The disclosure describes recombinant alphavirus RNA molecules and expression of heterologous proteins therefrom in animal cells. Recombinant alphaviruses of the present invention, when made to express an antigenic protein, can be administered as vaccines.

The present invention is related to DNA expression systems based on alphaviruses, which systems can be used to transform animal cells for use in the production of desired products, such as proteins and vaccines, in high yields.
The rapid development of biotechnology is to a large extent due to the introduction of recombinant DNA technique, which has revolutionized cellbiological and medical research by opening new approaches to elucidate the molecular mechanisms of the cell. With the aid of the techniques of cDNA cloning, large numbers of interesting protein molecules are characterized each year. Therefore, a lot of research activity is today directed to elucidate the relationship between structure and function of these molecules. Eventually this knowledge will increase our possibilities to preserve healthiness and combat diseases in both humans and animals. Indeed, there is today a growing list of new xe2x80x9cclonedxe2x80x9d protein products that are already used as pharmaceuticals or diagnostics.
In the recombinant DNA approaches to study biological questions, DNA expression systems are crucial elements. Thus, efficient DNA expression systems, which are simple and safe to use, give high yields of the desired product and can be used in a variety of host cells, especially also in mammalian cells, are in great demand.
Many attempts have been made to develop DNA expression systems, which fulfill these requirements. Often, viruses have been used as a source of such systems. However, up to date none of the existing viral expression systems fulfill all these requirements in a satisfying way. For instance, the Baculovirus expression system for cDNA is extremely efficient but can be used only in insect cells (see Reference 1 of the list of cited references; for the sake of convenience, in the following the cited references are only identified by the number they have on said list). As many important molecules will have to be produced and processed in cells of mammalian origin in order for them to become active, this system cannot be used in such cases. Furthermore, the Baculovirus cDNA expression system is not practically convenient for analysis of the relationship between structure and function of a protein because this involves in general the analysis of whole series of mutant variants. Today it takes about 6-8 weeks to construct a single Baculo recombinant virus for phenotype analyses. This latter problem is also true for the rather efficient Vaccinia recombinant virus and other contemporary recombinant virus cDNA expression systems (2,3). The procedure to establish stably transformed cell lines is also a very laborious procedure, and in addition, often combined with very low levels of protein expression.
Hitherto, most attempts to develop viral DNA expression systems have been based on viruses having DNA genomes or retroviruses, the replicative intermediate of the latter being double stranded DNA.
Recently, however, also viruses comprising RNA genomes have been used to develop DNA expression systems.
In EP 0 194 809 RNA transformation vectors derived from (+) strand RNA viruses are disclosed which comprise capped viral RNA that has been modified by insertion of exogenous RNA into a region non-essential for replication of said virus RNA genome. These vectors are used for expression of the function of said exogenous RNA in cells transformed therewith. The RNA can be used in solution or packaged into capsids. Furthermore, this RNA can be used to generate new cells having new functions, i.e. protein expression. The invention of said reference is generally claimed as regards host cells, (+) strand RNA viruses and the like. Nevertheless, it is obvious from the experimental support provided therein that only plant cells have been transformed and in addition only Bromo Mosaic virus, a plant virus, has been used as transformation vector.
Although it is stated in said reference that it is readily apparent to those skilled in the art to convert any RNA virus-cell system to a useful expression system for exogenous DNA using principals described in the reference, this has not been proven to be true in at least the case of animal cell RNA viruses. The reasons for this seem to be several. These include:
1) Inefficiencies in transfecting animal cells with in vitro transcribed RNA;
2) Inefficiency of apparently replication competent RNA transcripts to start RNA replication after commonly used transfection procedures;
3) The inability to produce high titre stocks of recombinant virus that does not contain any helper virus;
4) The inability to establish stable traits of transformed cells expressing the function of the exogenous RNA.
In Proc. Natl. Acad. Sci. USA, Vol 84, 1987, pp 4811-4815 a gene expression system based on a member of the Alphavirus genus, viz. Sindbis virus, is disclosed which is used to express the bacterial CAT (chloramphenicol acetyltransferase) gene in avian cells, such as chicken embryo fibroblasts.
Xiong et al., Science, Vol 243, 1989, 1188-119; also disclose a gene expression system based on Sindbis virus. This system is said to be efficient in a broad range of animal cells. Expression of the bacterial CAT gene in insect, avian and mammalian cells inclusive of human cells is disclosed therein.
Even though it is known from prior art that one member of the Alphavirus genus, the Sindbis virus, can tolerate insertion and direct the expression of at least one foreign gene, the bacterial chloramfenicol acetyl transferase (CAT) gene, it is evident from the results described that both systems described above are both ineffective in terms of exogenous gene expression and also very cumbersome to use. Hence, neither system has found any usage in the field of DNA expression in animal cells today.
In the first example a cDNA copy of a defective interfering (DI) virus variant of Sindbis virus was used to carry the CAT gene. RNA was transcribed in vitro and used to transfect avian cells and some CAT protein production could be demonstrated after infecting cells with wild-type Sindbis virus. The latter virus provided the viral replicase for expression of the CAT construct. The inefficiency of this system depends on 1) low level of initial DI-CAT RNA transfection (0.05-0.5% of cells) and 2) inefficient usage of the DI-CAT RNA for protein translation because of unnatural and suboptimal protein intitation translation signals. This same system also results in packaging of some of the recombinant DI-CAT genomes into virus particles. However, this occurs simultaneously with a very large excess of wild-type Sindbis virus production. Therefore, the usage of this mixed virus stock for CAT expression will be much hampered by the fact that most of the replication and translation activity of the cells infected with such a stock will deal with the wild-type and not with recombinant gene expression.
Much of the same problems are inherent to the other Sindbis expression system described. In this an RNA replication competent Sindbis DNA vector is used to carry the CAT gene. RNA produced in vitro is shown to replicate in animal cells and CAT activity is found. However, as only a very low number of cells are transfected the overall CAT production remains low. Another possible explanation for this is that the Sindbis construct used is not optimal for replication. Wild-type Sindbis virus can be used to rescue the recombinant genome into particles together with an excess of wild-type genomes and this mixed stock can then be used to express a CAT protein via infection. However, this stock has the same problems as described above for the recombinant DI system. The latter paper shows also that if virus is amplified by several passages increased titres of the recombinant virus particles can be obtained. However, one should remember that the titre of the wild-type virus will increase correspondingly and the original problem of mostly wild-type virus production remains. There are also several potential problems when using several passages to produce a mixed virus stock. As there is no selected pressure for preservation of the recombinant genomes these might easily 1) undergo rearrangements and 2) become outnumbered by wild-type genomes as a consequence of less efficient replication and/or packaging properties.
