricted to bacterial and insect enzymes have been discovered, rapid progress on the knowledge of short chain dehydrogenases/reductases resulted in an increasing number of structures, which could be assigned to the SDR family. Currently, about 1.600 putative members are known, from which up to 100 may be derived from human, such as hydroxysteroid dehydrogenases (HSD). An approach to identify SDR proteins is described in W. N. Grundy et al., Biochemical and Biophysical Research Communications 231 (1997) 760-766 and in T. L. Bailey et al., J. Steroid Biochem. Molec. Biol. 62 (1) (1997) 29-44. Therein homologies are searched for via a hidden Markov model, i.e. a self-training model, and thus classified to a certain protein family. A classification based on the function is not made in these models. Since the SDR enzymes are involved in various metabolitic pathways and show different activities, such as oxidoreductases, lyases, or epimerases and, as discussed above, show only a low identity of 20-30%, it has been difficult, to assign new members unambiguously to the SDR family and to find modulators therefor. However, since HSD and other SDR play a critical role in higher vertebrates, it is desirable to discover further members of the SDR family and establish modulators for known and new SDR enzymes. It was therefore an object of the present invention to provide an algorithm which allows for the identification or verification of SDR family members with high confidence levels. It was a further object of the invention to provide an algorithm which provides a search hierarchy with various levels. It was another object of the present invention to provide modulators for SDR family members. Still another object of the invention was to provide pharmaceutical agents based on members of the SDR family.

<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a method for identifying or verifying members of the short chain dehydrogenase (SDR) family based on an algorithm using core SDR motifs for searching members of the SDR family. Further, the present invention relates to a method for providing modulators for such members of the short chain dehydrogenase (SDR) family, which enhance or inhibit the activity therefrom as well as a method for providing a pharmaceutical agent using modulators for members of the SDR family. In particular the present invention provides a combination of the steps (i) screening databases to search and find SDR sequences, (ii) store the data on an appropriate medium, rank and validate the hits and (iii) using the SDR sequences found to develop new drugs. detailed-description description="Detailed Description" end="lead"?

Method and system for creating marketplace visibility and administering freight shipments using fuzzy commodity transportation instruments

A utility for creating a real-time bid-ask transportation marketplace where all relevant information may be viewed and acted upon is disclosed. See FIG. 2. Users of the present invention tender shipments and offer capacity, which are analyzed and entered into transportation instruments. Contracts obligate the shipper to make a load available and the carrier to transport the load at a given time for a given price. Shipments may be managed throughout their entire life cycle using software tools that interact with the bid-ask marketplace.

1. A method of brokering transportation transactions, comprising: receiving into a staging area a plurality of dissimilar bids for shipping goods; receiving into said staging area a plurality of dissimilar offers for transporting goods; sorting and aggregating said shipping bids into a set of first fuzzy commodities; sorting and aggregating said carrier offers into a set of second fuzzy commodities; selecting matching sets of said first and second fuzzy commodities in said staging area to create transportation instruments; and creating underlying contracts to support the trading of said transportation instruments. 2. The brokering method of claim 1, further comprising: facilitating the administration of the underlying contracts by brokers or third party logistic providers. 3. The brokering method of claim 1, further comprises trading of said transportation instruments within spot, forward, shorthaul, series or derivative markets. 4. The brokering method of claim 1, wherein said trading step further comprises: measuring the objective performance of shippers and carriers and using that information in selecting a trade. 5. The brokering method of claim 4, wherein said ratings are used anonymously. 6. The brokering method of claim 1, wherein said trading step further comprises: evaluating the subjective performance of shippers and carriers and using that information in selecting a trade. 7. The brokering method of claim 6, wherein said ratings are used anonymously. 8. The brokering method of claim 1, further comprising: maintaining said bids or offers to remain open in the market for a predetermined time period; and automatically removing said bids or offers at the end of that time period. 9. The brokering method of claim 1, further comprising: maintaining said contingent bids or contingent offers to be open across multiple modes, lanes or markets of transportation, whereby upon first acceptance at a specific mode, lane or market; removing the remaining contingent bids or contingent offers across all other modes, lanes and markets. 10. The brokering method of claim 1, further comprising: removing open contingent bids or offers upon achieving an objective performance criterion. 11. The brokering method of claim 1, further comprising: using a Prioritized Scheduled Push (PSP) for updating one or more links either when a client submits a request to the server or periodically without any direct request from the client. 12. The brokering method of claim 1, further comprising the offer of contingent capacity for a multi-leg or backhaul; and coordinating said multi-leg or backhaul with said shipper(s) and carrier. 13. The brokering method of claim 10, wherein the booking of one leg causes the offer prices of remaining legs to be set to a different value. 14. The brokering method of claim 1, wherein multiple compatible partial loads are combined in booking an offered truck. 15. The brokering method of claim 1, further comprising: importing and exporting groups of bids or offers from Comma Separated Variable files or equivalents into and from said staging area, thereby allowing information from said staging area to be administered in a Comma Separated Variable file or spreadsheet format. 16. A method of matching a tendered shipment to offered conveyances, comprising: receiving into a staging area a tendered shipment; receiving into said staging area a plurality of dissimilar offers from carriers; sorting said shipment into a first fuzzy commodity; sorting and aggregating said carrier offers into a second fuzzy commodity; selecting one or more offers from the matching sets of said first and second fuzzy commodities in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipment. 17. A method of matching an offered conveyance to tendered shipments, comprising: receiving into a staging area an offered conveyance; receiving into said staging area a plurality of dissimilar tendered shipments; sorting said offered conveyance into a first fuzzy commodity; sorting and aggregating said tendered shipments into a second fuzzy commodity; selecting one or more shipments from the matching sets of said first and second fuzzy commodities in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipments. 18. A computer system for brokering a plurality of freight-shipments and carrier capacity, comprising: marketplace means for establishing a bid-ask (offer) marketplace including shipper bids and carrier offers, wherein said bids and offers are measured by mode, market, and lane and optionally accessorial services. 19. The computer system of claim 18, further comprising: display means for displaying a marketplace summary. 20. The computer system of claim 18, further comprising: display means for displaying market details in a bid-ask marketplace. 21. The computer system of claim 18, further comprising: display means for displaying most recent trades and trade volume 22. The computer system of claim 18, further comprising: acceptance means for enabling a customer to indicate acceptance of bids or offers. 23. The computer system of claim 18, further comprising: notification means for notifying one or more parties to a transaction. 24. The computer system of claim 18, further comprising: tracking/tracing means for determining the current location of a specific freight shipment. 25. The computer system of claim 18, further comprising: alert means for communicating fulfillment problems corresponding to a specific freight shipment. 26. The computer system of claim 18, further comprising: administration means for mitigating fulfillment problems corresponding to a specific freight shipment. 27. The computer system of claim 18, further comprising: trading of transportation within spot, forward, shorthaul, series or derivative markets. 28. The computer system of claim 18, further comprising: maintaining said bids or offers to remain open in the market for a predetermined time period; and automatically removing said bids or offers at the end of that time period. 29. The computer system of claim 18, further comprising: maintaining said contingent bids or contingent offers to be open across multiple modes, lanes or markets of transportation, whereby upon first acceptance at a specific mode, lane or market; removing the remaining contingent bids or contingent offers across all other modes, lanes and markets. 30. The computer system of claim 18, further comprising: removing open contingent bids or offers upon achieving an objective performance criterion. 31. The computer system of claim 18, further comprising: using a Prioritized Scheduled Push (PSP) for updating one or more links either when a client submits a request to the server or periodically without any direct request from the client. 32. The computer system of claim 18, further comprising the offer of contingent capacity for a multi-leg or backhaul; and coordinating said multi-leg or backhaul with said shipper(s) and carrier. 33. The computer system of claim 32, wherein the booking of one leg causes the offer prices of remaining legs to be set to a different value. 34. The computer system of claim 18, wherein multiple compatible partial loads are combined in booking an offered truck. 35. The computer system of claim 18, further comprising: importing and exporting groups of bids or offers from Comma Separated Variable files or equivalents into and from said staging area, thereby allowing information from said staging area to be administered in a Comma Separated Variable file or spreadsheet format. 36. The computer system of claim 18, further comprising a method of matching a tendered shipment to offered conveyances, comprising: receiving into a staging area a tendered shipment; receiving into said staging area a plurality of dissimilar offers for transporting goods; sorting said shipment into a first fuzzy commodity; sorting and aggregating said carrier offers into a second fuzzy commodity; selecting one or more offers from the matching sets of said first and second fuzzy commodity in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipment. 37. The computer system of claim 18, further comprising a method of matching an offered conveyance to tendered shipments, comprising: receiving into a staging area an offered conveyance; receiving into said staging area a plurality of dissimilar tendered shipments; sorting said offered conveyance into a first fuzzy commodity; sorting and aggregating said tendered shipments into a second fuzzy commodity; selecting one or more shipments from the matching sets of said first and second fuzzy commodity in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipments. 38. A computer system for trading transportation futures, comprising: receiving into a staging area a plurality of dissimilar bids for shipping goods; receiving into said staging area a plurality of dissimilar offers for transporting goods; sorting said shipping bids into a set of first futures; sorting and aggregating said carrier offers into a set of second futures; selecting matching sets of said first and second futures in said staging area to create a bid-ask marketplace for transportation future instruments; and creating underlying contracts to support the trading of said transportation future instruments. 39. A computer system for trading transportation options, comprising: receiving into a staging area a plurality of dissimilar bids for options on shipping goods; receiving into said staging area a plurality of dissimilar offers on options for transporting goods; sorting said shipping bids into a set of first options; sorting and aggregating said carrier offers into a set of second options; selecting matching sets of said first and second options in said staging area to create a bid-ask marketplace for transportation option instruments; and creating underlying contracts to support the trading of said transportation option instruments. 40. A computer system for trading transportation options on futures, comprising: receiving into a staging area a plurality of dissimilar bids for options on futures for shipping goods; receiving into said staging area a plurality of dissimilar offers on options on futures for transporting goods; sorting said shipping bids into a set of first options on futures; sorting and aggregating said carrier offers into a set of second options on futures; selecting matching sets of said first and second options on futures in said staging area to create a bid-ask marketplace for transportation option on future instruments; creating underlying contracts to support the trading of said option on futures transportation instruments, and bi-directional communication links coupled said computer system to the futures and options computer systems to create price consistency and to facilitate inter-market trading to manage risk taken in a position resulting from a trade in either market. 41. A method of calculating a standardized transportation line haul rate per mile, comprising: receiving into a staging area transportation data for a shipment; calculating standardized route miles from the zip codes of all stops in transit including origin and final destination and allowable practical routes for the type of cargo transported; calculating the line haul price from the total price less standardized charges for provided accessorials; and calculating the standardized line haul rate per mile by dividing the line haul price by the standardized route miles. 42. A computer system for calculating historical market data on transportation, comprising: means for receiving into a staging area a plurality of completed shipment transportation data, and; software program to calculate the standardized line haul rate per mile for each completed shipment. 43. The computer system of claim 42, further comprising: software program to sort and aggregate all shipments by lane, mode, market and date of shipment. 44. The computer system of claim 42, further comprising: means for receiving into a staging area a plurality of shipment requests and offered capacity transportation data; and software program to calculate the standardized line haul rate per mile for each tendered shipment or offered capacity. 45. The computer system of claim 42, further comprising: means for requesting the display of data sorted by lane, mode, market and date(s) of shipment. 46. The computer system of claim 42, further comprising: display means for displaying the requested data. 47. The computer system of claim 42, further comprising: output means for transferring the data to another computer system for further use.

<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to a method and apparatus to arrange for and manage freight shipments. Users of the present invention tender shipments and offer capacity, which are analyzed and entered into transportation instruments. These instruments are maintained in a real-time bid-ask marketplace where all relevant information may be viewed and acted upon by users. Users are shown a listing of available counterparties to a desired transaction, and upon agreeing to a set of terms, create a contractual obligation to perform according to the terms of the instrument. Contractual obligations may also be exchanged and sold amongst users. Shipments may be managed throughout their entire life cycle using software tools that interact with the bid-ask marketplace. 2. Background of the Art Today transportation brokers and/or third party logistics companies (“3PLs”) manage shipments on behalf of many shippers and carriers. Most transportation brokers, in a manner that is congruent to the thinking of brokers in the financial industry perhaps twenty years ago, believe that it is in their best economic interest to inhibit the visibility of transportation markets. They reason that they can maintain large spreads (i.e. their commissions) between the price that the shipper is wiling to pay and the fees paid to the carrier by not “commoditizing” transportation. Electronic trading within the financial community has indeed reduced spreads; however, the increase in sales volumes has resulted in overall increased profitability. Today there are other different ways for freight carriers and shippers to reach agreement. Competitive electronic marketplaces employ bulletin boards, static listings of available loads and capacity, and auctions. Shippers or carriers put loads or capacity out for bid and rule based exchanges utilize uniform rules and conditions to facilitate automated matching and services. Examples include DAT (a bulletin board/negotiating service), logistics.com (an auction service), and NTE (a form of a transportation exchange). Today's transportation agreements vary in relative “strength” from highly precise and enforceable dedicated contract carriage, to annual contracts and spot-market agreements with very loose terms and conditions. However, each suffers from drawbacks. Dedicated contract carriage and annual contracts are each respectively cumbersome to implement, and often require months of negotiation. The $950 billion transportation logistics industry represents about 10% of U.S. GDP. It is highly fragmented with limited market visibility and largely absent or dysfunctional information technology. Most business is conducted via telephone and FAX. Both shippers and carriers require user-friendly reliable market access and real-time information to provide the quality-of-service that their customers demand. Although there are a large number of transportation web sites, none of them effectively meets the objectives of customers, shippers, and carriers. Customers need accurate market data prior to making a decision on transportation, and wish to reduce their uncertainty in the marketplace. Furthermore, carriers need the ability to increase the probability of finding a backhaul. Shippers, meanwhile, want to lock in capacity for future anticipated needs. Finally, shippers also want the cost savings associated with collaborating with other shippers without having to identify or negotiate an agreement with every other shipper. Carriers want to predetermine their workload to minimize the cost of asset relocation. Carriers also need the ability to lower costs by offering a resource to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers. Shippers also need the ability to lower costs by offering a shipment to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers. To achieve these and other goals could require the cooperation of a competitor. To this end, some systems offer “collaborative logistics” in which dosed communities are formed to gain market efficiency. However, these systems are not real-time, and cannot process contingent orders, and they require the cooperation of the members of the community to share proprietary information. Often, members of the community are competitors of one another and are unwilling to compromise their competitive advantages to participate in the community. Also, these systems over-emphasize virtual world models at the expense of real-world operating environments in which equipment breaks down and there are delivery delays. Further, members who participate in these dosed systems often lack the best operating and dispatch people, because these people have migrated to better paying jobs with carrier or 3PLs for whom transportation is the core competency. Most other systems cover spot markets that represent only 20% of the for-hire truckload transportation market. Contract carriage represents about 80% of the for-hire truckload transportation market; thus, most other systems are aimed at the smaller market segment. Also, most other systems do not allow the hedging of price and availability risk by participating in forward or series purchases. Such systems thus entirely lack risk management. Third party logistics providers (3PLs) work on behalf of their customers i.e. shippers to both improve the reliability of transportation and minimize its cost. They do this by recommending shipping policy, selecting carriers to transport loads, and managing the entire life cycle of shipments. Unlike the financial industry in which perhaps more than 99.9% of all trades “clear” without incident, in transportation perhaps only 95% of all shipments are transported without the intervention of a “transportation expert” to remedy problems. When a transportation problem occurs, the 3PL provides a service to their customer and alleviates the problem. However, many other systems (some even proudly) do not allow the participation of brokers or 3PL companies to enable their users to avoid having to pay brokerage fees that are typically in the range of 8% to 12% of the total cost of transportation. The brokerage fees, which are proclaimed by these sites to be “recoverable” by using their system, are currently paid to the broker or 3PL who provide transportation management services using, for the most, part the inefficient technology of the “FAX and telephone” age. The cost of these services may be reduced considerably by using more advanced technology and competition. It is important to remember that many of these intermediaries, in addition to matching a shipper and carrier, provide valuable transportation management services and have and will try to protect their well-established customer relationships. Thus, these systems disintermediate existing players. As a result, they only penetrate a small piece of a well-entrenched market. Shippers and carriers need to improve their profitability by reducing the number of empty backhauls, delayed or lost shipments and warehouse bottlenecks, and the amount of effort required to manage core carrier relationships efficiently. Typically, when transportation managers have goods “ready-to-go” they send faxes or make multiple calls to their brokers at their 3PLs 100 , as shown in FIG. 1 . This process specifies the shipment and usually states what the shipper is willing to pay. The transportation manager is unable to “see the market”; i.e. they do not know the current spot price that other shippers are willing to pay or that carriers are willing to accept, or the availability of trucks. The brokers then send faxes or make multiple calls to dispatchers 101 at their carriers to check the price and availability of transportation to fill their need. The transportation manager does not participate in this process. After the shipment is booked, the broker must convey this information back to the shipper 103 and verify that the carrier has adequate insurance in force. This slow and people intensive process enables a broker to manage only 5 to 10 shipments a day. The above unsophisticated approach results from the fact that the current transportation industry contracting process was created in the “fax and telephone” age. Such a contracting process is antiquated, particularly when compared to the prevailing practices in the financial industry in which all participants are able to electronically view real-time markets and immediately execute orders when they see opportunities. Shippers and carriers cannot effectively manage risk using current transportation practices—most annual contracts are in reality just rate agreements that do not have firm commitments of shipments or trucks and only represent rates and other possible terms and conditions.

<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention provides a method of brokering transportation transactions, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into said staging area a plurality of dissimilar offers for transporting goods, sorting and aggregating said shipping bids into a set of first fuzzy commodities, sorting and aggregating said carrier offers into a set of second fuzzy commodities, selecting matching sets of said first and second commodities in said staging area to create transportation instruments; and creating underlying contracts to support the trading of the transportation instruments. In another aspect, the invention provides a computer system for brokering a plurality of freight-shipments and carrier capacity, including marketplace means for establishing a bid-ask (offer) marketplace including shipper bids and carrier offers, wherein the bids and offers are measured by mode, market, and lane and optionally accessorial services. In yet another aspect, the invention provides a computer system for trading transportation futures, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into the staging area a plurality of dissimilar offers for transporting goods, sorting the shipping bids into a set of first futures, sorting and aggregating the carrier offers into a set of second futures, selecting matching sets of the first and second futures in the staging area to create a bid-ask marketplace for transportation future instruments, and creating underlying contracts to support the trading of the transportation future instruments. In yet another aspect, the invention provides a computer system for trading transportation options on futures, including receiving into a staging area a plurality of dissimilar bids for options on futures for shipping goods, receiving into the staging area a plurality of dissimilar offers on options on futures for transporting goods, sorting the shipping bids into a set of first options on futures, sorting and aggregating the carrier offers into a set of second options on futures, selecting matching sets of the first and second options on futures in the staging area to create a bid-ask marketplace for transportation option on future instruments, creating underlying contracts to support the trading of the option on futures transportation instruments, and bi-directional communication links coupled the computer system to the futures and options computer systems to create price consistency and to facilitate inter-market trading to manage risk taken in a position resulting from a trade in either market. In yet another aspect, the invention provides a method of calculating a standardized transportation line haul rate per mile, including receiving into a staging area transportation data for a shipment, calculating standardized route miles from the zip codes of all stops in transit including origin and final destination and allowable practical routes for the type of cargo transported, calculating the line haul price from the total price less standardized charges for provided accessorials; and calculating the standardized line haul rate per mile by dividing the line haul price by the standardized route miles. In yet another aspect, the invention provides a computer system for calculating historical market data on transportation, including a means for receiving into a staging area a plurality of completed shipment transportation data, and a software program to calculate the standardized line haul rate per mile for each completed shipment. In yet another aspect, the invention provides a method of brokering transportation transactions, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into the staging area a plurality of dissimilar offers for transporting goods, sorting and aggregating the shipping bids into a set of first fuzzy commodities, sorting and aggregating the carrier offers into a set of second fuzzy commodities, selecting matching sets of the first and second fuzzy commodities in the staging area to create transportation instruments, and creating underlying contracts to support the trading of the transportation instruments.

Sampling device and sampling method

A sampling device (1) comprises a sampling container (4) for reception of a sample volume and a connecting piece (7) adapted to be connected to a fitting (2) mounted on a processing installation. The connecting piece (7) has a sample passage which is provided with a stationary valve port (12) and a corresponding valve member (14) which is displaceable between a closed and an open position. The stationary valve part (12) and the displaceable valve member (14) are integrated in the sampling container (4). The valve member (14) is spring-loaded towards its closed position and adapted to be automatically displaced to its open position upon connection of the connecting piece (7) with the fitting (2).

1. A sampling device (1) comprising a sampling container (4) for reception of a sample volume and a connecting piece (7) adapted to be connected to a fitting (2) mounted on a vessel (3) or pipe of a processing installation or the like, the connecting piece (7) having a sample passage which is provided with a stationary valve part (12) and a corresponding valve member (14) which is displaceable between a closed position, in which it abuts the stationary valve part (12) and closes the sample passage, and an open position, in which the sample passage is open, whereby the valve member (14) is spring-loaded towards its closed position and adapted to be automatically displaced to its open position upon connection of the connecting piece (7) with the fitting (2), characterized in that the stationary valve part (12) or the valve member (14) is formed as a conical face forming part of a wall surrounding the inside volume of the sampling container (4), and in that the other one of said stationary valve part (12) and said valve member, (14) has the form of a truncated cone carried by a spindle extending coaxially through the inside volume of the sampling container. 2. A sampling device according to claim 1, characterized in that the connecting piece (7) is adapted to be displaced in a longitudinal direction of the container (4) during at least part of the operation of connecting it to the fitting (2), and in that the displaceable valve member (14) is adapted to abut an edge (20) of the fitting (2) during at least part of said displacement. 3. A sampling device according to any one of the preceding claims, characterized in that the connecting piece (7) is adapted to be screwed onto the fitting (2). 4. A sampling device according to any one of the preceding claims, characterized in that the sampling container (4) comprises an outer container (5) and an inner container (6), the inner container (6) being arranged displaceably in a longitudinal direction of the outer container (5), in that the outer container (5) at a first end is formed integrally with the connecting piece (7) and at a second end is provided with a bottom (10), in that the inner container (6) at a first end is formed integrally with an annular sealing surface (15), thereby forming the displaceable valve member (14), and at a second end has a bottom (16) in which a spindle passage (17) is provided, in that a spindle (11) has a first end which is provided with the stationary valve part (12) and a second end which is fixed to the bottom (10) of the outer container (5), and in that the spindle passage (17) is arranged tightly around and displaceably along the spindle (11). 5. A sampling device according to claim 4, characterized in that the outer length of the outer container (5) is between 100 mm and 300 mm, in that the outer diameter of the outer container (5) is between ½ and ⅔ of the outer length of the outer container (5), and in that the smallest diameter of the annular sealing surface (15) of the inner container (6) is between ¼ and ⅔ of the outer diameter of the outer container (5). 6. A sampling device according to claim 4 or 5, characterized in that the outer container (5) and the inner container (6) are formed from Plexiglass, glass, tempered glass or the like, and in that the stationary valve part (12) is formed from polytetrafluoroethylene or the like. 7. A sampling device according to any one of the preceding claims, characterized in that the sampling device (1) comprises a fitting (2) mating the connecting piece (7) and having an installation end adapted to be installed onto the vessel or pipe of said processing installation or the like, and in that the fitting (2) has a tubular part (24) provided with an inner shielding meter (25) adapted to shield the stationary valve part (12) in the connected state of the connecting piece (7) and the fitting (2). 8. A sampling device according to claim 7, characterized in that the tubular part (24) of the fitting (2) is provided with a covering member (31) adapted to cover the displaceable valve member (14) in the connected state of the connecting piece (7) and the fitting (2). 9. A sampling device according to claim 8, characterized in that the shielding member (25) is adapted to seal against the covering member (31) in the disconnected state of the connecting piece (7) and the fitting (2). 10. A sampling device according to claim 8 or 9, characterized in that a circumferential contour (29) of the shielding member (25) fits a circumferential contour (30) of the stationary valve part (12), in that the shielding member (25) is adapted to be displaced with the stationary valve part (12) against a spring-load during at least part of the operation of connecting the connecting piece (7) to the fitting (2), in that a circumferential contour (32) of the covering member (31) fits a circumferential contour (33) of the displaceable valve member (14), in that the covering member (31) is fixedly mounted in the fitting (2), in that the circumferential contour (29) of the shielding member (25) fits the circumferential contour (32) of the covering member (31), and in that the circumferential contour (30) of the stationary valve part (12) fits the circumferential contour (33) of the displaceable valve member (14). 11. A sampling device according to any one of the claims 8 to 10, characterized in that the shielding member (25) has a conical sealing surface (34) corresponding to a conical sealing surface (35) on the covering member (31). 12. A sampling device according to any one of the claims 8 to 11, characterized in that the shielding member (25) is spring-loaded against the covering member (31) by means of a spring (28) located in a tube element (25) in which a spindle of the shielding member (25) is guided, in that the tube element (26) is guided axially in the tubular part (24) of the fitting (2), and in that the tube element (26) is fixed in a plate (27) adapted to abut an edge (38) of a flange (37) on the vessel or pipe of the processing installation or the like, in the mounted state of the fitting on said flange. 13. A sampling device according to any one of the claims 7 to 12, characterized in that the fitting comprises a shut-off valve (22). 14. A sampling device according to any one of the claims 9 to 13, characterized in that the sampling device (1) comprises a nozzle (41) adapted to be connected to the fitting (2) and having an internal projection (44) adapted to keep the shielding member (25) displaced against the spring-load in the connected state of the nozzle (41) to the fitting (2), and in that the nozzle (41) has a tube connection (43) for supply or discharge of cleaning or washing fluid. 15. A sampling method for taking a sample of a product from a processing installation or the like, comprising the steps of connecting a connecting piece (7) of a sampling device (1) to a fitting (2) mounted on a vessel (3) or pipe of the processing installation or the like, displacing a valve member (14) in a sample passage of the connecting piece (7) from a closed position, in which it abuts a stationary valve part (12) and thereby closes the sample passage, to an open position, in which the sample passage is open, allowing product to pass from the processing installation or the like, through the sample passage, and into a sampling container (4) of the sampling device (1), displacing the valve member (14) from its open position to its closed position, and disconnecting the connecting piece (7) from the fitting (2), whereby the valve member (14) in displaced from its closed position to its open position against a spring-load, and whereby the valve member (14) is automatically displaced to its open position upon connection of the connecting piece (7) with the fitting (2), characterized by that, in the open position of the valve member (14), the product passes through a conical annular passage formed between a conical face forming part of a wall surrounding the inside volume of the sampling container (4) and a truncated cone carried by a spindle extending coaxially through the inside volume of the sampling container. 16. A sampling method according to claim 15, characterized by that the valve member (14) is displaced to its open position by means of a covering member (31) fixedly mounted in the fitting (2) and abutting the valve member (14) during at least part of the operation of connecting the connecting piece (7) to the fitting (2), whereby a circumferential contour (32) of the covering member (31) fits a circumferential contour (33) of the valve member (14), by that a shielding member (25) mounted displaceably in the fitting (2) abuts the stationary valve part (12), whereby a circumferential contour (29) of the shielding member (25) fits a circumferential contour (30) of the stationary valve part (12), and whereby the shielding member (25) is displaced against a spring-load during at least part of the operation of connecting the connecting piece (7) to the fitting (2), and by that, after disconnecting the connecting piece (7) from the fitting (2), the circumferential contour (29) of the shielding member (25) fits the circumferential contour (32) of the covering member (31), and the circumferential contour (30) of the stationary valve part (12) fits the circumferential contour (33) of the displaceable valve member (14). 17. A sampling method according to claim 15 or 16, characterized by that a shut-off valve (22) of the fitting (2) is opened and subsequently closed in the fully connected state of the connecting piece (7) and the fitting (2) in order to allow the product to pass through the fitting (2). 18. A sampling method according to claim 16, characterized by that, in the disconnected state of the connecting piece (7) and the fitting (2), a nozzle (41) is connected to the fitting (2), whereby an internal projection (44) in the nozzle (41) keeps the shielding member (25) displaced against the spring-load in the connected state of the nozzle (41) to the fitting (2), and by that a cleaning or washing fluid is supplied to or discharged from the fitting through the nozzle (41).

Method of identification and quantification of biological molecules and apparatus therefore

A method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair is disclosed. The method comprises interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair, the corresponding second member or members of the binding pair being directly or indirectly tagged with a heavy atom; and determining a spatial distribution of the heavy atom over a surface of the solid support, thereby detecting the binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair.

1-105. (canceled) 106. A method for detecting binding between all least one first member of a binding pair and at least one corresponding second member of the binding pair, the method comprising: interacting a solid support auto which said at least one first member of the binding pair is immobilized and arrayed with said at least one corresponding second member of the binding pair, wherein said at least one corresponding second member of the binding pair is at least one of: (a) directly tagged; and (b) indirectly tagged; with a heavy atom; and determining a spatial distribution of said heavy atom over a surface of the solid support so as to detect binding between said al least one first member of the binding pair and said at least one corresponding second member of the binding pair. 107. The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support has a dynamic range of linearity of at least four orders-of-magnitude. 108. The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a sensitivity of detection equal to at least 1 in 10 binding events. 109. The method of claim 108, wherein said sensitivity is equal to at least 1 in 5 binding events. 110. The method of claim 109, wherein said sensitivity is about 1 in 1 binding events. 111. The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a signal-to-noise ratio of more than 20. 112. The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a signal-to-noise ratio of more than 50. 113. The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a signal-to-noise ratio of more than 80. 114. The method of claim 106, wherein said binding pair is selected from the group consisting of antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten, nucleic acid-complementary nucleic acid, nucleic acid-substantially complementary nucleic acid, ligand-receptor, receptor-ligand, enzyme-substrate, substrate-enzyme, enzyme-inhibitor and inhibitor-enzyme. 115. The method of claim 106, wherein said determining the spatial distribution for said heavy atom over said surface of the solid support is by particle scattering. 116. The method of claim 106, wherein said determining the spatial distribution for said heavy atom over said surface of the solid support is by electron scattering. 117. The method of claim 106, wherein said heavy atom is selected from the group consisting of gold, silver and iron. 118. A method for detecting binding between at least one first member of a binding pair and at least one corresponding second member of the binding pair, the method comprising: interacting a solid support onto which said at least one first member of the binding pair is immobilized and arrayed with said at least one corresponding second member of the binding pair; and determining a spatial distribution of said at least one corresponding second member of the binding hair at a signal-to-noise ratio of more than 10. 119. The method of claim 118, wherein said at least one first member comprises at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. 120. The method of claim 118, wherein said at least one second member comprises at least one macromolecule of a known identity selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates. 121. A method for at least one of identifying and quantifying at least one biological molecule in a sample, the method comprising; contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between said at least one biological molecule and said macromolecules of known identities; and detecting a spatial distribution of said at least one biological molecule over a surface of said microarray at a sensitivity equal to at least 1 in 10 binding events so as to provide at least one of an identification and a quantification of the least one biological molecule in the sample. 122. The method of claim 121, wherein said at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. 123. The method of claim 121, wherein said macromolecules of known identities are selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates. 124. The method of claim 121, wherein said detecting the spatial distribution of said at least one biological molecule over said surface of said microarray has a dynamic range of linearity of at least four orders-of-magnitude. 125. The method of claim 121, wherein said detecting the spatial distribution of said at least one biological molecule over said surface of said microarray has a signal-to-noise ratio of more than 20. 126. The method of claim 121, wherein said detecting the spatial distribution of said at least one biological molecule over said surface of said microarray is by directly or indirectly tagging said at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of said at least one heavy atom. 127. The method of claim 121, wherein the signal-to-noise ratio is greater thin 20. 128. A method for at least one of identifying and quantifying at least one biological molecule in a sample, the method comprising: attaching biological molecules present in the sample to a solid support; contacting said solid support with at least one macromolecule of a known identity under conditions so as to allow binding between said at least one biological molecule and said at least one macromolecule of known identity; and detecting a level of binding between said at least one biological molecule and said at least one macromolecule of known identity by directly or indirectly tagging said at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of said at least one heavy atom, thereby providing at least one of an identification and a quantification of the least one biological molecule in the sample. 129. The method of claim 128, wherein said at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. 130. The method of claim 128, wherein said at least one macromolecule of known identity is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. 131. The method of claim 128, wherein said detecting the level of binding between said at least one biological molecule and said at least one macromolecule of known identity has a dynamic range of linearity of at least four orders-of-magnitude. 132. The method of claim 128, wherein said detecting the level of binding between said at least one biological molecule and said at least one macromolecule of known identity is at a sensitivity equal to at least 1 in 10 binding events. 133. The method of claim 128, wherein said detecting the level of binding between said at least one biological molecule and said at least one macromolecule of known identity is at a signal-to-noise ratio of more than 20. 134. A method for at least one of identifying and quantifying biological molecules in a preparation, the method comprising: localizing and tagging the biological molecules in the preparation; preparing the preparation for vacuum; loading the preparation into the specimen chamber of an electron beam device; irradiating the preparation with an electron beam, thus obtaining an image of the tags; and analyzing the image of the biological molecules by image analysis software so as to provide at least one of an identification and a quantification of the biological molecules in said preparation. 135. A method for at least one of identifying and quantifying biological molecule in a preparation, the method comprising: localizing and tagging the biological molecules in the preparation; lading the preparation into the specimen clamber of an electron beam device; irradiating the preparation with an electron beam, thus obtaining an image of the tags; and analyzing the image of the biological molecules by image analysis software so as to provide at least one of an identification and a quantification of the biological molecules in sail preparation. 136. An apparatus for analysis of at least two samples comprising biological molecules, comprising: an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from said electron source on the surface of a first sample of said at least two samples; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a second sample of said at least two samples in one chamber while simultaneously inspecting said first sample; at least one electron detector; a measuring system for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive said second sample and to provide at least one degree of motion to said second sample while the first sample is being scanned; and an image analysis system for carrying, out image analysis of the molecules of the first and second samples.

<SOH> BACKGROUND OF THE INVENTION <EOH>In the past, genes, proteins, carbohydrates and cells where mainly studied at isolation, greatly limiting the ability to elucidate a realistic picture of the complex array of biochemical processes taking place in living cells. Genomics, proteomics, glycomics and cellomics techniques, which evolved in this sequence during the last decade, aim at analyzing biochemical processes, as complex as these may be, in a more integrated fashion, aiming at looking at all or substantially all of the biochemical changes, large scale changes, as well as minute changes, that take place in living cells under various conditions. Presently, genomics, proteomics, glycomics and cellomics rely on several technologies that are insufficiently quantitative, insufficiently sensitive and are characterized by a relatively low signal-to-noise (S/N) ratio and as such fail to provide a complete insight of cell function. These include the nowadays routine technology of nucleic acid microarrays (e.g., DNA microarrays, also referred to in the art as DNA chips) for analyzing nucleic acid molecules, such as DNA and RNA; the emerging technology of protein (e.g., antigen or antibody) microarrays (also known as protein chips); the recently revived and improved technology of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), both latter techniques serve for analyzing protein molecules; the recently introduced carbohydrate microarrays for the analysis of carbohydrate molecules and various cellomics assays for the analysis of integrated cells. DNA microarrays or DNA chips comprise a plurality of DNA strands (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate In a typical assay in which a DNA microarray is used in the analysis of nucleic acids, single stranded molecules (targets or probes, respectively), typically oligonucleotides or cDNA, tagged with fluorescent markers, are interacted with the substrate, resulting in hybridization of targets and probes according to the DNA parity rules. Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light. Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the sequence of the targets and/or the expression level of a variety of genes. The basic limitation associated with the use of nucleic acid microarrays is the ‘flood’ of poor quality data. Currently about 90% of the data is insignificant. In most cases, weakly expressed genes that can be very important in a biological pathway, are not detected. This limitation arises from the poor signal-to-noise ratio (S/N) and insufficient sensitivity of this technique. It further leads to poor reproducibility. It is difficult to quantify the result of an experiment. The results of seemingly identical experiments also vary considerably. Furthermore, in many cases, the genes of most importance produce a weak signal that is not at all detected. Hence, there is a great need to increase the sensitivity and dynamic range of microarrays and reduce their inherent noise and background levels. These challenges are possibly achievable by substantially miniaturizing the microarrays. The miniaturization is important since it will provide a possibility to imprint larger portions of the genome on the same array (perhaps even the entire human genome). An additional reason for miniaturization is the long period of time required for the target molecules to cover an array by diffusion. The smaller the array, the shorter this time is, in a quadratic manner. nevertheless, the presently employed analyzing techniques, i.e., the use of fluorescent tags and laser scanning, impose great limitations on further miniaturization, both with respect to spatial resolution and with respect to scanning time. An additional type of limitations of the presently employed microarrays arise from the bleaching of the fluorescent tags once analyzed. After an array is scanned, it is bleached, meaning that the fluorescent tags emit less than the required intensity of light. This property significantly reduces the ability of a user to repeat a measurement of a pre-measured experiment. Mainly due to the above limitations, there is no standard by which microarray experiments are performed and/or analyzed, leaving a too large room for personal know-how. In many cases, experiments executed in different laboratories cannot be repeated or even compared. Hence, it is evident that there is a great need for a microarray detection system whose quantification is limited only by the biology. Preferably this system will be based on single molecule detection. This system should be free of the limitations associated with fluorescence-based readings and advantageously should have the following properties: (i) high sensitivity; (ii) high S/N ratio; (iii) compatibility with miniaturization of the microarray, and with smaller sample sizes; (iv) it should not bleach, providing the opportunity to rescan a sample or its regions of interest more than once; and (v) it should be able to incorporate assisted hybridization processes (not only diffusion). One objective of the present invention is to disclose a microarray, scanning method and system, capable of performing high throughput detection on the level of a single molecule. This system is sensitive and reproducible enough to set the industry standard. One option that may be considered is the use of a Scanning Force Microscope in the analysis of microarrays. However, the inherently low throughput of this system prevents it from being practical. The present invention solves the above mentioned limitations by means of scanning electron microscopy. An additional limitation, complementary to quantifying genes in microarrays, is the quantitative study of proteomics (the study of proteins). Proteins determine many biological processes and are very important to drug discovery and many other applications. One tool in proteomics is high resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and the image analysis thereof Electrophoresis is the migration of charged molecules in a solution, in response to an electric field. The rate of migration depends on the strength of the field, on the charge, size and shape of the molecules, as well as on the parameters of the medium through which the molecules are moving. 2D gel electrophoresis is a method to separate molecules that differ in any combination of size or charge. The solution is supported by a gel (agarose, polyacrylamide), which prevents undesired migration (convection, diffusion) and sieves the molecules, thus contributing to their separation on the basis of their sizes. In the present application, this system is referred to as 2D PAGE. The scope is not limited to any particular gel. 2D PAGE systems typically resolve about 1000 proteins according to their isoelectric propertied through a pH gradient in one direction and thereafter according to their size, in the presence of SDS, in a second direction, perpendicular to the first. The abundance of proteins in a cell is within a range from single to millions of molecules. There are many proteins in the gel that are not resolved, partly due to the lack of sensitivity of the separation and partly due to the lack of sensitivity of the detection. There are two typical phases in protein analysis: (i) separation of the proteins, e.g., via 2D PAGE; and (ii) analyzing the types of the separated proteins, typically by mass spectrometry. What is clearly missing is an intermediate stage where the number of proteins in each spot is counted. Preferably the counting method will be able to distinguish between the different proteins. Preferably it will rely on single molecule detection. Currently there is no technology that provides a satisfactory quantitative answer to the issue of how many of the separated proteins exist in each spot. An additional question is how many types of proteins exist in each spot. Thus, there is a need for a technology that counts the number of proteins before they are inserted in the mass spectrometer. The above-mentioned limitations of genomics and proteomics technologies are amplified in the emerging technology of protein microarrays. Protein microarrays or protein chips comprise a plurality of proteins (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate. In a typical assay in which a protein microarray is used in the analysis of proteins, protein molecules (targets or probes, respectively), typically antigens or antibodies, tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity. Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light. Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets and/or the level of their expression. In many cases, comparative competition assays are performed, where a change in the pattern of fluorescence is indicative of the identity of the targets and/or the level of their expression. Protein microarrays will go a long way towards elucidating aspects of cellular functions that DNA chips cannot provide, since measuring mRNA levels alone ignores issues which are of great influence on cellular function, such as, but not limited to, protein lifetime, protein post transnational modifications, etc. Protein chips find uses in two major fields: drug discovery and diagnostics. In drug discovery, processes such as drug candidate discovery and candidate optimization can be greatly assisted should highly sensitive and reliable protein chips and analysis methods were available. In diagnostics, determining titers of viruses and other pathogens, presence, absence or level of cancer and other markers, antibody profiles, etc., could be greatly assisted should highly sensitive and reliable protein chips and analysis methods were available. It is apparent that proteomic tools are essential to obtain information that is unavailable when performing analysis on the gene level. Expressed genes can be subjected to significant post-translational regulation, and proteins undergo significant post-translational modifications (such as phosphorylation, acetylation, etc.) that significantly affect their function at the cellular level. In many cases, no correlation exists between the level of a specific messenger RNA and the level/activity of its encoded protein, because, most of the control of the expression of that protein takes place at the post translational phase. To this effect, see, for example, S. P. Gygi, et al., Mol. Cell. Biol. 19 (1999) 1720-1730). Recently it was shown that it is possible to array different proteins, or protein ligands on a microscope slide to study a variety of protein functions (Macbeath et al., Nature 289, 2000). The basic challenge is that the proteins bound on surface retain their activity and/or their antigenic epitopes. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other macromolecules, e.g., other proteins, or with small molecules in solution. In this study the proteins attached to the slides were probed with fluorescently-labeled proteins. Screening for protein-protein interactions, substrates of protein kinases and targets of small molecules was demonstrated. It often occurs that the less highly expressed proteins are those that are of most interest since their response to various physiological stimuli is the most interesting and informative. Unfortunately, it seems that the low abundance proteins, such as hormones, cytokines, small G-proteins, DNA binding proteins etc., are not easily detected by the present proteomics techniques (S. P. Gygi, Proc. Natl. Acad. Sci. USA 97 (2000) 9390-9395). Detection on the single or close to single molecule level usually requires painstaking techniques that require tedious sample preparation and imaging and are hard to apply to high throughput methods. Carbohydrate microarrays comprise a plurality of carbohydrates (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate. In a typical assay in which a carbohydrate microarray is used in the analysis of carbohydrates, molecules such as antibodies directly or indirectly tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity. Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light. Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets/probes and/or the level of their expression. The limitations described hereinabove with respect to nucleic acid and protein microarrays clearly apply also to carbohydrate microarrays. Cell microarrays comprise a plurality of cells immobilized on a surface of a substrate, which cells can be screened for various properties in a living or fixated state. The limitations described hereinabove with respect to nucleic acid and protein microarrays clearly apply also to carbohydrate microarrays. There is thus a widely recognized need for, and it would be highly advantageous to have, a technique for the implementation of genomics, proteomics glycomics and cellomics devoid of the above limitations. In particular, it would be highly advantageous to have a technique for implementing genomics, proteomics glycomics or cellomics that reaches single molecule detection levels, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple sample preparation, readily applicable for high throughput screening.