Another important aspect of viral DNA expression vectors is use thereof to express antigens of unrelated pathogens and thus they can be used as vaccines against such pathogens.
Development of safe and effective vaccines against viral diseases has proven to be quite a difficult task. Although many existing vaccines have helped to combat the worldwide spread of many infectious diseases, there is still a large number of infectious agents against which effective vaccines are missing. The current procedures of preparing vaccines present several problems: (1) it is often difficult to prepare sufficiently large amounts of antigenic material; (2) In many cases there is the additional hazard that the vaccine preparation is not killed or sufficiently attenuated; (3) Effective vaccines are often hard to produce since there is a major difficulty in presenting the antigenic epitope in an immunologically active form; (4) In the case of many viruses, genetic variations in the antigenic components results in the evolution of new strains with new serological specificities, which again creates a need for the development of new vaccines.
Two types of viral DNA vectors have been developed in order to overcome many of these problems in vaccine production. These either provide recombinant viruses or provide chimaeric viruses. The recombinant viruses contain a wild-type virus package around a recombinant genome. These particles can be used to infect cells which then produce the antigenic protein from the recombinant genome. The chimaeric viruses also contain a recombinant genome but this specifies the production of an antigen, usually as part of a normal virus structural protein, which then will be packaged in progeny particles and e.g. exposed on the surface of the viral spike proteins. The major advantages of these kind of virus preparations for the purpose of being used as a vaccine are 1) that they can be produced in large scale and 2) that they provide antigen in a natural form to the immunological system of the organism. Cells, which have been infected with recombinant viruses, will synthesize the exogenous antigen product, process it into peptides that then present them to T cells in the normal way. In the case of the chimaeric virus there is, in addition, an exposition of the antigen in the context of the subunits of the virus particle itself. Therefore, the chimaeric virus is also-called an epitope carrier.
The major difficulty with these kind of vaccine preparations are, how to ensure a safe and limited replication of the particles in the host without side effects. So far, some success has been obtained with vaccinia virus as an example of the recombinant virus approach (69) and of polio virus as an example of a chimaeric particle (70-72). As both virus variants are based on commonly used vaccine strains one might argue that they could be useful vaccine candidates also as recombinant respectively chimaeric particles (69-72). However, both virus vaccines are combined with the risk for side effects, even severe ones, and in addition these virus strains have already been used as vaccines in large parts of the population in many countries.
As is clear from the afore mentioned discussion there is much need to develop improved DNA expression systems both for an easy production of important proteins or polypeptides in high yields in various kinds of animal cells and for the production of recombinant viruses or chimaeric viruses to be used as safe and efficient vaccines against various pathogenes.
Thus, an object of the present invention is to provide an improved DNA expression system based on virus vectors which can be used both to produce proteins and polypeptides and as recombinant virus or chimaeric virus, which system offers many advantages over prior art.
To that end, according to the present invention there is provided an RNA molecule derived from an alphavirus RNA genome and capable of efficient infection of animal host cells, which RNA molecule comprises the complete alphavirus RNA genome regions, which are essential to replication of the said alphavirus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof.
Alphavirus is a genus belonging to the family Togaviridae having single stranded RNA genomes of positive polarity enclosed in a nucleocapsid surrounded by an evelope containing viral spike proteins.
The Alphavirus genus comprises among others the Sindbis virus, the Semliki Forest virus (SFV) and the Ross River virus, which are all closely related. According to a preferred embodiment of the invention, the Semliki Forest virus (SFV) is used as the basis of the DNA expression system.
The exogenous RNA sequence encodes a desired genetic trait, which is to be conferred on the virus or the host cell, and said sequence is usually complementary to a DNA or cDNA sequence encoding said genetic trait. Said DNA sequence may be comprised of an isolated natural gene, such as a bacterial or mammalian gene, or may constitute a synthetic DNA sequence coding for the desired genetic trait i.e. expression of a desired product, such as an enzyme, hormone, etc. or expression of a peptide sequence defining an exogenous antigenic epitope or determinant.
If the exogenous RNA sequence codes for a product, such as a protein or polypeptide, it is inserted into the viral RNA genome replacing deleted structural protein encoding region(s) thereof, whereas a viral epitope encoding RNA sequence may be inserted into structural protein encoding regions of the viral RNA genome, which essentially do not comprise deletions or only have a few nucleosides deleted.
The RNA molecule can be used per se, e.g. in solution to transform animal cells by conventional transfection, e.g. the DEAE-Dextran method or the calcium phosphate precipitation method. However, the rate of transformation of cells, and, thus the expression rate can be expected to increase substantially if the cells are transformed by infection with infectious viral particles. Thus, a suitable embodiment of the invention is related to an RNA virus expression vector comprising the RNA molecule of this invention packaged into infectious particles comprising the said RNA within the alphavirus nucleocapsid and surrounded by the membrane including the alphavirus spike proteins.
The RNA molecule of the present invention can be packaged into such particles without restraints provided that it has a total size corresponding to the wild type alphavirus RNA genome or deviating therefrom to an extent compatible with package of the said RNA into the said infectious particles.
These infectious particles, which include recombinant genomes packaged to produce a pure, high titre recombinant virus stock, provides a means for exogenous genes or DNA sequences to be expressed by normal virus particle infection, which as regards transformation degree, is much more efficient than RNA transfection.
According to a suitable embodiment of the invention such infectious particles are produced by cotransfection of animal host cells with the present RNA which lacks part of or the complete region(s) encoding the structural viral proteins together with a helper RNA molecule transcribed in vitro from a helper DNA vector comprising the SP6 promoter region, those 5xe2x80x2 and 3xe2x80x2 regions of the alphavirus cDNA which encode cis acting signals needed for RNA replication and the region encoding the viral structural proteins but lacking essentially all of the nonstructural virus proteins encoding regions including sequences encoding RNA signals for packaging of RNA into nucleocapsid particles, and culturing the host cells.
According to another aspect of the invention efficient introduction of the present RNA into animal host cells can be achieved by electroporation. For example, in the case of Baby Hamster Kidney (BHK) cells a transformation degree of almost 100% has been obtained for the introduction of an RNA transcript derived from SFV cDNA of the present invention. This makes it possible to reach so high levels of exogenous protein production in every cell that the proteins can be followed in total cell lysates without the need of prior concentration by antibody precipitation.
By electroporation, it is also possible to obtain a high degree of cotransfection in the above process for production of infectious particles comprising packaged RNA of the present invention. Essentially all animal cells will contain both the present RNA molecule and the helper RNA molecule, which leads to a very efficient trans complementation and formation of infectious partcles. A pure recombinant virus stock, consisting of up to 109-1010 infectious particles, can be obtained from 5xc3x97106 cotransfected cells after only a 24 h incubation. Furthermore, the so obtained virus stock is very safe to use, since it is comprised of viruses containing only the desired recombinant genome, which can infect host cells but can not produce new progeny virus.
Theoretically, a regeneration of a wild-type virus genome could take place when producing the recombinant virus in the contransfected cells. However, the possibility to avoid spread of such virus can be eliminated by incorporating a conditionally lethal mutation into the structural part of the helper genome. Such a mutation is described in the experimental part of this application. Thus, the virus produced with such a helper will be noninfectious if not treated in vitro under special conditions.
The technique of electroporation is well known within the field of biotechnology and optimal conditions can be established by the man skilled in the art. For instance, a BioRad Gene pulser apparatus (BioRad, Richmond, Calif., USA) can be used to perform said process.
The RNA molecule of the present invention is derived by in vivo or in vitro transcription of a cDNA clone, originally produced from an alphavirus RNA and comprising an inserted exogenous DNA fragment encoding a desired genetic trait.
Accordingly, the present invention is also related to a DNA expression vector comprising a full-length or partial cDNA complementary to alphavirus RNA or parts thereof and located immediately downstream of the SP6 RNA polymerase promoter and having a 5xe2x80x2ATGG, a 5xe2x80x2GATGG or any other 5xe2x80x2 terminus and a TTTCCA69ACTAGT (SEQ ID NO: 25) or any other 3xe2x80x2 terminus.
According to one aspect of the present invention portions of the viral cDNA are deleted, the deletions comprising the complete or part of the region(s) encoding the virus structural proteins, and the vector further comprises an integrated polylinker region, which may correspond to BamHI-SmaI-XmaI, inserted at a location which enables an exogenous DNA fragment encoding a foreign polypeptide or protein to be inserted into the vector cDNA for subsequent expression in an animal host cell.
According to another aspect of this invention, the vector is comprised of full-length cDNA wherein an exogenous DNA fragment encoding a foreign epitopic peptide sequence can be inserted into a region coding for the viral structural proteins.
It is appreciated that this cDNA clone with its exogenous DNA insert is very efficiently replicated after having been introduced into animal cells by transfection.
A very important aspect of the present invention is that it is applicable to a broad range of host cells of animal origin. These host cells can be selected from avian, mammalian, reptilian, amphibian, insect and fish cells. Illustrative of mammalian cells are human, monkey, hamster, mouse and porcine cells. Suitable avian cells are chicken cells, and as reptilian cells viper cells can be used. Cells from frogs and from mosquitoes and flies (Drosophilal) are illustrative of amphibian and insecticidal cells, respectively. A very efficient virus vector/host cell system according to the invention is based on SFV/BHK cells, which will be discussed more in detail further below.
However, even though a very important advantage of the present DNA expression vector is that it is very efficient in a broad variety of animal cells it can also be used in other eucaryotic cells and in procaryotic cells.
The present invention is also related to a method to produce transformed animal host cells comprising transfection of the cells with the present RNA molecule or with the present transcription vector comprised of cDNA and carrying an exogenous DNA fragment. According to a suitable embodiment of the invention, transfection is produced by the above mentioned electroporation method, a very high transfection rate being obtained.
A further suitable transformation process is based on infection of the animal host cells with the above mentioned infectious viral particles comprising the present RNA molecule.
The transformed cells of the present invention can be used for different purposes.
One important aspect of the invention is related to use of the present transformed cells to produce a polypeptide or a protein by culturing the transformed cells to express the exogenous RNA and subsequent isolation and purification of the product formed by said exepression. The transformed cells can be produced by infection with the present viral particles comprising exogenous RNA encoding the polypeptide or protein as mentioned above, or by transfection with an RNA transcript obtained by in vitro transcription of the present DNA vector comprised of cDNA and carrying an exogenous DNA fragment coding for the polypeptide or the protein.
Another important aspect of the invention is related to use of the present transformed cells for the production of antigens comprised of chimaeric virus particles for use as immunizing component in vaccines or for immunization purposes for in vivo production of immunizing components for antisera production.
Accordingly, the present invention is also related to an antigen consisting of a chimaeric alphavirus having an exogenous epitopic peptide sequence inserted into its structural proteins.
Preferably, the chimaeric alphavirus is derived from SFV.
According to a suitable embodiment, the exogenous epitopic peptide sequence is comprised of an epitopic peptide sequence derived from a structural protein of a virus belonging to the immunodeficiency virus class inclusive of the human immunodeficiency virus types.
A further aspect of the invention is related to a vaccine preparation comprising the said antigen as immunizing component.
In said vaccine the chimaeric alphavirus is suitably attenuated by comprising mutations, such as the conditionally lethal SFV-mutation described before, amber (stop codon) or temperature sensitive mutations, in its genome.
For instance, if the chimaeric virus particles containing the aforementoned conditional lethal mutation in its structural proteins (a defect to undergo a certain proteolytical cleavage in host cell during morphogenesis) is used as a vaccine then chimaeric virus particles are first activated by limited proteolytic treatment before being given to the organism so that they can infect recipient cells. New chimaeric particles will be formed in cells infected with the activated virus but these will again have the conditional lethal phenotype and further spread of infection is not possible.
The invention is also concerned with a method for the production of the present antigen comprising
a) in vitro transcription of the cDNA of the present DNA vector carrying an exogenous DNA fragment encoding the foreign epitopic peptide sequence and transfection of animal host cells with the produced RNA transcript, or
b) transfection of animal host cells with the said cDNA of the above step a), culturing the transfected cells and recovering the chimaeric alphavirus antigen. Preferably, transfection is produced by electroporation.
Still another aspect of the invention is to use a recombinant virus containing exogenous RNA encoding a polypeptide antigen for vaccination purpose or to produce antisera. In this case the recombinant virus or the conditionally lethal variant of it is used to infect cells in vikvo and antigen production will take place in the infected cells and used for antigen presentation to the immunological system.
According to another embodiment of the invention, the present antigen is produced in an organism by using in vivo infection with the present infectious particles containing exogenous RNA encoding an exogenous epitopic peptide sequence.