<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a method for identification and quantification of biological molecules in a sample that overcomes the drawbacks of the existing methods The term biological molecule includes any molecule with biological relevance. This includes, but is not limited to: polysaccharides, small chemical molecules such as lipids, peptides, hormones and other messengers, ATP GTP etc., drugs, non proteinaceous antigens and any homo- (e.g., protein-protein as example) and hetero- (e.g., drug-protein, DNA-RNA, DNA-protein, etc.) complexes, as well as chemically modifications and derivatisations whether naturally occurring or man made, of all these different molecules. It is another object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is based on single-molecule detection, has a higher sensitivity and signal-to-noise ratio and broader dynamic range than existing methods. It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out without the bleaching that characterizes fluorescence-based detection, thus enabling measuring the same sample several times. It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out with an Environmental Scanning Electron Microscope (ESEM), thus enabling the investigation of a sample at almost atmospheric pressure. It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out with a Wafer Inspection Scanning Electron Microscope (WISEM), thus greatly reducing the cost of instrumentation required to implement the method. It is still a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out on a miniaturized microarray and allows to imprint larger portions of the human genome, or even the entire human genome, on the same array. According to one aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair, the corresponding second member or members of the binding pair being directly or indirectly tagged with a heavy atom; and determining a spatial distribution of the heavy atom over a surface of the solid support, thereby detecting the binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair. Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a sensitivity of detection equals to or greater than 1 of 10 binding events, e.g., i of 5 binding events, most preferably, about 1 of 1 binding events. Yet preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably greater than 50, more preferably greater than 80. According to another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a dynamic range of linearity of at least four orders-of-magnitude. Preferably, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support. Still preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude. Yet preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a sensitivity of detection equals to or greater than I of 10 binding events, e.g., equals to or greater than I of 5 binding events, optimally the sensitivity is about 1 of 1 binding events. Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably greater than 50, more preferably, greater than 80. According to still another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a sensitivity of detection equals to or greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, more preferably, the sensitivity equals to about 1 of 1 binding events. In a preferred embodiment, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support. Preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80. According to yet another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80. Preferably, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support. Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude. Yet preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a sensitivity of detection equals to or greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, most preferably, the sensitivity is about 1 of 1 binding events. According to further features in preferred embodiments of the invention described below, the binding pair is selected from the group consisting of antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten, nucleic acid-complementary nucleic acid, nucleic acid-substantially complementary nucleic acid, ligand-receptor, receptor-ligand, enzyme-substrate, substrate-enzyme, enzyme-inhibitor and inhibitor-enzyme. According to still further features in the described preferred embodiments determining the spatial distribution of the heavy atom over the surface of the solid support is by particle scattering. Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is by electron scattering. According to still further features in the described preferred embodiments the corresponding second member or members of the binding pair is indirectly tagged with a heavy atom. According to still further features in the described preferred embodiments the heavy atom is selected from the group consisting of gold, silver and iron. According to an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a dynamic range of linearity of at least four orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events. Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20. Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom. According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a sensitivity equals to or greater than 1 of 10 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20. Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom. According to yet an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a signal-to-noise ratio greater than 20, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events. Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom. According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events. Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20. According to further features in preferred embodiments of the invention described below, the at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. According to still further features in the described preferred embodiments the macromolecules of known identities are selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates. According to another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a dynamic range of linearity of at least four orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity equals to or greater than 1 of 10 binding events. Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20. Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom. According to still another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a sensitivity equals to or greater than 1 of 10 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20. Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom. According to a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a signal-to-noise ratio greater than 20, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity greater than or equals to 1 of 10 binding events. Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom. According to still a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample. Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude. Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity greater than or equals to 1 of 10 binding events. Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20. According to further features in preferred embodiments of the invention described below, the at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. According to still further features in the described preferred embodiments the at least one macromolecule of known identity is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate. According to another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing and tagging the biological molecules in the preparate; preparing the preparate for vacuum; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image of the tags; and analyzing the image to quantity the biological molecules by image analysis software. According to another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing and tagging the biological molecules in the preparate; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image of the tags; and analyzing the image to quantify the biological molecules by image analysis software. According to still another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing the biological molecules in the preparate; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image representing the biological molecules; and analyzing the image to quantify the biological molecules by image analysis software. According to still another aspect of the present invention there is provided an apparatus for inspection of a preparate of biological molecules comprising an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from the electron source on the surface of the preparate; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a first preparate in one chamber while simultaneously inspecting a second preparate; at least one electron detector; means for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive the preparate and to provide at least one degree of motion to the preparate while the preparate is being scanned; and means for carrying out image analysis of the molecules on the preparate. The biological molecules may be polynucleotides, e.g. DNA, cDNA, RNA, clusters, or proteins such as antigens, antibodies. The term biological molecules also refers but is not limited to: polysaccharides, small chemical molecules such as lipids, peptides, hormones and other messengers, ATP antibodies, GTP, etc., drugs, non proteinaceous antigens and any homo-(protein-protein as example) and hetero- (drug-protein, DNA-RNA, DNA-protein etc.) complexes as well as chemically modifications and derivatisations whether naturally occurring or not of all these different molecules. The preparate for the polynucleotides may be a microarray, e.g., a DNA chip, and for the proteins may be a 2D PAGE, a protein chip, e.g., an antigen or antibody chip, cell chip, cell preparate, and the like. The localization of the biological molecules may be carried out before or after tagging, depending on the type of the biological molecule and of the technique used. When the biological molecule in the preparate is a polynucleotide, the localization may be carried out, for example, by hybridization, either to a polynucleotide of known sequence (probe) when the polynucleotide immobilized in the microarray preparate is of unknown sequence (target), or to a polynucleotide of unknown sequence (target) when the polynucleotide immobilized in the microarray preparate is of a known sequence (probe). The same is true for protein microarrays, with respect to either antigens and/or antibodies, each of which can serve as a target or probe, and in any case can be immobilized to the microarray or be interacted therewith. When the biological molecule in the preparate is a protein, the localization may be carried out, for example, by separating the molecules by one- or two-dimensional electrophoresis, or by attaching the molecules to a blot membrane. When the preparate is a 2D PAGE or proteins extracted therefrom, the separation in the gel may be preferably performed on-line under the scanning electron beam. Identification of the proteins can be done by mass spectrometry. The localization in space may further consist of localizing the molecules by their affiliation to specific biological cells. Tagging of the biological molecules such as DNA, RNA and proteins, may be carried out with heavy metals such as silver or gold, for example using colloidal gold or gold clusters, or doping with metal-enriched organic compounds, wherein the metal is, for example, Fe. The heavy metal colloids (e.g., gold), preferably of diameter range of 1-200 nm, more preferably, less than 20 nm, create a high intensity back scattered electron signal and, therefore, high image contrast. In one embodiment, there is one tag per target molecule. More specifically the tagging may be carried out with biotin followed by gold tagged avidin. Tagging may also be made with electro-luminescent molecules whereby the electron beam creates a light signal that is detected. Tagging may also be done with more than one type of tags to make a distinction between two preparates. According to one embodiment multi-labeling or Multi-tagging is achieved. This is achieved, for example, by using gold colloids of a plurality of sizes. According to another embodiment, multi-labeling is achieved by using a combination of gold colloids and fluorescent labels. According to a yet another embodiment, the multi-labeling is achieved by using a plurality of metals that are read by the X-RAY reading apparatus of the SEM, such as Energy Depressive Spectrum and so forth. According to one embodiment, the DNA molecules are not tagged and the SEM is sensitive enough to detect density differences between hybridized and non-hybridized regions. Direct detection with no tagging enables the identification of an additional variety of substances such as viral particles. The preparates are prepared for vacuum by known standard methods that include drying, fixation and coating with a conductive layer such as carbon, to prevent charge accumulation., protection with a membrane and freezing to prevent out-gassing. The preparates are examined in a particles beam device, preferably, an electron beam device, namely an electron microscope such as a scanning electron microscope (SEM). Presently, most preferably, the preparates are scanned and are analyzed using a wafer inspection SEM (WISEM) typically used in the microelectronics industry. The irradiation of the preparate is carried out in such a way as to form sufficient contrast of the electrons that are back scattered from the tags in comparison with those that are emitted/scattered from the background. In another embodiment, the SEM system is an environmental scanning electron microscope (ESEM) that works at almost atmospheric pressure, thus minimizing the need to prepare the preparate for vacuum. According to a yet another embodiment, the SEM system that allows the proteins to remain in their native wet state and still imaged. This embodiment utilizes a device and method that uses membrane partition. To this end, see U.S. Provisional Patent Application No. 60/250,879, which is incorporated herein by reference. The image analysis may comprise any one of: performing edge detection algorithm to identify the colloids in each region-of-interest (ROI) and counting the colloids; counting fluorescence signals; and identifying X-ray spectrum of each particle for identification by comparison to a reference spectrum. The invention further relates to an apparatus for inspection of a preparate of biological molecules according to the above method, the apparatus comprising an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from the electron source on the surface of the preparate; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a first preparate in one chamber while simultaneously inspecting a second preparate; at least one electron detector; means for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive the preparate and to provide at least one degree of motion to the preparate while the preparate is being scanned by the electron beam; and means for carrying out image analysis of the molecules on the preparate. In one preferred embodiment, the charged particle beam column of is a microcolumn. In a further embodiment, the invention provides a method for the inspection of biological molecules on a preparate using an electron beam, the method comprising localizing the biological molecules in space and tagging them with markers; preparing the preparate for vacuum; taking out the preparate to be analyzed from the preparate cassette; pre-aligning preparate and read preparate number; reading a recipe that contains the information to be detected; loading the preparate on X-Y-T stage (T means tilt) of an electron beam device; aligning the preparate; moving XYT stage to analysis position; positioning the electron beam on the substrate accurately by measuring the position of the substrate; scanning the preparate at low resolution to create a preparate map, while enhancing contrast; determining the regions-of-interest (ROI) spots on the map that should be scanned in a high resolution; scanning the ROIs with the electron beam as the substrate is continuously moving with at least one degree of motion in an x-y plane; detecting electrons emanating from the substrate as a result of previous step and forming an image; enhancing the image contrast; storing both modified and bare image; analyzing the ROIs; and displaying the results. The present invention successfully addresses the shortcomings of the presently known configurations by providing a technique for the implementation of genomics, proteomics, glycomics and cellomics that reaches the highest sensitivity of ultimately single molecule detection, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple preparate preparation, readily applicable for high throughput screening.

Cell phone bungie cord

The “Cell Phone Bungle” is a precision made and engineered device to allow the user to avoid damaging or losing their cell phone or other personal items. It is made of high quality velcro fasteners to secure the cell phone to the bungle and also secure the bungle to a belt loop, belt, gym bag, purse, etc. The bungle is made from vinyl coated memory cord which is considered to be one of the best on the market. The Velcro fastener which secures the bungle to the person of article has a chrome plated swivel fastener to avoid a tangled bungle cord. It is capable of holding up to one pound and maintaining its adhesive qualities from 0 to 110 degrees. This product has been thoroughly tested and inspected by an engineer to guarantee is quality and durability.

1. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” is made of the best materials available to me at the time of manufacture. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle's” memory cord to be approximately 3.0″ in length while relaxed and up to 23.0″ in the extended “in use” position. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” to fit or attach to any small item, ie. beepers, flash lights, remote controls, hunting items, small tools, wallets. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will hold or secure items up to one pound. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will attach to belt loops, belts, gym bags, purse straps or others items used to secure personal belongings as long as less that 1.25″ in diameter. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will maintain its holding ability in temperatures from 0 degrees to 110 degrees. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” that the holding strap is made from Velcro 4.0, to 10″ in length. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will significantly cut the loss or breakage of the items it is used to protect. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” uses a swivel chrome fastener to prevent knotting of bungle cord. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will STOP THAT DROP creating a significant decline in the loss and damage of cell phones and other small personal items when used as directed. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will significantly decline the loss and damage of cell phones and other small personal items when used during biking, recreation vehicles, equine events, fishing, boating, on the Job, construction work, motorcycling, etc. I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” can relieve the stress of constantly looking for your cell phone or possible loss or damage to your cell phone.

Grooved crucible ceramic clay

The nature of the technical disclosures of my invention is simple and unique. A ¼×0.0381 inches groove around a ceramic clay crucible is simple to adapt. I invented a mold which has the feature of ¼×0.0381 inches groove around, to make the casting of a grooved ceramic clay crucible feasible. This is new in the art of casting because the ceramic clay crucibles used in Goldsmith industry do not have a groove around them. The purpose of the groove is to enable anyone who is working in casting precious metals under excessive heat to handle the crucible which is used in the process more efficiently and securely. This groove would certainly improve the melting and casting procedures simply because the strap around the newly grooved crucible would be firmly fastened, thus preventing any spills, and at the same time protecting the worker.

1. A designed Groove for holding a strap around a ceramic clay crucible.

<SOH> BACKGROUND OF INVENTION <EOH>1. Field of Invention Technically eliminates the hazards of spilling hot precious melted metals. Thus giving protection and safety to the one who is handling the molding procedure and at the same time preserving precious melted metals directly in the mold “no waste”. 2. Background Art Goldsmith industries which include molding had been much the same over the decades. Safety first and spilling precious melted metals are my concerns. My invention is to solve these problems which are facing those who work in the industry.

<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The advantages of the invention are: 1. Safety procedures are my primary concerns. 2. Not wasting precious melted metals when put in a mold. 3. Proper handling of the whole process. Brief description of the several views of the figures; listing of all figures by numbers (e.g. FIG. 1A ) and with corresponding statements explaining what each figure depicts. 1. FIG. 1 —A side view of a grooved crucible. 2. FIG. 2 —Shows how the strap is fastened firmly around the groove of the crucible. 3. FIG. 3 —Illustration of the invention. 4. FIG. 4 —Application of the invention. detailed-description description="Detailed Description" end="lead"?

2-3-disubstituted quinuclidiness as modulators of monoamine transporters and theraperutic and diagnostic methods based thereon

The present invention relates to a class of compounds of formula (I) and (II): wherein R1 is hydrogen; linear or branched C1-C15 alkyl; C1-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra, penta substituted aryl or heteroaryl; COOR3; —(CH2)n-aryl; —COO—(CH2)nR3; —(CH2)n—COOR3; —C(O)R3; —C(O)NHR3; or an unsubstituted or substituted oxadiazole; and R2 is hydrogen; linear or branched C1-C15 alkyl; C1-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra, penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; onnthracene; or —(CH2)n-Ph; wherein the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C1-C5 alkyl, C1-C5 alkenyl, H, F, Cl, Br, I, —NO2, NHR, or —OR, R is C1-C7 alkyl; R3 is C1-C5 alkyl, C1-C5 alkenyl, benzyl, substituted aryl or heteroaryl; and n=1-7. These compounds are discovered, synthesized and confirmed as potent inhibitors of dopamine (DA), serotonin (5-HT), and norepinephrine inhibitors. These compounds are therefore particularly useful in the treatment conditions or diseases wherein modulation of the monoamine neurotransmitter system involving dopamine (DA), serotonin (5-HT), and norepinephrine plays a role.