An apparatus, method, and non-transitory machine-readable medium provide for accurate automatic determination of alarm-operator action linkage for operator assessment and alarm guidance using custom graphics and control charts. The apparatus includes a memory and a processor operably connected to the memory. The processor receives process control system data regarding a field device in an industrial process control and automation system; extracts information from the process control system data; generates a tuple based on the extracted information; and performs a rectifying operation in the industrial process control and automation system based on the generated tuple of the field device.

Effective process alarm analysis and identification of corrective action sequences is important for efficient manual and autonomous operation of an industrial plant. Alarms often re-occur on the same device tag or, at times, groups of devices go into alarm state together. The operator actions to resolve the process alarms can be assessed from the system data to identify the competency gaps and guide the operators to resolve alarms faster and better.

System and method for accurate automatic determination of “alarm-operator action” linkage for operator assessment and alarm guidance using custom graphics and control charts


In one embodiment, a cable reel axle shaft is configured with a mounting member and an encased rotary coupling. In particular, a first end of the axle shaft at the mounting member comprises a stationary radio frequency (RF) connection (e.g., a stator), and another end of the axle shaft comprises a rotating RF connection (e.g., a rotor). The rotor-stator break may then be located within the axle shaft, illustratively within the member. In this manner, a rotary coupling is extended and integrated into the center of the structural axle shaft for the cable reel, such that an RF connection may be maintained throughout adjustment of an accompanying variable-length RF antenna while efficiently handling the changes in required RF cable length. This provides numerous benefits over individual components, such as decreased size and weight, increased RF performance, greater survivability, and ease of operation (e.g., to spool and unspool an RF cable).

There exist many situations where it is desirable to have a variable length radio frequency (RF) connection. For instance, many RF antennas, such as for news vans or other mobile antenna platforms, have a variable-length (e.g., height) RF antenna, where one end of an RF cable is attached to the variable-length RF antenna, and another end is attached to a stationary opposite end.
To date, to allow the RF cable to accommodate the adjustable-length RF antenna, a variety of techniques have been used. In one technique, the maximum amount of needed RF cable according to the maximum length of the antenna is connected, and when the length of the antenna is anything other than the maximum length, the excess cable is manually coiled on a vertical surface in the shape of a side-ways figure eight held up by a pair of lobes or hooks. Excess cable can also be stored horizontally by coiling it inside a protective enclosure (often referred to as a "cable coffin"), as neatly as the operator is capable of accomplishing. In another technique, a cable reel may be used to spool and unspool the RF cable in response to the adjusted length of the RF antenna, however, due to the rotation of the cable reel, an operator has to manually disconnect and reconnect the RF cable end whenever the antenna height is adjusted. Both of these two manual techniques pose great inconveniences to the operators, and result in increased set-up (emplacement) and tear-down times of the antennas.
In another known technique, the RF cable may be wound around the adjustable-length RF antenna in a generally helical (spiraling/coiling) manner, much like a television news truck. As the RF antenna is extended (e.g., raised), the RF coil expands like a spring around the antenna (e.g., a mast on which the antenna rests). As the RF antenna is shortened (e.g., lowered), the RF coil compresses. While this arrangement may be suitable for certain situations, such as low power and/or low frequency transmissions, as will be appreciated by those skilled in the art, RF cable length is a great contributor to line loss, typically expressed in decibels (dB). In addition to cable length, line loss in a given cable is a function of the dielectric material between the center and outer conductor, the diameter of the center conductor, the diameter of the outer conductor, and also the frequency. Lower frequency communications such as VHF (below 300 MHz frequency) have less line loss for a given length than higher, microwave frequencies (e.g., C-Band or X-Band). For example, using a premium, high-performance, large diameter cable, approximate loss may be characterized for high frequency transmissions as 1 dB of signal loss for every 20 feet of RF cable. Due to the spiraling nature of this particular alternative, the length of the RF cable is roughly eight to ten times the maximum length (e.g., height) in order to allow the proper expansion of the RF cable spiral to match the length of the variable-length antenna. In addition to other signal losses, such as from weather, interference from jamming, line of site, curvature of the earth, etc., the signal loss due to the extra cable length may be significant and unsatisfactory (e.g., particularly to maintain a high baud rate over the RF transmission).
Additionally, slip-rings may be used for certain classes of cable reels, such as multi-conductor power, discrete controls, and low frequency communications, often found on harbor and boom cranes. For this a slip-ring is typically enclosed in an environmental housing, sized for the number of channels (conductors) and current carrying capability required by the load. Typical slip-ring housings for a cable reel (e.g., a 10-inch wide cable reel supporting 1-inch diameter cable), however, are approximately 6-8 inches in diameter and 8-10 inches high. This extra size and weight may pose particular difficulty to mobile applications. Slip-ring frequency capability is limited by the geometry of the ring and brush assembly, as may be appreciated by those skilled in the art, has a cut-off limit of approximately 100 kHz. For this reason slip rings are typically not used for RF communications.
For higher frequency RF communications an RF rotary coupling is typically used. These devices are designed specifically for higher frequencies and use contacting (single channel) or wave-guide (multi-channel) architectures. As with slip rings, implementation of an RF rotary coupling with a cable reel requires a suitable environmental enclosure. The weight and volume requirements for a 1-inch diameter high-performance coaxial cable are approximately the same as a slip-ring for a power cable of the same size. The additional volume and weight required poses great difficulty to mobile applications.