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is of formulae (I) or (II): wherein R1 is a hydrogen; linear or branched C1-C15 alkyl; C2-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; —(CH2)n-aryl; COOR3; —COO—(CH2)nR3; —(CH2)n—COOR3; —C(O)R3; —C(O)NHR3; or an unsubstituted or substituted oxadiazole; R2 is a hydrogen; linear or branched C1-C15 alkyl; C2-C15 alkenyl; C3-C10 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; anthracene; or —(CH2)n-Ph; and R3 is C1-C5alkyl, C2-C5 alkenyl, benzyl, substituted aryl or heteroaryl; and wherein R1 and R2 are independently selected; n=1-7, the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C1-C5 alkyl, C2-C5 alkenyl, H, F, Cl, Br, I, —NO2, NHR, or —OR, wherein R is C1-C7 alkyl. 2. A compound according to claim 1, wherein the compound is of formula (I) and is selected from the group consisting of the (±)-; (+)- and (−) isomers. 3. A method of preparing a compound according to claim 1, wherein the method comprises: (a) preparing a quinuclidinone having a first substituent under Mannich reaction conditions; (b) reacting the product of step (a) to add a second substituent to the quinuclidinone thereby producing the compound. 4. The method of claim 3, further comprising (c) reducing the compound obtained in step (b) to produce a disubstituted quinuclidine of formula (I). 5. The method of claim 4, further comprising chiral separation of the product of step (c) to obtain a compound of formula (I) in non-racemic enantiomer form. 6. The method of claim 5, wherein the chiral separation produces a (+)- enantiomer or (−)- enantimer. 7. A method of treatment of a condition or disease wherein dopamine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound according to claim 1. 8. A method of treatment of a condition or disease wherein serotonin flow plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound according to claim 1. 9. A method of treatment of a condition or disease wherein norepinephrine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment a compound according to claim 1. 10. A method for the treatment of cocaine abuse in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 11. A method for the treatment of depression in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 12. A method for the treatment of anxiety in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 13. A method for the treatment of an eating disorder in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 14. A method for the treatment of Parkinson's disease in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 15. A method for the treatment of Alcoholism in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 16. A method for the treatment of a neurological disorder in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 17. A method for the treatment of chronic pain in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 18. A method for the treatment of obsessive compulsive disorder in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1. 19. A compound according to claim 1, wherein the compound is 2-Butyl-3-phenylquinuclidine. 20. A compound according to claim 1, wherein the compound is 2-Butyl-3-(4-methylphenyl)quinuclidine. 21. A compound according to claim 1, wherein the compound is 2-Butyl-3-(4-chlorophenyl)quinuclidine 22. The compound of claim 19, wherein the compound is in substantially pure (+)- or (−)- form. 23. The compound of claim 20, wherein the compound is in substantially pure (+)- or (−)- form. 24. A compound according to claim 1, wherein the compound is compound 16 or compound 17 as shown in Table 2. 25. The compound of claim 21 in substantially pure (+)- or (−)- form. 26. A compound according to claim 1, wherein the compound is selected from the compounds listed in Table 2. 27. A method of diagnosis of a condition wherein at least one of dopamine, serotonin and norepinephrine flow plays a role, the method comprising contacting a sample of body fluid with a compound according to claim 1, wherein the compound is labeled. 28. The method of claim 27 wherein the compound is labeled with a radioactive agent. 29. The method of claim 27, wherein the compound is labeled with a fluorescent agent. 30. The method of claim 27, wherein the compound is labeled with an electromagnetic moiety. 31. The method of claim 27, wherein the compound is conjugated to an antibody. 32. A compound according to claim 1, wherein the compound is labeled with a label selected from the group consisting of a radioactive agent and a fluorescent agent. 33. A method of treatment of a condition involving an antigen, wherein the method comprises administering to a subject a compound according to claim 1, wherein the compound is conjugated to an antibody that binds to the antigen. 34. The method of claim 33, wherein the compound of claim 1 is labeled.

<SOH> BACKGROUND OF THE INVENTION <EOH>1. Related Applications This application is based on U.S. Provisional Application Ser. No. 60/226,581, filed Aug. 21, 2000, the contents of which are hereby incorporated by reference in their entirety. 2. Field of the Invention The present invention relates to discovery, synthesis and enantiomer separation of compounds 2,3-disubstituted quinuclidines as potent inhibitors for dopamine, serotonin and norepinephrine transporters and therapeutic uses of such compounds. 3. Summary of the Related Art The specific reuptake of the monoamine neurotransmitters, dopamine (DA), serotonin (5-HT), and norepinephrine (NE) from the synaptic cleft is the primary physiological mechanism for the termination of monoaminergic neurotransmission. Blocking the uptake increases synaptic availability of the neurotransmitters, thereby potentiating the signal (Kitayama, S. Dohi, T. Jpn. Pharmacol. 1996, 72, 195-208). This has been exploited to develop treatments for a large number of neurological disorders. The selective serotonin transporter (SERT) inhibitor, such as fluoxetine (Prozac) is used for the treatment of depression. The selective dopamine transporter (DAT) inhibitor, benzotropine, is used clinically for the treatment of Parkinson's disease. Other potent and selective DAT inhibitors such as RTI-113 and GBR 12909 are now in clinical trials for the treatment of cocaine abuse. Norepinephrine transporter (NET) inhibitors such as desipramine are effective in the treatment of depression. The present invention relates to a novel class of compounds, 2,3-disubstituted quinuclidines as potent inhibitors of dopamine, serotonin and norepinephrine transporters and their therapeutic use. Potent, long-duration DAT inhibitors with no or little abuse liability themselves can be used for the treatment of cocaine abuse. One aspect of the present invention can be used as novel therapeutic agents for the treatment of cocaine abuse. Cocaine abuse is one of the greatest concerns of the American public today, and has therefore become a focus of medical, social, and political debate. Cocaine is one of the most addictive substances known, and cocaine addicts may lose their ability to function at work or in interpersonal situations. Although cocaine potently inhibits the reuptake of both norepinephrine (NE) and serotonin (5-HT), many lines of evidence indicate that its ability to act as a reinforcer stems from its ability to inhibit the reuptake of dopamine (DA) into dopaminergic neurons. Cocaine exerts this effect via specific interaction with DA transporter (DAT) proteins (cocaine receptor) located on DA nerve terminals. This increase of dopaminergic transmission in the reward mediating brain mesolimbic system is the essence of the dopamine hypothesis for cocaine action. However, recent studies have shown that the simultaneous flow of dopamine, serotonin and norepinephrine plays an important role in the molecular mechanisms involved in addiction to cocaine. A common molecular aspect to the flow of dopamine, serotonin and norepinephrine involves monoamine transporters. Therefore, it would be greatly beneficial if a class of small molecule compounds could be identified or designed to modulate the activity of monoamine transporters, thereby simultaneously modulating the uptake of dopamine serotonin and norepinephrine by monoamine transporters. Such novel compounds and therapeutic and diagnostic methods based thereon will be greatly beneficial in the treatment of numerous neurological disorders. Of particular interest are lead compounds capable of antagonizing all or some of cocaine action.

<SOH> SUMMARY AND OBJECTS OF TILE INVENTION <EOH>It is an object of the invention to provide compounds which inhibit abnormal dopamine signaling in the synaptic space in neurons. It is another object of the invention to provide compounds which are antagonistic of cocaine. Another object of the invention is to provide a method for modulation of brain dopamine flow in a subject in need of such control. The method comprises administering to the subject a compound identified according to the above-described method. Yet another object of the invention is to provide a method of inhibiting cocaine action in a subject in need of such inhibition comprising administering to the subject a compound identified according to the method described above. A still further object of the invention is to provide a method of promoting dopamine reuptake action in a subject in need of such action comprising administering to said subject a compound identified according to the method described above. In one aspect, the invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is of formulae (I) or (II): wherein R 1 is a hydrogen; linear or branched C 1 -C 15 alkyl; C 1 -C 15 alkenyl; C 3 -C 6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; —(CH 2 ) n -aryl; COOR 3 ; —COO—(CH 2 ) n R 3 ; —(CH 2 ) n —COOR 3 ; —C(O)R 3 ; —C(O)NHR 3 ; or an unsubstituted or substituted oxadiazole; R 2 is a hydrogen; linear or branched C 1 -C 15 alkyl; C 1 -C 15 alkenyl; C 3 -C 6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; anthracene; or —(CH 2 ) n -Ph; and R 3 is C 1 -C 5 alkyl, C 1 -C 5 alkenyl, benzyl, substituted aryl or heteroaryl; and n=1-7; and wherein the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C 1 -C 5 alkyl, C 1 -C 5 alkenyl, H, F, Cl, Br, I, —NO 2 , NHR, or —OR, wherein R is C 1 -C 7 alkyl. The compounds of formula (I) are preferably prepared and isolated in an enantiomeric form selected from the group consisting of the (±)-; (+)- and ( − ) isomers. Another aspect of the invention provides a method of preparing a compound according to the invention, wherein the method comprises:(a) preparing a quinuclidinone having a first substituent under Mannich reaction conditions; and (b) reacting the product of step (a) to add a second substituent to the quinuclidinone thereby producing the compound. The method of the invention, further comprises (c) reducing the compound obtained in step (b) to produce a disubstituted quinuclidine of formula (I). The invention also provides a method of treatment of a condition or disease wherein dopamine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above. The invention also provides a method of treatment of a condition or disease wherein serotonin flow plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above. The invention also provides a method of treatment of a condition or disease wherein norepinephrine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above. One particularly advantageous aspect of the invention provides a method for the treatment of cocaine abuse in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound of formulae (I) or (II) as described above. The compounds of the invention are greatly advantageous in the treatment of various neurological disorders that involve the dopamine, serotonin and/or norepinephrine monoamine transmitter reuptake. The compounds of the invention are particularly useful in the treatment of condition such as clinical depression, anxiety, Alcoholism, eating disorders and Parkinson's disease. The compounds of the invention are also useful in the treatment of chronic pain and obsessive compulsive disorders by modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to a subject a compound according of formulae (I) or (II). Preferred compounds according to the invention include 2-Butyl-3-phenylquinuclidine, preferably in substantially pure (±)- enantiomeric form, and 2-Butyl-3-(4-methylphenyl)quinuclidine, preferably in substantially pure (±)- or (+)- enantiomeric form. Other preferred compounds of the invention are listed in Table 2. In another aspect, the invention provides a method of diagnosis of a condition wherein modulation at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake plays a role, the method comprising contacting a sample of body fluid with a compound of formulae (I) or (II), wherein the compound is labeled. Preferred labeling agents include radioactive agents, fluorescent agents and labeling agents containing a traceable electromagnetic moiety.

Compact Sewage Secondary Treatment System

A treatment tank (1) for the secondary treatment of sewage, for providing the processes of aeration, nitrification and denitrification, in a single structure, for which a single, small horsepower, effluent pump (26) is the only moving part. In the treatment tank (1), the sewage is subjected to two separate biological treatments, in two separate chambers under different conditions. One biological treatment is carried out under anoxic conditions in a pipe coil (3). Anoxic conditions are ensured by keeping the pipe coil (3) full at all times; the pipe coil axis is vertical, and the pump forces the fluid flow upwardly through the coil. The second biological treatment is carried out under aerobic conditions in a trickle down filter (8). In a preferred embodiment, a welded pipe coil (3) used both to provide the anoxic conditions and to provide a tank containing the trickle down filter (8). The secondary treatment tank is generally used as part of a raw sewage treatment system, which will include a recycle loop which ensures that even when there is no raw sewage entering the system there is always a flow of liquid through the treatment tank.

1. A secondary sewage treatment apparatus comprising in combination in sequence: (a) a sewage inflow means; (b) a circulating pump means; (c) a coil of pipe having an inlet and an outlet, the outlet being disposed vertically higher than the inlet; (d) a treatment tank containing a trickle down filter; (e) a collection means to receive the flow of sewage from the trickle down filter; and (f) an effluent outflow means; wherein: (i) the circulating pump means provides a flow of sewage from the sewage inflow means to the pipe coil inlet at a pressure sufficient to provide a flow of sewage at the pipe coil outlet; (ii) the pipe coil outlet is constructed and arranged to pass sewage to the trickle flow filter; (iii) the collection means is constructed and arranged to receive a flow of treated sewage from the trickle down filter; and (iv) the outflow means is constructed and arranged to receive the flow of treated sewage collected by the collection means. 2. A secondary sewage treatment apparatus according to claim 1 wherein the pipe coil is substantially cylindrical with its axis vertical. 3. A secondary sewage treatment apparatus according to claims 1 wherein the pipe coil is substantially cylindrical with its axis vertical, and the pipe coil is welded together to provide a cylindrical wall for the treatment tank. 4. A secondary sewage treatment apparatus according to claim 1 or 2 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is nested inside the treatment tank. 5. A secondary sewage treatment apparatus according to claim 1 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is nested inside the treatment tank. 6. A secondary sewage treatment apparatus according to claim 1 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is wound around the outside of the treatment tank. 7. A secondary sewage treatment apparatus according to claim 2 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is wound around the outside of the treatment tank. 8. A secondary sewage treatment apparatus according to claim 1 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means. 9. A secondary sewage treatment apparatus according to claim 3 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means. 10. A secondary sewage treatment apparatus according to claim 1 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means. 11. A secondary sewage treatment apparatus according to claim 3 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means. 12. A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate at least one half of the flow of treated sewage. 13. A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate at least one half of the flow of treated sewage. 14. A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate about one half of the flow of treated sewage. 15. A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate about one half of the flow of treated sewage. 16. A secondary sewage treatment apparatus according to claim 12 wherein the sewage inflow means includes a mixing tank where inflowing sewage is mixed with re-circulated treated sewage from the outflow means. 17. A secondary sewage treatment apparatus according to claim 13 wherein the sewage inflow means includes a mixing tank where inflowing sewage is mixed with re-circulated treated sewage from the outflow means. 18. A secondary sewage treatment apparatus according to claim 1 wherein the effluent outflow means further includes a tank with a hydraulic balancing means constructed and arranged to maintain a minimum of sewage recirculating through the treatment tank, even when there is no flow of raw sewage into the treatment system. 19. A secondary sewage treatment apparatus according to claim 3 wherein the effluent outflow means also includes a tank with a hydraulic balancing means constructed and arranged to maintain a minimum of sewage recirculating through the treatment tank, even when there is no flow of raw sewage into the treatment system.

Lighting system and method

A lighting system and method (1) is disclosed having a pulse-width modulation (PWM) lighting system and method for use in environments where the PWM ratio is variable, for example though not exclusively, in 42V and/or dual-voltage electrical systems in automotive applications.

1. A lighting system for driving light bulbs having a lower voltage tolerance than the voltage supply to the system, comprising: a pulse wave modulator converter supplied with a required voltage from a system source voltage supply of the system for regulating an average output signal of the pulse wave modulator converter supplied to a register to selectively connect the output signal of the pulse wave modulator converter to a light bulb selected by the converter supply from a plurality of light bulbs, each light bulb having a lower voltage tolerance than the system voltage, the register and converter synchronised via a clock source, wherein the converter drives a single light bulb at any one time even with a variable PWM mark-space ratio. 2. A lighting system as claimed in claim 1, wherein the system further comprises a light bulb driver connected between each light bulb and register. 3. A lighting system as claimed in claim 1 or 2 wherein the pulse wave modulator comprises a comparator with one input receiving a required voltage, and another input receiving the output signal of the converter from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal. 4. A lighting method for driving light bulbs having a lower voltage tolerance than the voltage supply to the system, comprising the steps or: supplying a required voltage from system source voltage from a system source voltage supply; regulating an average output signal from a pulse wave modulator converter supplied with the system source voltage from the source system supply; supplying the regulated average output signal to a register, the register and converter synchronised via a clock source; and selectively connecting the output signal of the pulse wave modulator converter from the register to a single light bulb at any one time from a plurality of light bulbs, even with a variable PWM mark-space ratio, wherein each light bulb having a lower voltage tolerance than the system voltage supply. 5. A lighting method as claimed in claim 4, wherein the step of selectively connecting the output signal of the pulse wave modulator converter from the register to a single light bulb further comprises a light bulb driver connected between each light bulb and register. 6. A lighting method as claimed in claim 4 or 5 wherein the step of regulating an average output signal from the pulse wave modulator converter further comprises comparing at a comparator in the pulse wave modulator converter one input receiving the required voltage, with another input receiving the output signal from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal. 7. A lighting system substantially as hereinbefore described and with reference to the drawings. 8. A lighting method substantially as hereinbefore described and with reference to the drawings. 9. A lighting system as claimed in claim 2 wherein the pulse wave modulator comprises a comparator with one input receiving a required voltage, and another input receiving the output signal of the converter from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal. 10. A lighting method as claimed in claim 5 wherein the step of regulating an average output signal from the pulse wave modulator converter further comprises comparing at a comparator in the pulse wave modulator converter one input receiving the required voltage, with another input receiving the output signal from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal.

<SOH> BACKGROUND OF THE DISCLOSURE <EOH>Lighting of incandescent lamps or light bulbs in, for example, automotive applications typically involves a driving a 12V filament. However, developments in the automotive industry have been made to introduce vehicles with 42V electrical systems and dual voltage electrical systems. Vehicles with dual voltage electrical systems require two batteries having nominal voltages of 14V and 42V (12V and 36V rated batteries respectively). The 12V battery typically has a high amp-hour rating and is used to provide energy to 14V loads such as lighting circuits, which are difficult to implement at higher voltages, and other applications for example driving 12V resistive loads, like small heaters, and 12V inductive loads, like motors, relays, solenoids. The 36V battery typically has a high cranking current capability and is coupled to a 42V generator and higher voltage loads, which may include the engine starter motor. However, compared with single voltage electrical systems, having an additional power rail such as the 12V power rail in the dual-voltage electrical system increases cost, adds additional weight and reduces efficiency in the system. Vehicles with 42V electrical systems require a 36V rated battery that is coupled to a 42V generator, as in the dual voltage electrical system. Clearly, in 42V electrical systems all electrical systems, including the lighting system, must be driven by the 36V rated battery. There are currently several lighting arrangements for 42V and dual voltage electrical systems. A first lighting arrangement that is presently not very practical is to drive incandescent lamps with 42V filaments. However, filaments for 42V/36V are too long and fragile. The 42V/36V bulb filaments are relatively thin filaments when compared to filaments in 12V bulbs, for example, for the same power at 42V/36V filament of the same diameter would be 9 times the length, or alternatively ⅓ the diameter of a 12V filament. 43V/36V bulbs are presently unpractical to use because the thin filaments results in a lifetime that would be unacceptably low in the automotive environment. Another lighting arrangement that has been proposed for 42V and dual voltage electrical systems is a DC-DC converter to step 42V down to 14V/12V. Although this system provides the convenience of 12V bulbs, it is an expensive solution to implement the DC-DC converter and such a system consumes, for example, more than 300 W. For these reasons, using a DC-DC converter is also presently not practical to implement in an automotive environment. There are also lighting systems that us PWM for driving lamps. Such a system is disclosed in SU909805. PWM systems typically operate at 120 Hz to replicate 50 to 60 Hz AC operation. However, especially where multiple lights are switched on together, very high peak currents, which may rupture the bulb filament, are reached in the wiring of the lighting system resulting in excessive heat and electromagnetic interference. In 42V applications, for example, when a bulb is first switched on a large current surge flows in a 42V/36V PWM system, which may be three times as large as 12V DC system, which may rupture the bulbs filament. For these reasons, such PWM lighting systems are unacceptable in an automotive environment. Therefore, there is a need in the art for a cost effective and power efficient way to power a bulb with a lower voltage tolerance, for example 12V or 6-8V, from a higher voltage supply, for example 42V/36V as used in automotive systems and/or 24V as used in truck systems, in an environment wherein the PWM mark-space ratio is variable such as for example in a 42V or dual-voltage electrical automotive systems.

<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Embodiments of the invention will now be more fully described, by way of example, with reference to the drawings, of which: FIG. 1 is a schematic block diagram of lighting apparatus according to a an embodiment of the invention; FIG. 2 is a schematic block diagram of a lighting apparatus according to an embodiment of the invention; and FIG. 3 is a graph charting the timing of signals according to an embodiment of the invention. detailed-description description="Detailed Description" end="lead"?

Haemophilus influenzae lipopolysaccharide inner-core oligosaccharide epitopes as vaccines for the prevention of haemophilus influenzae infections

The present invention relates to a lipopolysaccharide moiety comprising a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extension, and to vaccines obtaines therefrom which are cross-reactive for Haemophilus influenzae strains. The invention also relates to defined mutations in the biosynthetic machinery for lipopolysaccharide (LPS;) expression in Haemophilus influenzae useful to obtain the abovementioned moiety. The invention also relates to using conjugates of the LPS from the mutant strains so obtained to elicit a heterologous immune response against a wide range of disease-causing H. influenzae strains. More specifically, the invention relates to vaccines for prevention of bacterial infections comprising core lipopolysaccharide of Haemophilus influenzae. A lipopolysaccharide moiety comprising a conserved triheptosylinner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions.

1. A lipopolysaccharide moiety consisting essentially of a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions. 2. A lipopolysaccharide moiety consisting essentially of a triheptosyl inner-core moiety of lipopolysaccharide having the structure I: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, and Kdo is 3-deoxy-D-manno-2-octulosonic acid. 3. A lipopolysaccharide moiety consisting essentially of a triheptosyl inner-core moiety of lipopolysaccharide having the structure II: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, and P-Petn is pyrophosphoethanolamine. 4-18. (canceled) 19. The lipopolysaccharide moiety of claim 2, wherein HepII is substituted at the 6 position by Petn or a functional equivalent. 20. The lipopolysaccharide moiety of claim 2, substantially free of oligosaccharide chain extensions which are cross-reactive with mammalian tissue. 21. The lipopolysaccharide moiety of claim 2, substantially free of oligosaccharide chain extensions which mimic human tissue antigens. 22. The lipopolysaccharide moiety of claim 2, wherein an O-acyl is substituted at any position within the triheptosyl inner-core moiety thereof. 23. An immunogenic composition for conferring protection in an animal host against a disease caused by Haemophilus influenzae, comprising the lipopolysaccharide moiety defined in claim 2. 24. The immunogenic composition of claim 23 which is a conjugated vaccine. 25. Use of at least one gene in a biosynthetic pathway for the production of lipopolysaccharide in Haemophilus influenzae to obtain a Haemophilus influenzae strain comprising the lipopolysaccharide moiety defined in claim 2. 26. Use of at least one inmunogenic epitope to elicit a functional cross-reactive antibody against Haemophilus influenzae wherein the epitope comprises the lipopolysaccharide moiety defined in claim 2. 27. The use of claim 26 wherein the Haemophilus influenzae is non-typeable Haemophilus influenzae. 28. A functional antibody which is cross-reactive against Haemophilus influenzae and which is elicited by the lipopolysaccharide moiety defined in claim 2. 29. A method for the production of a functional cross-reactive antibody against Haemophilus influenzae which comprises: (a) generating antibodies to the lipopolysaccharide moiety defined in claim 2, (b) testing such antibodies against a plurality of Haemophilus influenzae strains, and (c) selecting those antibodies which are cross-reactive. 30. The method of claim 29, wherein the Haemophilus influenzae is non-typeable Haemophilus influenzae. 31. A method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition defined in claim 23. 32. A method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition defined in claim 24. 33. The method of claim 31, wherein the Haemophilus influenzae is non-typeable Haemophilus influenzae. 34. The method of claim 31, wherein the disease is selected from the group consisting of otitis media, meningitis, pneumonia, and respiratory tract infection. 35. The method of claim 33, wherein the disease is selected from the group consisting of otitis media, meningitis, pneumonia, and respiratory tract infection.

<SOH> BACKGROUND OF THE INVENTION <EOH>Haemophilus influenzae is a major cause of disease worldwide. Six capsular serotypes (“a” to “f”) and an indeterminate number of acapsular (non-typeable) strains of H. influenzae are recognised. Type b capsular strains are associated with invasive diseases, including meningitis and pneumonia, while non-typeable H. influenzae (NTHi) is a primary cause of otitis media in children and respiratory tract infections in adults. Otitis media is a common childhood disease which accounts for the highest frequency of paediatric visits in the United States (Stool et al., Pediatr. Infect. Dis. Suppl., 8:S11-S14, 1989). The development of a vaccine for NTHi diseases has proved difficult because of a lack of understanding of the antigens that confer protective immunity. Efforts in developing a NTHi vaccine have been focused on cell surface antigens such as outer membrane proteins and pili or fimbria (Kyd et al., Infect. Immun., 63:2931-2940, 1995; Deich et al., Vaccine Res., 2:31-39, 1995). Recent advances in molecular genetics, molecular structure analysis and immunochemistry provide powerful tools which have permitted the identification of carbohydrate: structures as candidate vaccine antigens. Gram-negative bacteria have an outer membrane comprised of components including proteins, lipoproteins, phospholipids, and glycolipids. The glycolipids comprise primarily endotoxin lipopolysaccharides (LPS). LPS are molecules comprised of a) a Lipid A portion which consists of a glucosamine disaccharide that is substituted with phosphate groups and long chain fatty acids in ester and amide linkages; b) a core polysaccharide which is attached to Lipid A by an eight carbon sugar, Kdo (ketodeoxyoctonate), and heptose, glucose, galactose, and N-acetylglucosamine; and, optionally, c) o-specific side chains comprised of repeating oligosaccharide units which, depending on the genera and. species of bacteria, may contain mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a dideoxyhexose (abequose, colitose, tyvelose, paratose, trehalose). LPS which lacks repeating O-side chains is sometimes referred to as short chain lipopolysaccharide, or as lipooligosaccharide (LOS). In this application, the term lipopolysaccharide (or LPS) includes short chain lipopolysaccharide and lipooligosaccharide (and LOS). The major antigenic determinants of gram-negative bacteria are believed to reside in the complex carbohydrate structure of LPS. These carbohydrate structures vary significantly, even among different species of the same genus of gram-negative bacteria, primarily because of variations in one or more of the sugar composition, the sequence of oligosaccharides, the linkage between the monomeric units of the oligosaccharides and between the oligosaccharides themselves, and substitutions/modifications of the oligosaccharides (particularly the terminal oligosaccharide). For this reason, development of a vaccine having a broad spectrum effect against Haemophilus influenzae (particularly against NTHi) has been unsuccessful. LPS is a bacterial component which has potential as a vaccine immunogen because of the antigenic determinants (“epitopes”) residing in its carbohydrate structures. However, the chemical nature of LPS detracts from its use in vaccine formulations; i.e., active immunization with LPS is unacceptable due to the inherent toxicity, in some animals, of the Lipid A portion. The pathophysiologic effects induced (directly or indirectly) by Lipid A of LPS in the bloodstream include fever, leucopenia, leucocytosis, the Shwartzman. reaction, disseminated intravascular coagulation, abortion, and in larger doses, shock and death. It has been established that vaccines comprised of capsular polysaccharides are effective at preventing human disease caused by the homologous encapsulated bacteria. These carbohydrate antigens are often poorly immunogenic in humans due to a lack of T-cell dependent response. However, by conjugating the specific polysaccharide antigen to a suitable protein carrier, the immunogenicity of the carbohydrate antigen can be greatly enhanced in patients who do not respond to the polysaccharide alone. Glycoconjugate vaccines based on the specific capsular polysaccharide of type b H. influenzae (Hib), e.g. ProHiBit™, and ActHib™, have already proven successful in the control of invasive Hib disease in infants. Capsular polysaccharide-protein conjugate Hib vaccines do not provide protection against disease caused by acapsular (non-typeable) strains of H. influenzae (i.e. against disease caused by NTHi) because they are only protective against infections caused by H. influenzae strains bearing the type b capsule. Lipopolysaccharide (LPS) is a major NTHi cell surface antigen. LPS of Haemophilus influenzae has only been found to contain lipid A and oligosaccharide (OS) components. Because the lipid A component of LPS is toxic, it must be detoxified prior to conjugation to an immunogenic carrier, as discussed above. Barenkamp et al. (Pediatr. Infect. Dis. J., 9:333-339, 1990) demonstrated that LOS stimulated the production of bactericidal antibodies directed against NTHi. McGehee et al. (Am. Journal Respir. Cell Biol., 1:201-210, 1989) showed that passive immunization of mice with monoclonal antibodies directed against LOS from NTHi enhanced the pulmonary clearance of NTHi. Green et al. (Vaccines, 94:12S-129, 1994) disclose a NTHi vaccine comprising a conjugate of NTHi oligosaccharide and the mutant nontoxic diphtheria protein CRM.sub.197. The lipid A moiety was removed from LOS by treatment with acid, followed by derivatizing the resulting OS with adipic acid dihydrazide (ADH) and coupling to CRM.sub.197. Despite the showing of Barenkamp et al. that LOS stimulated production of bactericidal antibodies against NTHi, the conjugates of Green et al. were determined to be poorly immunogenic after injection into mice. Moreover, the conjugates did not elicit bactericidal antibodies against NTHi. Gu et al. (U.S. Pat. No. 6,207,157) is concerned with the detoxification of isolated NTHI LOS by removal of ester-linked fatty acids therefrom, so that it may be made suitable for vaccine preparation. However, Gu does not describe any other modifications to or desired chemical attributes of NTHi LOS. There is currently no vaccine available to provide broad spectrum protection against infections caused by Haemophilus influenzae . Thus, there is a need for a vaccine having broad spectrum efficacy against Haemophilus influenzae , particularly NTHi. In order to utilise an antigen for vaccine development, four essential criteria must be fulfilled. That is, the immunogenic epitope must be: 1. genetically stable; 2. conserved in all clinically relevant strains across the species; 3. accessible (in vitro and in vivo) to host immune mechanisms; and, 4. able to induce protective antibodies in vivo. There is a need to identify LPS carbohydrate epitopes of H. influenzae , particularly NTHi, which satisfy these criteria.

<SOH> SUMMARY OF THE INVENTION <EOH>The subject invention is directed at immunity providing B-cell activating molecules derived from H. influenzae lipopolysaccharide (LPS), said molecules comprising one or more epitopes of a conserved inner-core oligosaccharide portion of the lipopolysaccharide. The invention discloses a strategy to identify and characterise said epitopes that are representative of LPS expressed by H. influenzae strains across the range of disease causing isolates. The invention is also directed at methods for obtaining such epitopes, both synthetically and through genetic engineering techniques. Furthermore, the invention relates to methods for preparing conjugates of molecules comprising said epitopes with suitable carriers, optionally in liposome formulations. In one aspect, the invention provides a lipopolysaccharide moiety comprising a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions. In another aspect, the invention provides a lipopolysaccharide moiety comprising a triheptosyl inner-core moiety of Haemophilus influenzae lipopolysaccharide having the following structure [I]: wherein R is hydrogen or phosphoethanoelamine, Glcs is D-glucopyranose, and Kdo is 3-deoxy-D-manno-2-octulosnic acid. In another aspect, the invention provides a lipopolysaccharide moiety comprising a triheptosyl inner-core moiety of Haemophilus influenzae lipopolysaccharide having the following structure [II]: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, and P-Petn is pyrophosphoethanolamine. In another aspect, the invention provides an immunogenic composition for conferring protection in an animal host against a disease caused by Haemophilus influenzae , comprising either of the lipopolysaccharide moieties described above. In another aspect, the invention provides a use of at least one gene in a biosynthetic pathway for the production of lipopolysaccharide in Haemophilus influenzae to obtain a Haemophilus influenzae strain comprising either of the lipopolysaccharide moieties described above. In another aspect, the invention provides a use of at least one immunogenic epitope to elicit a functional cross-reactive antibody against Haemophilus influenzae wherein the epitope comprises either of the lipopolysaccharide moieties described above. In another aspect, the invention provides a functional antibody which is cross-reactive against Haemophilus influenzae and which is elicited by either of the lipopolysaccharide moieties described above. In another aspect, the invention provides a method for the production of a functional cross-reactive antibody against Haemophilus influenzae which comprises: (a) generating antibodies to either of the lipopolysaccharide moieties described above, (b) testing such antibodies against a plurality of Haemophilus influenzae strains, and (c) selecting those antibodies which are cross-reactive. In another aspect, the invention provides a method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition described above.

Bacterial Toxin Adsorbing Material, Method of Removing the Toxin by Adsorbing, and an Adsorber Formed by Filling the Adsorbing Material Therein

The invention aims at providing an adsorbent for bacterial toxins, a method for removal of such toxins by adsorption, and an adsorber packed with said adsorbent. Provided are an adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms, a method for removal of bacterial toxins using said adsorbent, and an adsorber packed with said adsorbent.

1. An adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms. 2. The adsorbent according to claim 1, wherein the water-insoluble porous material comprises a polystyrene-based material. 3. The adsorbent according to claim 1, wherein the water-insoluble porous material comprises an active carbon and/or a substance similar thereto. 4. The adsorbent according to claim 1, wherein the water-insoluble porous material comprises an acrylic material. 5. The adsorbent according to any one of claims 1 to 4, which is intended for use in adsorbing at least one bacterial toxin selected from the group consisting of superantigens, toxins having enterotoxin activity, and staphylococcus-derived pathogenic factors. 6. A method for removal of bacterial toxins by adsorption, which comprises the step of contacting the adsorbent according to any one of claims 1 to 5 with a bacterial toxin-containing fluid. 7. An adsorber for bacterial toxins, which comprises the adsorbent according to any one of claims 1 to 5 as packed in a container having a fluid inlet and a fluid outlet and equipped with a device for preventing the adsorbent from flowing out of the container.

<SOH> BACKGROUND ART <EOH>A bacterial toxin may be defined as “a substance which is a bacterial metabolite or constituent and causes, in a trace amount, an unfavorable response in the living body”. Bacterial toxins are roughly classified into two classes, endotoxins and exotoxins, according to the site of occurrence. The former are also called as protein toxins, and the latter as lipopolysaccharides (hereinafter referred to as “LPSs”). There are a large number of bacterial toxins, and the number of such toxins so far known now stands at about 200. Typical examples are cholera toxin, staphylococcal α toxin, botulinum toxin, tetanus toxin, enterotoxin (10 species are already known, namely staphylococcal enterotoxin A, B, C1, C2, C3, D, E, G, H, and I; hereinafter referred to as SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, and SEI, respectively), verotoxins, diphtheria toxin, pertussis toxin, Toxic shock syndrome toxin-1 (hereinafter referred to as TSST-1), and endotoxins. Among these, enterotoxins and TSST-1 are also called as superantigens. In the case of an ordinary antigen, it is taken up by an antigen-presenting cell and fragmented (into peptides composed of 10 to 15 amino acids) therein, and an antigen fragment, in the form bound to a pocket site of a MHC (major histocompatibility complex) class II molecule, is presented on the antigen-presenting cell surface. This is recognized by the α chain and β chain of the TCR (T cell receptor) of a specific T cell clone, and the T cell is activated and the immune response goes on. On the other hand, in the case of a superantigen, the antigen is not fragmented but binds directly to a MHC class II molecule on the antigen-presenting cell. This is further recognized by the TCR on a T cell, whereby the T cell is activated. On that occasion, the antigen is recognized through a specific Vβ region of the TCR. Unlike ordinary antigens, that antigen is recognized by almost all members of a T cell population expressing this specific Vβ region, and T cells are thereby activated and causing cytokine production. Thus, when an individual is exposed to a superantigen, a huge number of T cells are activated as compared with the ordinary specific immune response and the release of a cytokine, for instance, occurs in a short period of time, supposedly causing an abnormal reaction(s) in the living body. Known as the superantigen are TSST-1, enterotoxins and exfoliative toxin A (exfoliative A: ETA) produced by Staphylococcus aureus , which is a gram-positive bacterium, and exotoxins produced by streptococci (streptococcal pyrogenic exotoxin A, B, C: SPE-A, SPE-B, SPE-C), among others. An enterotoxin is one of toxins produced by bacteria. It has various biological activities, such as emetic, pyrogenic and mitogenic activities, and causes food poisoning symptoms or Toxic shock syndrome (hereinafter referred to as TSS). “Enterotoxin” is a term formed from “entero”, which means the intestine, and toxin. Although it essentially means a toxin causing diarrhea, it has not yet established that an enterotoxin causes diarrhea; hence what it really means is unclear. Toxins having enterotoxin activity include toxins having various biological activities, such as emetic, pyrogenic and mitogenic activities, and causing food poisoning symptoms or TSS. Known among them are enterotoxins produced by staphylococci, heat-labile enterotoxin (hereinafter referred to as LT) produced by Campylobacter species, and LT and heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli , among others. Staphylococci are widely distributed on or in the skin, nasal cavity, oral cavity, pharynx, urinary organs and intestinal tract of various animals including humans and, further, in air, sewage, river, food, and so forth and include a large number of species. Among such a large number of Staphylococcus species, it is coagulase-positive Staphylococcus aureus (hereinafter referred to as S. aureus ) that shows pathogenicity in humans. S. aureus causes various infectious diseases, such as TSS and staphylococcal scaled skin syndrome (SSSS) and, as pathogens, producing problems such as hospital infections. Furthermore, Staphylococcus epidermidis , for instance, may cause endocarditis, meningitis, septicemia, etc., and Staphylococcus saprophticus may cause urinary tract infection, although they are coagulase-negative. Known staphylococcal pathogenic factors include various toxins, enzymes and other biologically active substances, such as Clumping factor, fibrinogen-binding protein, fibrinogen-binding protein A, fibrinogen-binding protein B, collagen-binding protein, coagulase, polysaccaride/adhesin, polysaccaride intracellular adhesin, 220-kDa adhesin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI, TSST-1, exfoliative toxin A, exfoliative toxin B, protein A, lipase, V8 protease, fatty acid modifying enzyme, panton-valentine leucocidin, leucocidin R, capsular polysaccaride, staphylokinase, α-toxin, β-hemolysin, γ-hemolysin, δ-hemolysin, phospholipase C, metalloprotease (elastase), and hyaluronidase. These bacterial toxins cause a great variety of diseases, from such relatively slight ones as food poisoning and traveler's diarrhea to such severe ones possibly leading to death as lethal diarrhea, tetanus, pertussis, botulism, diphtheria, cholera, TSS, and septicemia. In the case of septicemia and TSS, for instance, antibiotics, γ-globulin preparations and so forth are used in the treatment thereof, but the mortality is still high. As for food poisoning, it is difficult to perfectly prevent the occurrence of food poisoning how much care is taken from the hygienic point of view and, further, even when a food sufficiently heated is taken, it may cause food poisoning if a heat-stable toxin, such as an enterotoxin, is already contained therein. As regards the removal of superantigens by adsorption, Japanese Kokai Publication Hei-8-319431 discloses an adsorbent having a specific side chain. However, this cannot be said to be satisfactory from the capacity viewpoint. The present inventors have previously found that a material with a compound having a log P value (P being the partition coefficient in the octanol-water system) of not less than 2.50 as immobilized thereon can well adsorb TSST-1 (one of bacterial toxins) (Japanese Kokai Publication Hei-10-290833). For preparing such adsorbent material, however, a number of steps are required. When, for instance, antibodies specific to various toxins respectively are used, it is indeed possible to remove toxins from body fluids such as blood, plasma and serum, culture supernatants, foodstuffs, and drinks, but these have disadvantages, namely they are expensive and, when sterilized, they are denatured and their absorptive ability is markedly reduced. As regards endotoxins, various adsorbents are known for removing them from body fluids. For example, Japanese Kokoku Publication Hei-1-16389 discloses an adsorbent comprising polymyxin, which is known as an antidote against endotoxins, immobilized on an appropriate carrier. This adsorbent is effective against infections with gram negative bacteria but the removal of endotoxins alone cannot be expected to be highly effective against infections with gram positive bacteria or multiple infections with gram positive and gram negative bacteria. Furthermore, in recent years, it has been revealed that TSST-1, as a superantigen, activates the immune system and enhances the endotoxin toxicity to a level thousands of times higher. Therefore, the occurrence of an endotoxin at a low concentration, at which septicemia will not be caused clinically, is indicated to cause septicemia. Accordingly, the advent of a highly effective adsorbent for bacterial toxins, which can be produced in an inexpensive and simple and easy manner, is earnestly desired.

<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide an adsorbent for bacterial toxins which is free of such drawbacks of the prior art, a method for removal of such toxins by adsorption, and an adsorber packed with said adsorbent. As a result of intensive investigations for overcoming the above-mentioned disadvantages, the present inventors could discover that when a water-insoluble porous material, which has a mode of pore radius of 20 angstroms to 1,000 angstroms, is used, bacterial toxins can be adsorbed at very high rates. The present invention, therefore, is directed to an adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms.

Process and mould for thermoforming containers

The present invention relates to a method ol manufacturing water-soluble containers using a horizontal intermittent motion thermolorming machine which comprises the steps of: a) locating a first water-soluble film overa mould, said mould containing a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3 mm and between an edge of the mould and the closest cavity is at least 1.5 mm; b) thermoforming the first film to produce a plurality of pockets; c) at least partially filling the pockets with a composition; and d) sealing the plurality ol the at least partially filled pockets. The cavities are positioned in the array such that there are a plurality of continuous strips of uninterrupted planar surface of the mould from a leading to a trailing edge ol the mould, for receiving support means fitted to the machine for supporting the film.