Cable reel axle shaft with integrated radio frequency rotary coupling


A carrying case for planar, rectangular articles of minimal thickness, such as the housings in which audio compact discs are packaged, is disclosed. The carrying case includes a housing comprised of six rectangular side panels, five of which are integrally bound into a fixed rectilinear relation with one-another, leaving a remaining sixth side open, the sixth side panel being hingedly fixed adjacent the housing's open side to one of the five fixed side panels. Hook and loop fabric fasteners on the housing, and on a hinged closure flap of the sixth panel, permit the sixth side panel to cover the open sixth side securely. A plurality of drawers is slidably disposed within the housing upon opposed pairs of drawer supports comprised of tracks and runners. Each drawer has a bottom, and upstanding front, rear and side walls. Each also includes a plurality of thin, upstanding rectilinearly-oriented fences dividing the drawer's interior space, into nine compartments. An opening is provided in the drawer bottom within each compartment, and an opposed pair of semicircular cutouts in the front wall of each drawer serves as a drawer pull.

1. Field Of The Invention
The present invention relates generally to carrying cases for planar articles, and more specifically to a carrying case for audio compact discs.
2. Description Of The Related Art
Consumers of every technological era have been challenged by the need to store and transport all manner of articles comprising media upon which sounds, images or data are recorded. Today's consumer is particularly challenged with the need for article carrying cases having particular suitability for storing such media in a manner convenient to access while driving an automobile.
The need and practicality of carrying music media for use in an automobile arose with the advent of the 8-track tape cartridge, and was followed by the more convenient minicassette. For the storage and transport of these media, a myriad of designs have been proposed upon several primary themes including folders, tote bags, briefcases and suitcases, some being multiply divided to retain the media in a particular order.
Currently, with the introduction of compact audio disc media, the mobile consumer has been offered a whole new frontier of music transport and storage density. However, such compact discs, as they are presently packaged, are sometimes difficult to manipulate. Further, some of the indicia on the housings thereof identifying the nature and content of the audio recording therewithin is in small type and therefore very difficult to read. This presents a formidable practical problem in a whole class of storage and transport formats wherein a narrow edge of the housing bearing such small type is the only surface presented to the user for viewing and choosing a selection. This problem is exemplified in the device disclosed in U.S. Pat. No. 4,762,225 issued to Henkel in 1988. Therein, to avoid reading small type, a user must shuffle through the individual disc housings to confirm a selection.
By far, a better manner of presentation appears to be one in which the user is permitted to view one of a compact disc housing's broad faces where larger typefaces are commonly used along with additional identifying indicia such as photographic or other graphic representations. Such presentation of the broad face of a compact disc housing has been employed in apparatus disclosed in U.S. Pat. No. 4,892,189 issued to Kunimune in 1990. Therein, twin, hinged, opposed mating shells having four housing receptacles apiece are proposed for storing, transporting and displaying audio compact discs. However, this configuration has an inherent limitation in that only two such opposed mating shells may be used with one another. Further, to access the compact disc housings within a case such as that in the Kunimune patent, one must swing one shell into a ninety-degree orientation to the other, thus requiring a relatively large volume of space for operating the case as it is intended to be used. This is a drawback in that, for example, it would require one to refrain from piling other articles on top of such a case if access to the contents thereof was later desired.
Thus, there exists a need for a compact disc storage and carrying case able to present compact disc housings in an easily-readable orientation. Further, such a case should be able to store a fair number of such items, but it should not require an undue amount of space for proper operation and access to its contents. And, ideally, this case should be capable of being stowed in more than one orientation while still permitting its contents to be accessed.


An implant is set forth having at least one first element and a second element, wherein the first element is provided in a pre-determined relative location to the second element, in particular at least partly in a recess of the second element. The first element has a pre-positioning location having play with respect to the second element and allowing a fully area gas sterilization of both elements and can be displaced from this pre-positioning location into an end positioning location corresponding to a fixed connection of both elements.

1. Field of the Invention
The invention relates to an implant and to a method of manufacturing an implant packed in a sterile manner.
2. Description of the Related Art
Ultra-high molecular weight polyethylene (UHMWPE) is a material widespread in orthopaedic endoprosthetics which has proven itself as a sliding partner with respect to metallic and ceramic materials. A problem with these material pairings is the wear unavoidable in use and the particle-induced osteolysis triggered thereby which in the worst case can result in a loosening of the implant.
A suitable measure for the optimization of the UHMWPE with the aim of reducing the arising wear is high cross-linking. This is achieved by electron radiation or gamma radiation and a heat treatment of the material, whereby the ultra-high molecular weight polyethylene is made wear resistant and given long term stability. The high cross-linking takes place by a cross-linking of the UHMWPE molecule chains and by a de-saturation of the free radicals.
The sterilization method of gamma radiation usual in orthopaedic endoprosthetics cannot be used for highly cross-linked UHMWPE, because unwanted free radicals would again thereby arise. These free radicals promote an oxidization effect and an embrittlement of the associated material. Highly cross-linked UHMWPE is therefore not sterilized with ionizing radiation, but with gas, for example with ethylene oxide or gas plasma.
With implant components which consist exclusively of highly cross-linked polyethylene, satisfactory results are achieved by means of high cross-linking and heat treatment of the UHMWPE blank, subsequent mechanical processing, packaging and gas sterilization.
However, implants can consist of a plurality of elements put together during the manufacturing process. With such composite implants, the problem of effective sterilization arises. A sterilization with gamma rays would cover the whole implant and thus oxidize elements made of UHMWPE. A gas sterilization, in contrast, only acts on all those surfaces of the implant which are accessible to the gas during the sterilization. To also make surfaces covered by additional elements accessible to the sterilizing gas, it is known in this connection to carry out the connection of implant elements with play. However, such play is problematic on the basis of clinical experience, since micro-movements of implant elements made possible in this manner are under suspicion of producing unwanted wear and of countering a permanent ingrowth of the bone.

Implant and a method of manufacturing an implant packed in a sterile manner


An optical system includes a window made of a curved piece of a transparent material having an inner surface and an outer surface. The inner surface has a nominal inner surface shape defined by a first conicoidal relationship, and the outer surface has a nominal general aspheric surface shape. The optical system also typically includes a sensor and an optical train on the side of the inner surface of the window. The accuracy of the shape of the inner surface is tested by directing a coherent light beam through a remote focus of the inner surface, reflecting the light beam from the inner surface toward an adjacent focus of the inner surface, reflecting the light beam from a spherical reflector at the adjacent focus of the inner surface and back toward the inner surface, reflecting the light beam from the inner surface back toward the remote focus, and interferometrically comparing the reflected beam arriving at the remote focus with a reference beam.

This invention relates to an optical system having a window therein, and in particular to such an optical system used in an aircraft or missile wherein the window is a conformal window.
An optical sensor receives radiated energy from a scene and converts it to an electrical signal. The electrical signal is provided to a display or further processed for pattern recognition or the like. Optical sensors are available in a variety of types and for wavelengths ranging from the ultraviolet, through the visible, and into the infrared. Optical sensors are used in a variety of commercial and military applications. In some applications the optical sensors are fixed in orientation, and in others the optical sensor is movable such as by a pivoting motion to allow sensing over a wide angular range.
The optical sensors generally employ a photosensitive material that faces the scene and produces an electrical output responsive to the incident energy. The photosensitive material and remainder of the sensor structure are rather fragile, and are easily damaged by dirt, erosion, chemicals, or high air velocity. In service, the sensor is placed behind a window through which it views the scene and which protects the sensor from such external effects. The window must be transparent to the radiation of the operating wavelength of the sensor and resist attack from the external forces. The window must also permit the sensor to view the scene over the specified field of regard.
The window would ideally introduce no wavefront aberration at the center of the field of view, other than possibly spherical aberration, particularly if the sensor is an imaging sensor. The thicker and more highly curved is the window, the more likely is the introduction of significant wavefront aberration. A wide variety of sensor windows have been used in various aircraft applications. In many cases such as low-speed commercial helicopters, flat windows are acceptable. Windows that are shaped as segments of spheres are used in aircraft and missile applications, but for these windows the wavefront aberration tends to be high if the gimbal location is not at the spherical center of the window. In all of these window types, if the window must be wide or must project a substantial distance into an airflow to permit a large field of regard, the aerodynamic drag introduced by the window is large.
For applications involving aircraft and missiles operating at high speeds, the window should be relatively aerodynamic such that the presence of the window extending into the airstream does not introduce unacceptably high and/or asymmetric aerodynamic drag to the vehicle. A conformal window is therefore beneficial to reducing drag and increasing the range of the aircraft. Some existing conformal windows introduce large wavefront aberrations into the sensor beam, particularly for high azimuthal pointing angles of the sensor.
An important consideration in achieving acceptable cost of the optical system is that the conformal window must be easily tested for its accuracy of shape, and must also be readily aligned upon mounting in the flight vehicle. The more complex the shape of the conformal window, the greater the challenge in testing and alignment.
There is a need for an improved window to be used in conformal window applications in high-speed missiles and aircraft. The present invention fulfills this need, and further provides related advantages.

Optical system with a window having a conicoidal inner surface, and testing of the optical system


An optical time domain reflectometry method and apparatus that employs Hadamard transforms to increase the signal-to-noise ratio. A sequence of N pulses of light corresponding to a Hadamard derived code are launched down a fiber optic cable for each of N such codes. Selected combinations of pulses of reflected light are transformed by a Hadamard derived matrix to a set of equations that are solved simultaneously to determine the power reflected from each of N points. Where a Hadamard code is used the average mean square error is reduced by a factor of 1/N. Where a simplex code is employed the average mean square error is reduced by a factor of 4N/(1+N).sup.2. In addition, N measurements of the power reflected at each point are computed and averaged.