1. A process for producing a water-soluble container using a horizontal intermittent motion thermoforming machine which comprises the steps of: a) locating a first water-soluble film over a mould, said mould containing a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3 mm and between an edge of the mould and the closest cavity is at least 1.5 mm; b) thermoforming the first film to produce a plurality of pockets; c) at least partially filling the pockets with a composition; and d) sealing the plurality of the at least partially filled pockets, wherein the cavities are positioned in the array such that there are a plurality of continuous strips of uninterrupted planar surface of the mould from a leading to a trailing edge of the mould, for receiving support means fitted to the machine for supporting the film. 2. A process as claimed in claim 1 in which step d) comprises placing a second water-soluble film on top of the at least partially filled pockets and sealing the films together. 3. A process as claimed in any one of the preceding 4. A process as claimed in any one of the preceding claims in which the depth of the cavities lies in the range of 10 to 80% of the shortest dimension of the mouth cavity. 5. A process as claimed in any one of the preceding claims in which the depth of the cavities lies in the range of 40 to 60% of the shortest dimension of the mouth cavity. 6. A process as claimed in any one of the preceding claims in which the cavity bases are planar. 7. A process as claimed in any one of claims 1 to 4 in which the cavity bases are rounded. 8. A process as claimed in claim 6 in which the rounded bases have a radius of 20 mm. 9. A process as claimed in any one of the preceding claims in which corners formed where the cavity side walls meet each other are rounded. 10. A process as claimed in claim 9 in which the side wall corners have a radius of 10 mm. 11. A process as claimed in any one of the preceding claims in which edges formed where the cavity side walls meet an upper surface of the mould are rounded. 12. A process as claimed in claim 11 in which the side wall-mould upper surface edges have a radius of lmm. 13. A process as claimed in any one of the preceding claims in which bottom corners, formed where the cavity side walls meet the cavity base, are rounded. side wall-base bottom corners have a radius of 10 mm. 15. A process as claimed in claim 13 or claim 14 in which air bores are located in the side wall base bottom corners. 16. A process as claimed in claim 15 in which the air bores have a diameter of 0.1 mm to 1 mm. 17. A process as claimed in claim 16 in which the air bores have a diameter of 0.4 mm to 0.5 mm. 18. A process as claimed in any one of the preceding claims in which the shortest dimension of the planar surface between two adjacent cavities lies in the range of 4 mm to 10 mm and between an edge of the mould and the closest cavity lies in the range of 2 mm to 5 mm. 19. A process as claimed in any one of the preceding claims in which a continuous strip of uninterrupted planar surface is provided between adjacent rows of cavities. 20. A process as claimed in any one of the preceding claims in which a continuous strip of uninterrupted planar surface is provided between every other pair of adjacent rows of cavities. 21. A mould for use in a thermoforming process for manufacturing water-soluble containers from water-soluble films, in which said mould contains a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3mm and between an edge of the mould and the closest cavity is at least 1.5 mm, and uninterrupted planar surface of the mould from a leading to a trailing edge of the mould. 22. A mould as claimed in claim 21 in which the depth of the cavities lies in the range of 10 to 80% of the shortest dimension of the cavity mouth. 23. A mould as claimed in claim 21 or 22 in which the depth of the cavities lies in the range of 40 to 60% of the shortest dimension of the cavity mouth. 24. A mould as claimed in any one of claims 21 to 23 in which the cavity bases are planar. 25. A mould as claimed in any one of claims 21 to 24 in which the cavity bases are rounded. 26. A mould as claimed in claim 25 in which the rounded bases have a radius of 20mm. 27. A mould as claimed in any one of claims 21 to 26 in which corners formed where the cavity side walls meet each other are rounded. 28. A mould as claimed in claim 27 in which the side wall corners have a radius of 10 mm. 29. A mould as claimed in any one of claims 21 to 28 in which edges formed where the cavity side walls meet an upper surface of the mould are rounded. 30. A mould as claimed in claim 29 in which the side wall-mould upper surface edges have a radius of lmm. 31. A mould as claimed in any one of claims 21 to 30 in which bottom corners, formed where the cavity side walls meet the cavity base, are rounded. 32. A mould as claimed in claim 31 in which the side wall-base bottom corners have a radius of 10 mm. 33. A mould as claimed in claim 31 or claim 32 in which air bores are located in the side walls base bottom corners. 34. A mould as claimed in claim 33 in which the air bores have a diameter of 0.1 mm to 1 mm. 35. A mould as claimed in claim 34 in which the air bores have a diameter of 0.4 mm to 0.5 mm. 36. A mould as claimed in any one of claims 21 to 35 in which the shortest dimension of the planar surface between two adjacent cavities lies in the range of 4mm to lOmm and between an edge of the mould and the closest cavity lies in the range of 2 mm to 5 mm. 37. A mould as claimed in any one of claims 21 to 36 in which a continuous strip of uninterrupted planar surface is provided between adjacent rows of cavities. 38. A mould as claimed in any one of claims 21 to 37 in which a continuous strip of uninterrupted planar surface is provided between every other pair of adjacent rows of cavities. 39. A mould as claimed in any one of claims 21 to 38 in which air bores are lacated. 40. A container formed by the process of any one of the preceding claims.

Solution for preparing stool specimens for diagnostic purposes

The present invention relates to a new solution for the preparation of stool samples for diagnostic tests. The solution allows samples to be prepared simply before use in an immunological detection process and also provides a high level of sensitivity, specificity and reproducibility of the tests. The solution must contain at least one buffer substance, one detergent and one blocking reagent. The Invention also relates to a process for the analysis of a stool sample for diagnostic purposes using the solution according to the invention.

1. Process for the analysis of a stool sample for the diagnosis of an H. pylori infection comprising the following steps: a) bringing the sample into contact with a solution that has a pH in a range from 7.0 to 8.0 containing: at least one buffer substance selected from PBS, glycine buffer (0.1 M glycine, 140 mM NaCI), HEPES ([4-(2-hydroxyethyl)-piperazino]-ethane sulfonic acid) and MOPS (3-Morpholino-1-propane sulfonic acid); the zwitterionic detergent Chaps (3-[(3-chloramidopropyl)-dimethylammonium]-1-propane sulfonate) in a concentration of 0.01 to 1.5% (v/v), preferably in a range from 0.05 to 0-3% (v/v), very particularly preferred in a range from 0.1 to 0.15% (v/v); and the blocking reagent mouse serum in a concentration of 0.05 to 5%, preferably in a range from 0.1 to 1%, very particularly preferred in a range from 0.4 to 0.6%; if necessary, gentamicin sulfate and/or Proclin TM 300 as the stabiliser; and if necessary a complexing agent, preferably selected from EDTA and EGTA, particularly preferred EDTA, preferably in a concentration of 1 mM; b) carrying out an immuno assay with the sample treated according to step a); and c) taking of a measurement signal that was obtained within the scope of the immuno assay. 2. Process according to claim 1, characterized in that the immuno assay is an ELISA. 3. Process according to claim 1, characterized in that the stool sample treated with the solution is applied to a filter strip.

Biopanning and rapid analysis of selective interactive ligands (brasil)

The present invention concerns novel methods of identifying peptide sequences that selectively bind to targets. In alternative embodiments, targets may comprise cells or clumps of cells, particles attached to chemicals compounds, molecules or aggregates, or parasites. In preferred embodiments, target cells are sorted before exposure to the phage library. The general method, Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) provides for rapid and efficient separation of phage that bind to targets, while preserving unbound phage. BRASIL may be used in preselection procedure to subract phage that bind non-specifically to a first target before exposing the subtracted library to a second target. Certain embodiments concern targeting peptides identified by BRASIL and methods of use of such peptides for targeted delivera of therapeutic agents or imaging agents or diagnosis or treatment of diseases. Novel compositions comprising a first phase, second phase, target and a phage library are also disclosed.

1. A composition comprising: a) a target for phage binding; b) a phage display library; c) a first phase; and d) a second phase; wherein the density of the target is greater than the density of the second phase and the density of the second phase is greater than the density of the first phase. 2. The composition of claim 1, wherein the target comprises isolated cells or small clumps of cells. 3. (Cancelled) 4. (Cancelled) 5. The composition of claim 1, wherein the target is a parasite. 6. (Cancelled) 7. The composition of claim 1, wherein the target comprises a particle attached to a chemical, a compound, a molecule or an aggregate of molecules. 8. (Cancelled) 9. The composition of claim 1, wherein the first phase is an aqueous phase. 10. (Cancelled) 11. The composition of claim 1, wherein the second phase is an organic phase. 12. The composition of claim 1, wherein the density of the second phase is about 1.02 to 1.04 gm/ml. 13-21. Cancelled 22. A method comprising: a) exposing a target to a phage display library in an first phase; b) exposing the first phase to a second phase; and c) separating phage bound to the target from unbound phage; wherein bound phage enter the second phase and unbound phage remain in the first phase. 23. (Cancelled) 24. (Cancelled) 25. The method of claim 22, further comprising centrifuging the phage bound to the target through an organic phase to form a pellet. 26. (Cancelled) 27. (Cancelled) 28. The method of claim 22, wherein the target is a parasite. 29. (Cancelled) 30. The method of claim 22, wherein the target comprises a particle attached to a chemical, a compound, a molecule or an aggregate of molecules. 31. (Cancelled) 32. The method of claim 22, wherein the density of the first phase is about 1.00 gm/ml and the density of the second phase is about 1.02 to 1.04 gm/ml. 33-35. Cancelled 36. The method of claim 25, further comprising recovering bound phage from the pellet. 37-40. Cancelled 41. The method of claim 22, further comprising i) prescreening the library against a first target; ii) collecting unbound phage; and iii) screening the unbound phage against a second target. 42. The method of claim 41, wherein the first target comprises normal cells and the second target comprises diseased cells. 43. (Cancelled) 44. The method of claim 41, wherein the first target is a non-pathogenic organism and the second target is a pathogenic organism. 45. (Cancelled) 46. (Cancelled) 47. The method of claim 41, wherein the first target comprises quiescent cells and the second target comprises activated cells. 48. (Cancelled) 49. (Cancelled) 50. A targeting peptide prepared by BRASIL (Biopanning and Rapid Analysis of Selective Interactive Ligands). 51. An expression vector comprising a nucleic acid encoding a targeting peptide according to claim 50. 52. The expression vector of claim 51, further comprising a nucleic acid encoding a therapeutic protein or peptide. 53. The expression vector of claim 52, wherein the therapeutic protein or peptide is a a pro-apoptosis agent, an anti-angiogenic agent, an angiogenic agent, a hormone, a cytokine, a chemokine, a growth factor, a cytotoxic agent, an antibiotic, a survival factor, an anti-apoptotic agent, a hormone antagonist, an antibody or a Fab fragment of an antibody. 54-58 Cancelled 59. An isolated peptide of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a sequence selected from any of SEQ ID NO:6, [SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of] SEQ ID NO:13 through [SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289] SEQ ID NO:39, SEQ ID NO:114, SEQ ID NO:128 through SEQ ID NO:137, SEQ ID NO:201 through SEQ ID NO:207 or SEQ ID NO:259. 60-62. Cancelled 63. The isolated peptide of claim 59, wherein said peptide comprises at least 5 contiguous amino acids of a sequence selected from any of SEQ ID NO:6, [SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of] SEQ ID NO:13 through [SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289] SEQ ID NO:39, SEQ ID NO:114, SEQ ID NO:128 through SEQ ID NO:137, SEQ ID NO:201 through SEQ ID NO:207 or SEQ ID NO:259. 64. The isolated peptide of claim 59, wherein said peptide is attached to a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a cell, a bacterium, a yeast cell or a mammalian cell. 65. (Cancelled) 66. The isolated peptide of claim 59, wherein said peptide is attached to a virus, a bacteriophage, a bacterium, a yeast cell, a liposome, a microparticle, a magnetic bead, a cell or a microdevice. 67. The isolated peptide of claim 59, wherein said peptide is attached to a eukaryotic expression vector. 68. A method of targeted delivery comprising: a) selecting a peptide by BRASIL; b) attaching said peptide to a therapeutic agent; and c) providing said peptide and said agent to a subject. 69. (Cancelled) 70. A method of diagnosing a disease state comprising: a) selecting a peptide by BRASIL, wherein said peptide is targeted to cells associated with a disease state; b) administering said peptide to a subject; and c) determining the distribution of said peptide in said subject. 71. The method of claim 70, wherein said disease state is selected from the group consisting of diabetes, inflammatory disease, arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection, viral infection, cardiovascular disease and degenerative disease. 72. (Cancelled) 73. (Cancelled) 74. The method of claim 22, further comprising sorting target cells before they are exposed to the phage library. 75. The method of claim 74, wherein the cells are sorted by FACS (flourescent activated cell sorting). 76. The method of claim 75, wherein the cells are obtained from a subject with leukemia patient and leukemia cells are selected for exposure to the phage library. 77. The method of claim 76, wherein the library is presubtracted against normal cells from the same subject. 78-87. Cancelled

<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention concerns the fields of molecular medicine and targeted delivery. More specifically, the present invention relates to compositions and methods for identification and use of peptides that selectively target organs or tissues. In particular, the methods and compositions concern biopanning and rapid analysis of selective interactive ligands (BRASIL). 2. Description of Related Art Therapeutic treatment of many human disease states is limited by the systemic toxicity of the therapeutic agents used. Cancer therapeutic agents in particular exhibit a very low therapeutic index, with rapidly growing normal tissues such as skin and bone marrow affected at concentrations of agent that are not much higher than the concentrations used to kill tumor cells. Treatment of cancer and other organ or tissue confined disease states would be greatly facilitated by the development of compositions and methods for targeted delivery to a desired organ or tissue of a therapeutic agent. Diagnostic imaging would also be facilitated by the targeted delivery of imaging agents to desired organs, tissues or diseased cells. Recently, an in vivo selection system was developed using phage display libraries to identify organ or tissue targeting peptides in a mouse model system. Phage display libraries expressing transgenic peptides on the surface of bacteriophage were initially developed to map epitope binding sites of immunoglobulins (Smith and Scott, 1985, 1993). Such libraries can be generated by inserting random oligonucleotides into cDNAs encoding a phage surface protein, generating collections of phage particles displaying unique peptides in as many as 10 9 permutations. (Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al 1998b). Intravenous administration of phage display libraries to mice was followed by the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996). Phage were recovered that were capable of selective homing to the vascular beds of different mouse organs or tissues, based on the specific targeting peptide sequences expressed on the outer surface of the phage (Pasqualini and Ruoslahti, 1996). A variety of organ and tumor-homing peptides have been identified by this method (Rajotte et al., 1998, 1999; Koivunen et al., 1999; Burg et al., 1999; Pasqualini, 1999). Each of those targeting peptides bound to different receptors that were selectively expressed on the vasculature of the mouse target tissue (Pasqualini, 1999; Pasqualini et al., 2000; Folkman, 1995; Folkman 1997). Tumor-homing peptides bound to receptors that were upregulated in the tumor angiogenic vasculature of mice (Brooks et al., 1994b; Pasqualini et al., 2000). In addition to identifying individual targeting peptides selective for an organ or tissue (Pasqualini and Ruoslahti, 1996; Arap et al, 1998a; Koivunen et al., 1999), this system has been used to identify endothelial cell surface markers that are expressed in mice in vivo (Rajotte and Ruoslahti, 1999). Attachment of therapeutic agents to targeting peptides resulted in the selective delivery of the agent to a desired organ or tissue in the mouse model system. Targeted delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in tumor angiogenic vasculature resulted in a marked increase in therapeutic efficacy and a decrease in systemic toxicity in tumor-bearing mouse models (Arap et al., 1998a, 1998b; Ellerby et al., 1999). This relative success notwithstanding, cell surface selection of phage libraries has been plagued by technical difficulties. A high number of non-binder and non-specific binder clones are recovered when phage libraries are incubated with cell suspensions or monolayers. Removal of this background phage binding by repeated washes is both labor-intensive and inefficient. Cells and potential ligands are frequently lost during the many washing steps required. Thus, there is a need for a rapid and efficient method for in vitro biopanning that retains the selectivity and specificity of in vivo methods, while providing decreased non-specific background. Previous studies with phage display libraries have relied on a mouse model system to identify targeting peptides and their receptors, under the assumption that human targeting peptides are homologous. However, cell surface receptors may have a very different distribution and function in humans than in mice. Further, the mouse model system has been exploited to characterize targeting peptides for only a handful of specific organs. A need exists in the art for methods and compositions for identification of targeting sequences selective for human organs, tissues or cell types that can be of clinical use for targeted delivery of therapeutic agents