This invention relates to methods and apparatuses for carrying out optical time domain reflectometry, particularly to the use of weighing techniques to improve the signal-to-noise ratio of reflected optical power measurements in optical time domain reflectometry.
In optical time domain reflectometry, as applied to a fiber optic cable, a pulse of light is launched, or transmitted, down the fiber from one end and the amount of light power reflected back to that end is measured as a function of time. Since the velocity of light in the fiber is known, the power reflected from any given distance down the fiber can therefore be determined. The operations of launching a pulse into the fiber and detecting the optical power level leaving the fiber are repeated for a large number of different time delay values, and the resulting data is used to form a display that represents fiber condition as a function of distance. This allows the quality of the fiber to be determined and, often more importantly, permits the existence of any significant discontinuity in the fiber or fiber system to be identified and its effect to be measured.
The reflected power comes from either Fresnel reflections, off a connector for example, or from Rayleigh backscatter. Rayleigh backscatter results from randomly dispersed anomolies in the index of refraction of the fiber. In contrast to the Fresnel reflections, Rayleigh backscatter is very weak, typically 40 dB or more less than the launch power. Consequently, the ability of an optical time domain reflectometer ("OTDR") to measure Rayleigh backscatter is limited by the noise in the receiver itself, and various techniques have been used to increase the signal-to-noise ratio.
In addition, the distance resolution of an OTDR customarily depends on the length of the pulse in the fiber (pulse period times the velocity of the light in the fiber). More specifically, the resolution is one half the pulse length. However, the amplitude resolution is directly proportional to the total energy of the pulse (pulse period times the power of the energy source). Since the light source has a fixed maximum power, there is a tradeoff between distance resolution and amplitude resolution. This tradeoff can be alleviated by increasing the signal-to-noise ratio of the reflected energy, thereby increasing the amplitude resolution or, alternatively, permitting the distance resolution to be increased by decreasing the pulse length.
Techniques used to increase the signal-to-noise ratio include high speed averaging, spread spectrum techniques using Golay codes and coherent optical detection. Correlation techniques using various codes are described in P. Healy, "Complementary Correlation OTDR With Three Codewords", Electron. Lett., 1990, 26, pp. 70-71; P. Healy, "Complementary Code Sets for OTDR", Electron. Lett., 1989, 25, pp. 692-693; P. Healy, "Optical Orthogonal Pulse Compression Codes by Hopping", Electron. Lett., 1981, 17, pp. 970-971. The use of Golay codes is described in Cheng et al. U.S. Pat. No. 4,743,753. While these techniques have been effective, further increases in sensitivity and signal-to-noise ratio are still needed.
In the invention described hereafter weighing designs have been employed to improve the signal-to-noise ratio of an optical time domain reflectometer. A weighing design is a scheme for accurately weighing a number of objects by weighing them in groups rather than one at a time. Where the error in weighing is independent of the weight of the objects, the mean square error .epsilon. can be reduced by a weighing design. Where i objects are weighed separately, the measured weight .mu..sub.i of the ith object is the actual weight .tau..sub.i plus some error e.sub.i. That is: EQU .mu..sub.i =.tau..sub.i +e.sub.i
The best estimate of the weight .tau..sub.i can therefore be expressed as .tau..sub.i +e.sub.i. It follows that: EQU e.sub.i =.tau..sub.i -.tau..sub.i
The expected value of the square of the error, that is, the mean square error, is expressed as E{e.sub.i.sup.2 }, where E denotes expected value. For a single measurement the mean square error is equal to the variance .sigma..sup.2. Here, it can also be expressed as: EQU E{(.tau..sub.i -.tau..sub.i).sup.2 }=.upsilon..sup.2
For example, suppose that the weighing is performed using a chemical balance with two pans, and that four objects are to be weighed, as follows: EQU .mu..sub.1 =.tau..sub.1 +.tau..sub.2 +.tau..sub.3 +.tau..sub.4 +e.sub.1 EQU .mu..sub.2 =.tau..sub.1 -.tau..sub.2 +.tau..sub.3 -.tau..sub.4 +e.sub.2 EQU .mu..sub.3 =.tau..sub.1 +.tau..sub.2 -.tau..sub.3 -.tau..sub.4 +e.sub.3 EQU .mu..sub.4 =.tau..sub.1 -.tau..sub.2 -.tau..sub.3 +.tau..sub.4 +e.sub.4
This means that in the first weighing all four objects are placed in the left hand pan, in the second weighing objects 1 and 3 are placed in the left hand pan, and 2 and 4 in the right hand pan, and so forth.
The best estimate of the weights can be found by solving these equations simultaneously for .tau..sub.1, .tau..sub.2, .tau..sub.3 and .tau..sub.4. For example, it can be shown that: EQU .tau..sub.1 =1/4(.mu..sub.1 +.mu..sub.2 +.mu..sub.3 +.mu..sub.4)
and that: EQU .tau..sub.1 =.tau..sub.1 +1/4(e.sub.1 +e.sub.2 +e.sub.3 +e.sub.4)
Consequently, the mean square error is: ##EQU1## In general, it can be shown for this case that: EQU E{(.tau..sub.i -.tau..sub.i).sup.2 }=1/4.sigma..sup.2
That is, by weighing the four objects together, the mean square error can be reduced by a factor of 1/4.
The signal-to-noise ratio for a weighing design is defined as the ratio of the true weight of the ith object divided by the standard deviation .sigma. of the error in the measurement. That is: ##EQU2##
Another weighing design is based on a spring balance having only one pan. Only coefficients 0 and 1 can be used; that is, an object is either weighed or it is not. For example, one method of weighing is: ##EQU3## By solving these equations simultaneously for .tau..sub.1 through .tau..sub.3, it can be shown that the mean square errors are reduced. For example: ##EQU4##
Weighing designs are described by a square, N.times.N matrix, where the rows of the matrix correspond to the weighing event and the columns correspond to the position of the object in the weighing event. The matrix therefore acts as a transform that changes a set of measurements to a set of equations. In a chemical balance weighing design the values in the matrix may take on +1, 0 or -1. In a spring balance weighing design the values may only be 0 or +1.
The best weighing designs use Hadamard matrices for chemical balance designs and simplex matrices ("S-matrices") for spring balance designs. A Hadamard matrix H.sub.N of order N is an N.times.N matrix of +1's and -1's with the property that the scalar product of any two distinct rows is 0. A normalized Hadamard matrix is one in which all elements of the first row and the first column are +1. If H.sub.N is a normalized Hadamard matrix, an S-matrix is the (N-1).times.(N-1) matrix of 0's and +1's obtained by (a) omitting the first row and the first column of H.sub.N, and (b) changing the +1's to 0 and the -1's to 1's. The rows of an S-matrix are codewords in a simplex code. The rows of a Hadamard matrix are referred to herein as codewords in a Hadamard code and are orthogonal.
Ideally, a weighing design should minimize the mean square error .epsilon..sub.i for all i simultaneously. Where this is impossible, other criteria are used. A weighing design is said to be "A-optimal" if it minimizes the average of the mean square error, that is: ##EQU5##
It can be shown that the average mean square error for a Hadamard matrix is: ##EQU6## and that the average mean square error for an S-matrix is given by: ##EQU7## For large N, S-matrices are within a few percent of being A-optimal.
The forgoing information has been derived from M. Harwit and N. Sloan, Hadamard Transform Optics., 6-17 (1979, Academic Press, Inc.), ISBN 0-12-330050-9.

Method and apparatus for carrying out optical time domain reflectometry using weighing techniques


A multi-functional pillow device is disclosed. The multi-functional pillow device comprises a main body, two side bodies and a control unit. The main body has a through hole for face down sleep, a matching element for fitting with the through hole, and a circuit board inside the main body for connecting to a vibration unit and a rotation unit. Each of the side bodies is connected to the rotation unit and has a speaker connected to the circuit board, wherein the side bodies are adjusted to a vertical position or a horizontal position by the rotation unit. The control unit is disposed at the external of the main body and is connected to the circuit board for setting and controlling the vibration unit and the rotation unit, and for inputing audio data into the circuit board for playing from the speakers.