<SOH> SUMMARY OF THE INVENTION <EOH>The present invention solves a long-standing need in the art by providing compositions and in vitro methods for identifying targeting peptides that are selective for organs, tissues or cell types. In a preferred embodiment, such targeting peptides are identified by collecting samples of one or more organs, tissues, or cell types, separating the samples into isolated cells or small clumps of cells, suspending the cells or clumps in a first phase, exposing the cells or clumps of cells to a phage display library, layering the first phase over a second phase, and centrifuging the two phases so that the cells are pelleted at the bottom of a centrifuge tube. In a more preferred embodiment, the first phase is aqueous and the second phase is organic. In even more preferred embodiments, the cells are human cells. In certain embodiments, phage may be collected from the pellet by exposure to bacteria and phage clones may be plated, isolated and grown up in bulk culture. In alternative embodiments, phage inserts may be recovered from the pellet by PCR™ or other amplification techniques and the inserts sequenced to identify the targeting peptides. In certain embodiments, the organic phase comprises dibutylphtalate or a mixture of dibutylphthalate and cyclohexane. The methods disclosed herein are generally referred to herein as Biopanning and Rapid Analysis of Selective Interactive-Ligands (BRASIL). In alternative embodiments, the BRASIL method may be used to identify targeting peptides against virtually any chemical, molecule or complex of molecules. The separation of bound and unbound phage is preferably accomplished by partitioning bound phage from an aqueous phase into an organic phase. This requires that the target to which the phage bind be either denser than phage, larger than phage or preferably both. In preferred embodiments, the target is insoluble in the aqueous phase. In order to satisfy this requirement, chemicals, compounds, or molecules may be attached to a large insoluble particle, for example a glass, plastic, ceramic or magnetic bead. The skilled article will realize that the invention is not limited to beads and any large and/or dense particle may be used. The particle attached target may be exposed to a phage library in an aqueous phasae and phage binding to the target partitioned into an organic phase. Although the examples shown herein illustrate the use of centrifugation to partition bound phage into the organic phase, the skilled artisan will realize that other types of partitioning may be used within the scope of the invention. For example, for targets attached to magnetic beads, a magnetic field could be imposed to pull the phage bound to beads into an organic phase. In embodiments where cells are the targets, the cells may be mammalian cells, human cells, mouse cells or animal cells. Alternatively, cells may include any type of prokaryotic or eukaryotic cell, such as bacteria or unicellular microorganisms. In preferred embodiments, specific populations of cells may be prepared for use in BRASIL. For example, cells from leukemic patients may be sorted using a FACS (fluorescent activated cell sorter, Becton-Dickinson) to sort cancer cells from non-cancer cells. A phage library may be screened against cancerous cells only, either with or without a preselection subtraction against normal cells from the same patient. The skilled artisan will realize that cell sorting is not limited to leukemic samples, but rather may be practiced with any heterogenous population of cells. In certain embodiments, targeting peptides identified by BRASIL are of use for the selective delivery of therapeutic agents, including but not limited to gene therapy vectors and fusion proteins, to specific organs, tissues or cell types in subjects. The skilled artisan will realize that the scope of the claimed methods of use include any disease state that can be treated by targeted delivery of a therapeutic agent to a desired organ, tissue or cell type. The skilled artisan will understand that although the targeting peptides disclosed herein are particularly suited for use in human subjects, it is contemplated that they may be of use in other subjects such as mice, dogs, cats, horses, cows, sheep, pigs or any other mammal. Certain embodiments concern targeting peptides identified by the BRASIL method. One embodiment of the present invention concerns isolated peptides of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a targeting peptide sequence, selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. In a preferred embodiment, the isolated peptide is 50 amino acids or less, more preferably 30 amino acids or less, more preferably 20 amino acids or less, more preferably 10 amino acids or less, or even more preferably 5 amino acids or less in size. In other preferred embodiments, the isolated peptide of claim 1 comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a targeting peptide sequence, selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. In certain embodiments, the isolated peptide is attached to a molecule. In preferred embodiments, the attachment is a covalent attachment. In additional embodiments, the molecule is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, a nucleic acid or an antigen. Those molecules are representative only. Molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a subject. In preferred embodiments, the pro-apoptosis agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2 (SEQ ID NO:1), (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2 (SEQ ID NO:3) or (KLGKKLG) 3 (SEQ ID NO:4). In other preferred embodiments, the anti-angiogenic agent is thrombospondin, angiostatin, endostatin or pigment epithelium-derived factor. In further preferred embodiments, the cytokine is interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophage colony stimulating factor). Such examples are representative only and are not intended to exclude other pro-apoptosis agents, anti-angiogenic agents or cytokines known in the art. In other embodiments, the isolated peptide is attached to a macromolecular complex. In preferred embodiments, the attachment is a covalent attachment. In other preferred embodiments, the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a cell or a microdevice. These are representative examples only. Macromolecular complexes within the scope of the present invention include virtually any macromolecular complex that may be attached to a targeting peptide and administered to a subject. In other preferred embodiments, the isolated peptide is attached to a eukaryotic expression vector, more preferably a gene therapy vector. In another embodiment, the isolated peptide is attached to a solid support, preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (PPLC) matrix. Such attached peptides may be of use, for example, to purify or isolate an antibody, protein, peptide or other ligand that binds to the targeting peptide. In certain embodiments, this binding may be used to identify endogenous receptors, ligands or receptor:ligand pairs that are mimicked by the targeting peptide. Additional embodiments of the present invention concern fusion proteins comprising at least 3 contiguous amino acids of a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. Certain other embodiments concern compositions comprising the claimed isolated peptides or fusion proteins in a pharmaceutically acceptable carrier. Further embodiments concern kits comprising the claimed isolated peptides or fusion proteins in one or more containers. Additional embodiments concern kits comprising compositions and apparatus for performing BRASIL. Kit components may include, but are not limited to, any composition or apparatus that may be of use in performing BRASIL, such as solutions, buffers, media, organic phase, bacteria, phage libraries, control phage, centrifugation tubes, etc. Other embodiments concern methods of targeted delivery comprising selecting a targeting peptide for a desired organ or tissue, attaching said targeting peptide to a molecule, macromolecular complex or gene therapy vector, and providing said peptide attached to said molecule, complex or vector to a subject. Preferably, the targeting peptide is selected to include at least 3 contiguous amino acids from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. In certain preferred embodiments, the molecule attached to the targeting peptide is a chemotherapeutic agent, an antigen or an imaging agent. The skilled artisan will realize that within the scope of the present invention any organ, tissue or cell type can be targeted for delivery, using targeting peptides attached to any molecule, macromolecular complex or gene therapy vector. Certain embodiments of the present invention concern methods for imaging an organ, tissue, or cell type comprising selecting a peptide targeted to said organ or tissue, attaching an imaging agent to said peptide, administering said peptide to a subject and obtaining an image. In preferred embodiments, the targeted cells are associated with a disease or other condition. In other preferred embodiments, the targeting peptide comprises at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. In other embodiments, the present invention concerns methods of diagnosing a disease state, comprising selecting a peptide targeted to cells associated with such disease state, attaching an imaging agent to said peptide, administering said peptide and imaging agent to a subject suspected of having the disease, and diagnosing the presence or absence of the disease based on the distribution of said peptide and imaging agent within said subject. Preferably, the targeting peptide contains at least 3 contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. In preferred embodiments, the disease state is diabetes mellitus, inflammatory disease, rheumatoid arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection or viral infection. In a more preferred embodiment, the disease state is metastatic cancer. Additional embodiments concern methods for identifying a receptor for a targeting peptide, comprising contacting said peptide to an organ, tissue or cell containing said receptor, allowing said peptide to bind to said receptor, and identifying said receptor by its binding to said peptide. In preferred embodiments, the targeting peptide contains at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. The skilled artisan will realize that the contacting step can utilize samples of organs, tissues or cells, or may alternatively utilize homogenates or detergent extracts of the organs, tissues or cells. In certain embodiments, the cells to be contacted may be genetically engineered to express a suspected receptor for the targeting peptide. In a preferred embodiment, the targeting peptide is modified with a reactive moiety that allows its covalent attachment to said receptor. In a more preferred embodiment, the reactive moiety is a photoreactive group that becomes covalently attached to the receptor when activated by light. In another preferred embodiment, the peptide is attached to a solid support and the receptor is purified by affinity chromatography. In other preferred embodiments, the solid support comprises magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix. In certain embodiments, the targeting peptide inhibits the activity of the receptor upon binding to the receptor. The skilled artisan will realize that receptor activity can be assayed by a variety of methods known in the art, including but not limited to catalytic activity and binding activity. In another preferred embodiment, the receptor is an endostatin receptor, a metalloprotease or an aminopeptidase. Other embodiments of the present invention concern isolated nucleic acids of 300 nucleotides or less in size, encoding a targeting peptide. In preferred embodiments, the isolated nucleic acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or even 10 nucleotides or less in size. In other preferred embodiments, the isolated nucleic acid is incorporated into a eukaryotic or a prokaryotic expression vector. In even more preferred embodiments, the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a virus or a bacteriophage. In other preferred embodiments, the isolated nucleic acid is operatively linked to a leader sequence that localizes the expressed peptide to the extracellular surface of a host cell. Additional embodiments of the present invention concern methods of treating a disease state comprising selecting a targeting peptide that targets cells associated with the disease state, attaching one or more molecules effective to treat the disease state to the peptide, and administering the peptide to a subject with the disease state. Preferably, the targeting peptide includes at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289. In preferred embodiments the disease state is diabetes mellitus, inflammatory disease, rheumatoid arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection and viral infection. Another embodiment of the present invention concerns molecular adaptors for targeted gene therapy. In a preferred embodiment, the molecular adaptor comprises a Fab fragment of an antibody that is specific for a gene therapy vector, covalently attached to a targeting peptide sequence that provides selective targeting to a desired organ or tissue. The skilled artisan will realize that the present invention may include any gene therapy vector that is known in the art. The vector binding portion of the molecular adaptor is not limited to Fab fragments of antibodies, but may include any other molecule that can be used to attach a targeting peptide to a gene therapy vector. The only requirement is that the gene therapy vector should be selectively targeted to a desired organ or tissue in the presence of the molecular adaptor. Another embodiment of the present invention concerns compositions and methods of use of tumor targeting peptides against cancers. Tumor targeting peptides identified by the methods disclosed in the instant application may be attached to therapeutic agents, including but not limited to molecules or macromolecular assemblages and administered to a subject with cancer, providing for increased efficacy and decreased systemic toxicity of the therapeutic agent. Therapeutic agents within the scope of the present invention include but are not limited to chemotherapeutic agents, radioisotopes, pro-apoptosis agents, cytotoxic agents, cytostatic agents and gene therapy vectors. Targeted delivery of such therapeutic agents to tumors provides a significant improvement over the prior art for increasing the delivery of the agent to the tumor, while decreasing the inadvertent delivery of the agent to normal organs and tissues of the subject. In a preferred embodiment, the tumor targeting peptide is incorporated into the capsule of a phage gene therapy vector to target delivery of the phage to angiogenic endothelial cells in tumor blood vessels. A further embodiment of the present invention concerns methods for identifying new tumor targeting peptides, using phage display libraries that incorporate reporter genes. Administration of the reporter gene phage library to a subject with a tumor is followed by recovery of phage from the tumor and identification of tumor targeting peptides by sequencing selected portions of the phage genome that contain the nucleic acid sequence encoding the targeting peptide. While these embodiments of the present invention concern tumors, the skilled artisan will realize that within the scope of the present invention other disease states that are localized to specific organs or tissues may also be treated with enhanced therapeutic efficacy and decreased systemic toxicity using the methods and compositions disclosed herein. Yet another embodiment of the present invention concerns methods of identifying targeting peptides against antibodies from a subject with a disease state, comprising obtaining a sample of serum from the subject, obtaining antibodies from the sample, adding a phage display library to the antibodies and collecting phage bound to the antibodies. In preferred embodiments, the antibodies are attached to a solid support, more preferably attached to protein G attached to beads. In another preferred embodiment, a subtraction step is added where the phage display library is first screened against antibodies from a subject who does not have the disease state. Only phage that do not bind to these control antibodies are used to obtain phage binding to the diseased subject's antibodies. In other preferred embodiments, phage that bind to a target organ or tissue, for example to placenta, may be pre-screened or post-screened against a subject lacking that organ or tissue. Phage that bind to the subject lacking the target organ or tissue are removed from the library. Other embodiments concern methods of obtaining antibodies against an antigen. In preferred embodiments, the antigen comprises one or more targeting peptides. The targeting peptides are prepared and immobilized on a solid support, a sample containing antibodies is added and antibodies that bind to the targeting peptides are collected. In other preferred embodiments, a phage display library displaying the antigen binding portions of antibodies from a subject is prepared, the library is screened against one or more antigens and phage that bind to the antigens are collected. In more preferred embodiments, the antigen is a targeting peptide.

Ceramic heater and ceramic joined article

A ceramic heater capable of stably supporting a semiconductor safer and evenly heating the whole of a semiconductor wafer or the like without generating any warp in the semiconductor wafer or the like. The ceramic heater includes a disk-like ceramic substrate, a heating element formed on a surface of or inside the ceramic substrate, and through holes for letting lifter pins pass through the ceramic substrate. The number of the formed through holes is three or more, and the through holes are formed in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge of the ceramic substrate.

1. A ceramic heater comprising: a disk-like ceramic substrate; a heating element formed on a surface of or inside said ceramic substrate; and through holes for letting lifter pins pass through at said ceramic substrate, wherein three or more of said through holes are formed, and said through holes are formed in an area whose distance from the center of said ceramic substrate is ½ of more of the distance from the center of said ceramic substrate to the outer edge of said ceramic substrate. 2. The ceramic heater according to claim 1, wherein said through holes are formed at substantially regular intervals on a single circle which has a concentric circle relationship with said ceramic substrate. 3. A ceramic heater comprising: a disk-like ceramic substrate; a heating element formed on a surface of or inside said ceramic substrate; and through holes for letting lifter pins pass through at said ceramic substrate, wherein the diameter of each of said through holes on a heating face side for heating an object to be heated is larger than the diameter of said through hole on the side opposite to said heating face. 4. The ceramic heater according to claim 3, wherein each of said through holes comprises a columnar portion and a diameter-increasing portion, the diameter of said diameter-increasing portion becomes larger as the portion is closer to the heating face. 5. The ceramic heater according to claim 3, wherein the diameter on the heating face side of each of said through holes is from 1.2 to 10 times as large as the diameter on the side opposite to said heating face of said through holes. 6. A ceramic bonded body comprising: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of said ceramic substrate, wherein the center of an area surrounded by the interface between said ceramic body and said ceramic substrate, or the center of an area constituted by the interface between said ceramic body and said ceramic substrate is 3 to 200 μm apart from the center of the bottom face of said ceramic substrate. 7. A ceramic bonded body comprising: a disk-like ceramic substrate inside which a conductor is provided; and a cylindrical ceramic body having a cylindrical shape bonded to the bottom face of said ceramic substance, wherein the center of the circle surrounded by the interface between said cylindrical ceramic body and said ceramic substrate is 3 to 200 μm apart from the center of the bottom face of said ceramic substrate. 8. The ceramic bonded body according to claim 6, wherein said conductor is a heating element, and functions as a hot plate. 9. The ceramic bonded body according to claim 6, wherein said conductor is an electrostatic electrode, and functions as an electrostatic chuck. 10. The ceramic bonded body according to claim 6, wherein said ceramic substrate has a diameter of 250 mm or more. 11. The ceramic heater according to claim 4, wherein the diameter on the heating face side of each of said through holes is from 1.2 to 10 times as large as the diameter on the side opposite to said heating face of said through holes. 12. The ceramic bonded body according to claim 7, wherein said conductor is a heating element, and functions as a hot plate. 13. The ceramic bonded body according to claim 7, wherein said conductor is an electrostatic electrode, and functions as an electrostatic chuck. 14. The ceramic bonded body according to claim 7, wherein said ceramic substrate has a diameter of 250 mm or more.

<SOH> BACKGROUND ART <EOH>Conventionally, a heater, wafer prober or the like wherein a base material made of a metal such as stainless steel or aluminum alloy is used has been used in semiconductor producing/inspecting devices and so on, examples of which include an etching device and a chemical vapor phase growth device and the like. However, such a heater made of metal has the following problems. First, the thickness of the heater plate must be as thick as about 15 mm since the heater is made of a metal. Because in a thin metal plate, a bend, a strain and so on are generated on the basis of thermal expansion resulting from heating so that a silicon wafer put on the metal plate is damaged or inclined. However, if the thickness of the heater plate is made thick, a problem that the heater becomes heavy and bulky arises. The temperature of a face for heating an object to be heated such as a silicon wafer (referred to as a heating face hereinafter) and the like is controlled by changing the voltage or current quantity applied to the heating elements. However, since the metal plate is thick, the temperature of the heater plate does not follow the change in the voltage or current quantity promptly. Thus, a problem that the temperature is not easily controlled is caused. Thus, JP Kokai Hei 11-40330 and so forth suggest a ceramic substrate wherein a nitride ceramic or a carbide ceramic, with a high thermal conductivity and a high strength, is used as a substrate and heating elements formed by sintering metal particles are provided on a surface of a plate-form body made of such a ceramic. As illustrated in FIG. 15 , usually, in such a ceramic heater, heating elements 62 are formed inside a ceramic substrate 61 and further through holes for passing lifter pins through are formed in the vicinity of the center. This is because by letting the lifter pins pass through the through holes 65 and then moving the pins up and down, a semiconductor wafer can be relatively easily received from the previous line or the semiconductor wafer can be carried to the next line. Reference numeral 64 represents bottomed holes 64 for embedding temperature measuring elements such as thermocouples, and reference numeral 63 represents external terminals for connecting the heating elements 62 to a power source. As described above, the through holes 65 are arranged in the vicinity of the center of the ceramic substrate 61 . This is because in order to work the lifter pins by means of one motor, it is preferred that the positions of the lifter pins are closer to each other. In the case that this ceramic heater 60 is used to heat an object to be heated, such as a semiconductor wafer and the like, the temperature distribution in the surface of the ceramic heater 60 is reflected on the semiconductor wafer or the like if the object is heated in the state that the object contacts the heating face of the ceramic heater 10 . As a result, it is difficult that the semiconductor wafer or the like is evenly heated. In order that the surface temperature of the ceramic heater 60 is made to heat the semiconductor wafer or the like evenly, highly complicated control is required. Hence, the temperature control is not easy. Thus, when the semiconductor wafer is heated, there can be usually used a method of supporting the semiconductor wafer by means of the lifter pins provided for carrying the semiconductor wafer. That is, the lifters pins are held in the state that they project slightly from the surface of the ceramic substrate 61 , and the lifter pins are used to support the semiconductor wafer in the state that the semiconductor wafer is a given distance apart from the surface of the ceramic substrate 61 . The semiconductor wafer is then heated. According to this method of using the lifter pins, the semiconductor wafer is heated by radiation from the ceramic substrate 61 or convection current since the semiconductor wafer is held in the state that the semiconductor wafer is apart from the surface of the ceramic heater 61 by the given distance. Accordingly, the temperature distribution in the surface of the ceramic substrate 61 is not usually reflected directly on the semiconductor wafer, so that the semiconductor wafer is more evenly heated and any temperature distribution is not easily generated in the semiconductor wafer. However, when the lifter pins are used to intend to carry the semiconductor wafer, the semiconductor wafer may not be stably supported. In this case, there arises a problem that the semiconductor wafer is inclined to get out of position. At the time when a semiconductor wafer or a liquid crystal substrate is put thereon and heated (as well as the time of the state that a semiconductor wafer or a liquid crystal substrate is put on the heated ceramic substrate and until the temperature thereof returns to the original temperature, that is, a transition state), there is caused a problem that temperature difference is generated in the object to be heated, such as the semiconductor wafer or the liquid crystal substrate. Moreover, a problem that free particles adhere to the object to be heated is also encountered. Thus, the present inventors analyzed a cause for which an object to be heated, such a semiconductor wafer or a liquid crystal substrate, (which may be referred to as a semiconductor wafer or the like hereinafter), is inclined or temperature unevenness is generated when the semiconductor wafer or the like is heated. As a result, it has been found out that in the case that the lifter pins concentrate around the center, the object to be heated, such as the semiconductor wafer cannot be stably supported and the heat capacity per unit area (volume) in the central portion of the ceramic substrate 61 gets smaller than that in the peripheral portion so that the temperature of the central portion of the ceramic substrate 61 rises easily at the time of heating the ceramic substrate. It has also been found out that if the lifter pins are present in the vicinity of the center when a plate-form object to be heated, such as a semiconductor wafer or a liquid crystal substrate, is lifted up, the object warps and hence the peripheral portion of the object contacts the ceramic substrate 61 so that free particles are generated. When the ceramic heater 60 having these through holes 65 is used to heat an object to be heated such as a semiconductor wafer or a liquid crystal substrate, the temperature in the vicinity of the through holes 65 locally becomes low. That is, a cooling spot is generated. This results in a problem that the temperature of the semiconductor wafer, the liquid crystal substrate or the like falls in this portion so that the semiconductor wafer, the liquid crystal substrate or the like is not easily heated evenly. Furthermore, JP Kokai Hei 4-324276 suggests a ceramic heater in which aluminum nitride, which is a non-oxide ceramic having a high thermal conductivity and a large strength, is used as a substrate; heating elements and conductor filled through holes made of tungsten are made in this aluminum nitride substrate; and Nichrome wires as external terminals are brazed thereto. Since such a ceramic heater has a ceramic substrate having a large mechanical strength at high temperatures, the thickness of the ceramic substrate can be made small to make the heat capacity small. As a result, the temperature of the ceramic substrate can be caused to follow change in the voltage or electric current quantity promptly. A ceramic heater as described above adopts a means for bonding a cylindrical ceramic with a disk-like ceramic to protect wires such as external terminals from reactive gas, halogen gas and the like used in a semiconductor producing step, as described in Japanese Patent gazette Nos. 2525974 and 2783980, and JP Kokai 2000-114355. However, in the case that the ceramic heater described in Japanese Patent gazette No. 2525974 is used, it is exposed to reactive gas, halogen gas and the like for a long time and thermal stress concentrates on the bonding interface between the cylindrical ceramic and the disk-like ceramic, (which may be referred to as the interface hereinafter). Thus, by repeating temperature-rising and temperature-dropping thereof, thermal fatigue is generated, thereby causing a problem that cracks and the like are generated in the interface and air-tightness of the interface deteriorates so that the wires such as the external terminals are corroded. In the ceramic heater described in Japanese Patent gazette No. 2783980, in the interface thereof, the ceramic particles grow to extend to both sides of the interface, whereby the cylindrical ceramic is bonded to the disk-like ceramic. Therefore, the bonding strength of the interface is strong but thermal stress concentrates locally. Thus, by repeating temperature-rising and temperature-dropping thereof, thermal fatigue is generated so that cracks and the like may be generated in the interface, the cylindrical ceramic, or the disk-like ceramic. For recent semiconductor products, it is required to shorten a time necessary for throughput. Thus, it is strongly required to shorten a time for rising or dropping the temperature thereof. However, in the ceramic heaters described in Japanese Patent gazette No. 2525974, JP Kokai Hei 2000-114355 and so forth, a flange portion is formed in the cylindrical ceramic, thereby causing a problem that the thermal capacity increases and temperature-rising speed drops. In order to shorten the temperature-rising time, it is necessary to increase the temperature-rising speed. In order to shorten the temperature-dropping time, it is necessary to rise the temperature-dropping speed. However, if the temperature of the ceramic heater is abruptly risen or dropped, larger thermal stress is generated in the interface and the like. Thus, cracks and the like as described above become easy to be generated increasingly.

<SOH> SUMMARY OF THE INVENTION <EOH>The present invention has been made in view of the above-mentioned problems. An object thereof is to provide a ceramic heater capable of stably supporting an object to be heated, such as a semiconductor wafer, a liquid crystal substrate and the like, and evenly heating the semiconductor wafer or the like. Another object of the present invention is to provide a ceramic heater which prevents the generation of cooling spots, and causes no drop in the temperature of a semiconductor wafer, a liquid crystal substrate and the like in the vicinity of through holes formed in a ceramic substrate so that the object to be heated, such as the semiconductor wafer, the liquid crystal substrate and the like can be evenly heated. Still another object of the present invention is to provide a ceramic bonded body capable of keeping sufficient air-tightness and largely improving the reliability thereof because of the face that no thermal stress concentrates locally in the bonding interface between a ceramic body having a given shape such as a cylindrical shape or a columnar shape and a disk-like ceramic so that no cracks and the like are generated in this portion. In order to attain the above-mentioned objects, a ceramic heater according to the first aspect of the present invention comprises: a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein three or more of the above-mentioned through holes are formed, and the above-mentioned through holes are formed in an area whose distance from the center of the above-mentioned ceramic substrate is ½ or more of the distance from the center of the above-mentioned ceramic substrate to the outer edge of the above-mentioned ceramic substrate. According to the above-mentioned ceramic heater, the three or more through holes are present in the peripheral portion of the ceramic substrate; therefore, the lifter pins, which are passed through the through holes, are also present in the peripheral portion of the ceramic substrate and do not concentrate in the central portion so that a semiconductor wafer or the like supported by the lifter pins does not become unstable. As a result, even if impact and the like are caused when the ceramic heater is used, the semiconductor wafer or the like does not easily get out of position. Thus, an object to be heated, such as the semiconductor wafer, can be stably supported by the lifter pins. In the case that the semiconductor wafer or the like is heated to rise the temperature thereof, a difference in the thermal capacity per unit area (volume) between the central portion of the ceramic substrate and the peripheral portion thereof turns into a substantially negligible degree. As a result, the thermal capacity per unit volume (area) in the central portion of the ceramic substrate becomes almost equivalent to that in the peripheral portion. Thus, the semiconductor wafer or the like can be evenly heated even at the temperature-rising time thereof (at the transition time). Furthermore, in the case that the through holes are present in the vicinity of the center, a plate-form object to be heated, such as a semiconductor wafer or a liquid crystal substrate, warps when the plate-form object is pushed up by the lifter pins. Thus, the outer circumference of the plate-form object scrubs the ceramic substrate surface so that free particles are generated. However, according to the ceramic heater of the first aspect of the present invention, such a problem is not caused. It is desired that the through holes are formed at substantially regular intervals on a single circle having a concentric circle relationship with the above-mentioned ceramic substrate. Since the lifter pins passed through the through holes are widely dispersed on the ceramic substrate and are arranged at regular intervals, a semiconductor wafer or the like can be more stably supported. Moreover, the semiconductor wafer or the like can be kept more horizontally so that the distance between the ceramic substrate and the semiconductor wafer or the like is made constant. As a result, the semiconductor wafer or the like can be more evenly heated. A ceramic heater according to a second aspect of the present invention comprises: a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face. In case the ceramic heater is provided with the through holes for passing the lifter pins through, the temperature around side walls of the through holes drops since the side walls of the through holes usually contact gas having a lower temperature than the substrate itself. As a result, cooling spots are generated in the heating face. When a semiconductor wafer, a liquid crystal substrate or the like is put on this ceramic heater, in the cooling spots heat is taken away by the cooling spots; therefore, the temperature in this portion drops so that evenness in the temperature of the semiconductor wafer, the liquid crystal substrate and the like is lost. However, according to the ceramic heater of the second aspect of the present invention, the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face; thus, no solid constituting the substrate is present in portions where cooling spots are generated. As a result, the occupation ratio of space becomes large so that the heat capacity thereof gets small. Accordingly, the temperature of the semiconductor wafer, the liquid crystal substrate and the like of the portion in the vicinity of the formed through holes hardly drops, so that the object to be heated, such as the semiconductor wafer or the liquid crystal substrate and the like, can be more evenly heated. Regarding the through holes with a diameter on the heating face side being larger than that on the bottom face side, in case the through holes are constituted so as to have a columnar portion and a diameter-increasing portion having the diameter becoming larger as the portion is closer to the heating face, that is, so as to have a funnel shape, gas having accumulated heat remains in the funnel-shaped portion and cooling spots themselves are not enlarged. Thus, the object to be heated, such as the semiconductor wafer or the liquid crystal substrate, can be more evenly heated. Since the through holes having the above-mentioned shape can be relatively easily formed with a drill and the like, the through holes can be effectively formed. The wafer or the like is heated while the space of the above-mentioned diameter-increasing portion is not filled. This is because when the diameter-increasing portion is filled with a filling member, the ceramic and the filling member are rubbed with each other so that free particles are generated. Further, a ceramic bonded body according to a third aspect of the present invention includes: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of the above-mentioned ceramic substrate, wherein the center of an area surrounded by the interface between the above-mentioned ceramic body and the above-mentioned ceramic substrate or the center of an area constituted by the interface between the above-mentioned ceramic body and the above-mentioned ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the above-mentioned ceramic substrate. In the ceramic bonded body according to the third aspect of the present invention, the ceramic body may be a columnar body or a plate-form body, or may be a hollow body such as a cylindrical body, or may be a filled body having a ceramic-filled structure, which has no cavity inside. FIG. 28 is a sectional view which schematically illustrates a ceramic bonded body 700 using a ceramic body 281 made of a filled body. In the ceramic body 281 made of the filled body, external terminals 283 having sockets 285 , and conductive wires 235 are embedded, and further a lead wire 890 of a temperature measuring element 84 is also embedded. FIG. 29 is a sectional view which schematically illustrates a ceramic bonded body 800 using a ceramic body 381 made of a plate-form body. In the ceramic body 381 made of the filled body, external terminals 383 having sockets 385 , and conductive wires 335 are embedded, and further a lead wire 890 of a temperature measuring element 84 is also embedded. In the case of the columnar body, it may be a triangle pole body 150 or a square pole body 160 or may be a polygonal pole body 170 , as illustrated in FIGS. 30 (a) to 30 (c). In the third aspect of the present invention, the center of the area surrounded by the interface between the ceramic body and the ceramic substrate, or the center of the area constituted by the interface between the ceramic body and the ceramic substrate means the centroid of a figure constituted by being surrounded by the interface, or the centroid of a figure constituted by the interface itself. The centroid is defined as intersection points of straight lines for dividing a figure into two exact halves. In the case of a circle, the center of the circle is the centroid. The most preferred example of the present invention is a ceramic bonded body including: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of the ceramic substrate, wherein the center of a circle surrounded by the interface between the cylindrical ceramic body and the ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the ceramic substrate. Thus, this ceramic bonded body will be described hereinafter. For example, in the case of heating a ceramic bonded body wherein the center of a circle surrounded by the interface between a cylindrical ceramic body and a ceramic substrate, (which may be referred to the center A hereinafter), is consistent with the center of the bottom face of the ceramic substrate, (which may be referred to as the center B hereinafter), the direction along which the cylindrical ceramic body expands is consistent with the direction along which the ceramic substrate expands in the above-mentioned interface. As a result, thermal stress concentrates locally so that thermal fatigue is generated. Thus, cracks and the like are generated. However, according to the third aspect of the present invention, that is, the ceramic bonded body wherein the distance between the center A and the center B, (which may be referred to as the distance L), is from 3 to 200 μm apart, when it is heated, the direction along which the cylindrical ceramic body expands is different from the direction along which the ceramic substrate expands. As a result, thermal stress can be dispersed so that the generation of cracks and the like can be prevented. In the ceramic bonded body having a distance L of less than 3 μm, it is difficult to disperse thermal stress sufficiently. If the distance L exceeds 200 μm, thermal stress is conversely concentrated so that cracks are easily generated. Furthermore, the temperature distribution in the face for heating a semiconductor wafer gets large. It is desired that the above-mentioned conductor is a heating element and the ceramic bonded body functions as a ceramic heater. As described above, this ceramic bonded body has a structure capable of dispersing thermal stress so that the thermal stress does not concentrate locally. Thus, even if temperature-rising and temperature-dropping thereof are repeated, no thermal fatigue is generated. In the ceramic bonded body, a flange portion need not be formed in the bonding face between the cylindrical ceramic body and the ceramic substrate. Therefore, the thermal capacity does not increase so that the temperature-dropping speed is not lowered. For this reason, the ceramic bonded body can be suitably used as a ceramic heater. The heating element may be formed into a layer form or into a line form. Moreover, it is desired that the above-mentioned conductor is an electrostatic electrode and the above-mentioned ceramic bonded body functions as an electrostatic chuck. This is because any electrostatic chuck is used in a corrosive atmosphere in many cases and a constitution wherein the ceramic substrate and the cylindrical ceramic body are bonded to each other as described above is optimal for this chuck. Additionally, the ceramic substrate desirably has a diameter of 250 mm or more. If the diameter of the ceramic substrate is 250 mm or more, the effects of the third aspect of the present invention, i.e., the effects of dispersing thermal stress and preventing the generation of cracks and the like get larger. This fact can easily be understood from FIG. 32 , which shows results of Examples. That is, in the case of the distance L=0, the generation rate of the cracks is higher as the diameter is larger. When the diameter exceeds 250 mm, the rate abruptly becomes large. However, by setting L to 3 μm or 200 μm, the crack generation rate can be suppressed at a low level.

Diagnosis of illnesses or predisposition to certain illnesses

The present invention describes a set of oligomer probes (oligonucleotides and/or PNA oligomers), which serve for the detection of the cytosine methylation state in nucleic acids. These probes are particularly suitable for the diagnosis of existing diseases by analysis of a set of genetic and/or epigenetic parameters.

1. Nucleic acids comprising a sequence segment at least 18 bases long of a chemically pretreated DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712. 2. An oligomer (oligonucleotide or peptide nucleic acid (PNA) oligomer) for the detection of the cytosine methylation state in chemically pretreated DNA, containing at least one base sequence with a length of at least 9 nucleotides, which hybridizes to a chemically pretreated DNA (Seq. ID 1 to Seq. ID 40712). 3. The oligomer according to claim 2, whereby the base sequence comprises at least one CpG dinucleotide. 4. The oligomer according to claim 3, further characterized in that the cytosine of the CpG dinucleotide is found in approximately the middle third of the oligomer. 5. A set of oligomers according to claim 3, comprising at least one oligomer for at least one of the CpG dinucleotides of one of the sequences of Seq. ID 1 to Seq. ID 40712. 6. A set of oligomers according to claim 5 containing at least one oligomer for each of the CpG dinucleotides of one of the sequences of Seq. ID 1 to Seq. ID 40712. 7. A set of at least two nucleic acids according to claim 2, which are utilized as primer oligonucleotides for the amplification of DNA sequences according to at least one of the sequences Seq. ID 1 to Seq. ID 40712 or segments thereof. 8. A set of oligonucleotides according to claim 7, further characterized in that at least one oligonucleotide is bound to a solid phase. 9. A set of oligomer probes for the detection of the cytosine methylation state and/or of single nucleotide polymorphisms (SNPs) in chemically pretreated genomic DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712, comprising at least ten of the oligomers according to one of claims 2 to 4. 10. A method for the production of an arrangement of different oligomers (an array) fixed on a support material for the analysis of disorders related to the methylation state of the CpG dinucleotides of one of the sequences Seq. ID 1 to Seq. ID 40712, in which at least one oligomer according to one of claims 2 to 4 is coupled to a solid phase. 11. An arrangement of different oligomers (an array) according to one of claims 2 to 4, which is bound to a solid phase. 12. The array of different oligonucleotide and/or PNA oligomer sequences according to claim 11, further characterized in that these are arranged on a planar solid phase in the form of a rectangular or hexagonal grid. 13. The array according to claim 11, further characterized in that the solid phase surface is comprised of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold. 14. A DNA and/or PNA array for the analysis of disorders related to the methylation state of genes, which contains at least one nucleic acid according to one of claims 1 or 2. 15. A method for determining genetic and/or epigenetic parameters for the diagnosis of existing diseases or of the predisposition for specific diseases by analysis of cytosine methylations, is hereby characterized in that the following steps are conducted: a) in a genomic DNA sample, cytosine bases that are unmethylated at the 5′-position are converted by chemical treatment to uracil or another base unlike cytosine in its base-pairing behavior; b) from this chemically pretreated genomic DNA, fragments are amplified with the use of sets of primer oligonucleotides according to claim 7 or 8 and a polymerase, whereby the amplified products bear a detectable label; c) the amplified products are hybridized to a set of oligonucleotides and/or PNA probes containing at least one base sequence with a length of at least 9 nucleotides which hybridizes to a chemically pretreated DNA (Seq. ID 1 to Seq. ID 40712) or, however, to an array of different such oligonucleotides and/or PNA probes bound to a solid phase; d) the hybridized amplified products are then detected. 16. The method according to claim 15, further characterized in that the chemical treatment is conducted by means of a solution of a bisulfite, hydrogen sulfite or disulfite. 17. The method according to claim 15, further characterized in that more than ten different fragments are amplified, which are 100-2000 base pairs in length. 18. The method according to claim 15, further characterized in that the amplification of several DNA segments is conducted in one reaction vessel. 19. The method according to claim 15, further characterized in that the polymerase is a heat-stable DNA polymerase. 20. The method according to claim 18, further characterized in that the amplification is conducted by means of the polymerase chain reaction (PCR). 21. The method according to claim 15, further characterized in that the labels of the amplified products are fluorescent labels. 22. The method according to claim 15, further characterized in that the labels of the amplified products are radionuclides. 23. The method according to claim 15, further characterized in that the labels of the amplified products are removable molecular fragments with typical mass, which are detected in a mass spectrometer. 24. The method according to claim 15, further characterized in that the amplified products or fragments of the amplified products are detected in the mass spectrometer. 25. The method according to claim 23, further characterized in that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer. 26. The method according to 23, further characterized in that the detection is carried out and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI). 27. The method according to claim 15, further characterized in that the genomic DNA was obtained from cells or cell components that contain DNA, whereby sources for DNA comprise e.g., cell lines, biopsies, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and all possible combinations thereof. 28. A kit, comprising a bisulfite (=bisulfite (disulfite), hydrogen sulfite) reagent as well as oligonucleotides and/or PNA oligomers according to one of claims 2 to 4. 29. Use of a nucleic acid comprising a sequence segment at least 18 bases long of a chemically pretreated DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712, an oligonucleotide or PNA oligomer containing at least one base sequence with a length of at least 9 nucleotides which hybridizes to a chemically pretreated DNA (Seq. ID 1 to Seq. ID 40712), a kit comprising a bisulfite (=bisulfite (disulfite), hydrogen sulfite) reagent as well as oligonucleotides and/or PNA oligomers according to one of claims 2 to 4, or an array of such oligonucleotides and/or PNA oligomers fixed on a support material for the diagnosis and/or therapy of undesired drug interactions; cancer diseases; CNS malfunctions; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain lesions; psychotic disturbances and personality disorders; dementia and/or associated syndromes; cardiovascular disease; malfunction, damage or disorder of the gastrointestinal tract; malfunction, damage or disorder of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunctions, headaches; sexual malfunctions, by analysis of methylation patterns. 30. The method according to claim 24, further characterized in that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer. 31. The method according to claim 24, further characterized in that the detection is carried out and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI).

<SOH> FIELD OF THE INVENTION <EOH>The levels of observation that have been well studied in molecular biology according to developments in methods in recent years include the genes themselves, the transcription of these genes into RNA and the translation to proteins therefrom. During the course of development of an individual, which gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome. In this regard, pathogenic states are also expressed by a modified methylation pattern of individual genes or of the genome. The present invention describes nucleic acids, oligonucleotides, PNA oligomers and a method for the diagnosis of existing diseases or of predisposition for specific diseases.

Self-propelled screening apparatus

A self-propelled screening apparatus (1) having a chassis (11); a pair of endless tracks (12) supporting the chassis (11); a superstructure (13) rotatably mounted on the chassis (11); a prime mover provided in or on the superstructure (13) and arranged to provide motive power to operate the endless tracks (12) and where the prime mover is rotatable with the superstructure (13); and a screen box (16) mounted on the superstructure (13), also for rotation therewith. The screen box (16) has an input (17) for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material. The prime mover and the screen box (16) are arranged on opposite sides of the rotational axis of the superstructure (13), to provide a stable weight distribution.

1. A self-propelled screening apparatus which comprises: a chassis; a pair of endless tracks supporting the chassis; a prime mover provided in or on the superstructure and arranged to provide motive power to operate the endless tracks, said prime mover being rotatable with the superstructure; and a screen box mounted on this superstructure, also for rotation therewith, said screen box having an input for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material; in which the prime mover and the screen box are arranged on opposite sides of the rotational axis of the superstructure, to provide a stable weight distribution. 2. A self-propelled screening apparatus which comprises: a chassis; a pair of endless tracks supporting the chassis; a superstructure rotatably mounted on the chassis via a slewing ring; and a screen box mounted on the superstructure for rotation therewith, said screen box having an input for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material. 3. Apparatus according to claim 1, in which the superstructure comprises a housing or canopy in which an engine is mounted, and which serves as a source of power to operate the endless tracks. 4. Apparatus according to claim 3, in which the engine is arranged to drive driven components of the screen box. 5. Apparatus according to claim 1, in which the screen box has more than one screen deck, and has a loading chute at an inboard end, and an opposite discharge end over which oversized material can be discharged. 6. Apparatus according to claim 1, in which the screen box is adjustable inwardly or outwardly of the rotational axis of the superstructure, to move the discharge point relative to the apparatus. 7. Apparatus according to claim 6, in which the screen box is mounted on, or incorporates a telescopically adjustable frame. 8. Apparatus according to claim 1, and including a driver's cab from which an operator can control the movement of the apparatus and/or screening operation. 9. Apparatus according to claim 1, in which the apparatus is arranged to be capable of being operated by remote control. 10. Apparatus according to claim 1, in which the screenbox is independently adjustably relative to the superstructure. 11. Apparatus according to claim 1, in which the screenbox is pivotable through up to 90 degrees in either direction from a datum position relative to the superstructure. 12. Apparatus according to claim 2, in which the superstructure comprises a housing or canopy in which an engine is mounted, and which serves as a source of power to operate the endless tracks. 13. Apparatus according to claim 12, in which the engine is arranged to drive driven components of the screen box. 14. Apparatus according to claim 2, in which the screen box has more than one screen deck, and has a loading chute at an inboard end, and an opposite discharge end over which oversized material can be discharged. 15. Apparatus according to claim 2, in which the screen box is adjustable inwardly or outwardly of the rotational axis of the superstructure, to move the discharge point relative to the apparatus. 16. Apparatus according to claim 15, in which the screen box is mounted on, or incorporates a telescopically adjustable frame. 17. Apparatus according to claim 2, and including a driver's cab from which an operator can control the movement of the apparatus and/or screening operation. 18. Apparatus according to claim 2, in which the apparatus is arranged to be capable of being operated by remote control. 19. Apparatus according to claim 2, in which the screenbox is independently adjustably relative to the superstructure. 20. Apparatus according to claim 2, in which the screenbox is pivotable through up to 90 degrees in either direction from a datum position relative to the superstructure.