1. Field of the Invention
The present invention relates to a multi-functional pillow device, particularly to a pillow device which accommodates face down sleep, allows input of audio music for playing from its speakers, and is equipped with a vibration unit for massage.
2. Description of Related Art
Normally a prior art pillow is a plane device suitable for sleeping on one's back (i.e. face up sleep) or sleeping on one's side. However, the prior art pillow is not comfortable for sleeping on one's stomach (i.e. face down sleep). Besides, the pillow may only be used for taking a nap and sleeping without any additional function; therefore the prior art pillow is low in value. Moreover, if one wants to listen to music during sleep, one can only place a media player or an MP3 player beside the pillow, which could be troublesome and may disturb someone else.
Therefore, a pillow device with multiple functions which is not only comfortable for any sleeping positions but also plays music and songs as well as provides massage is substantially required.


This invention provides novel injectable compositions comprising botulinum toxin that may be administered to a subject for various therapeutic, aesthetic and/or cosmetic purposes. The injectable compositions contemplated by the invention exhibit one or more advantages over conventional botulinum toxin formulations, including reduced antigenicity, a reduced tendency to undergo unwanted localized diffusion following injection, increased duration of clinical efficacy or enhanced potency relative, faster onset of clinical efficacy, and/or improved stability.

Skin protects the body's organs from external environmental threats and acts as a thermostat to maintain body temperature. It consists of several different layers, each with specialized functions. The major layers include the epidermis, the dermis and the hypodermis. The epidermis is a stratifying layer of epithelial cells that overlies the dermis, which consists of connective tissue. Both the epidermis and the dermis are further supported by the hypodermis, an internal layer of adipose tissue.
The epidermis, the topmost layer of skin, is only 0.1 to 1.5 millimeters thick (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7 (1998)). It consists of keratinocytes and is divided into several layers based on their state of differentiation. The epidermis can be further classified into the stratum corneum and the viable epidermis, which consists of the granular melphigian and basal cells. The stratum corneum is hygroscopic and requires at least 10% moisture by weight to maintain its flexibility and softness. The hygroscopicity is attributable in part to the water-holding capacity of keratin. When the horny layer loses its softness and flexibility it becomes rough and brittle, resulting in dry skin.
The dermis, which lies just beneath the epidermis, is 1.5 to 4 millimeters thick. It is the thickest of the three layers of the skin. Most of the skin's structures, including sweat and oil glands (which secrete substances through openings in the skin called pores, or comedos), hair follicles, nerve endings, and blood and lymph vessels are found in the dermis (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7 (1998)). However, the main components of the dermis are collagen and elastin.
The hypodermis is the deepest layer of the skin. It acts both as an insulator for body heat conservation and as a shock absorber for organ protection (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7 (1998)). In addition, the hypodermis also stores fat for energy reserves. The pH of skin is normally between 5 and 6. This acidity is due to the presence of amphoteric amino acids, lactic acid, and fatty acids from the secretions of the sebaceous glands. The term "acid mantle" refers to the presence of the water-soluble substances on most regions of the skin. The buffering capacity of the skin is due in part to these secretions stored in the skin's horny layer.
Wrinkles, one of the telltale signs of aging, can be caused by biochemical, histological, and physiologic changes that accumulate from environmental damage to the skin. (Benedetto, "International Journal of Dermatology," 38:641-655 (1999)). In addition, there are other secondary factors that can cause characteristic folds, furrows, and creases of facial wrinkles (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological Surgery, 2nd ed., St. Louis, Mo.: Mosby Year Book: 5-15 (1990)). These secondary factors include the constant pull of gravity, frequent and constant positional pressure on the skin (e.g., during sleep), and repeated facial movements caused by the contraction of facial muscles (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological Surgery, 2nd ed., St. Louis, Mo.: Mosby Year Book: 5-15 (1990)).
Different techniques have been utilized in order to potentially mollify some of the signs of aging. These techniques range from facial moisturizers containing alpha hydroxy acids and retinol to surgical procedures and injections of neurotoxins. For example, in 1986, Jean and Alastair Carruthers, a husband and wife team consisting of an ocuplastic surgeon and a dermatologist, developed a method of using the type A form of botulinum toxin for treatment of movement-associated wrinkles in the glabella area (Schantz and Scott, In Lewis G. E. (Ed) Biomedical Aspects of Botulinum, New York: Academic Press, 143-150 (1981)). The Carruthers' use of the type A form of botulinum toxin for the treatment of wrinkles led to the seminal publication of this approach in 1992 (Schantz and Scott, In Lewis G. E. (Ed) Biomedical Aspects of Botulinum, New York: Academic Press, 143-150 (1981)). By 1994, the same team reported experiences with other movement-associated wrinkles on the face (Scott, Ophthalmol, 87:1044-1049 (1980)). This in turn led to the birth of the era of cosmetic treatment using the type A form of botulinum toxin.
The type A form of botulinum toxin is reported to be the most lethal natural biological agent known to man. Spores of C. botulinum are found in soil and can grow in improperly sterilized and sealed food containers. Botulism, which may be fatal, may be caused by the ingestion of the bacteria. Botulinum toxin acts to produce paralysis of muscles by preventing synaptic transmission by inhibiting the release of acetylcholine across the neuromuscular junction, and is thought to act in other ways as well. Its action essentially blocks signals that normally would cause muscle spasms or contractions, resulting in paralysis. During the last decade, botulinum toxin's muscle paralyzing activity has been harnessed to achieve a variety of therapeutic effects. Controlled administration of botulinum toxin has been used to provide muscle paralysis to treat a variety of medical conditions, for example, neuromuscular disorders characterized by hyperactive skeletal muscles. Conditions that have been treated with botulinum toxin include hemifacial spasm, adult onset spasmodic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, migraine headaches, strabismus, temporomandibular joint disorder, and various types of muscle cramping and spasms. More recently, the muscle-paralyzing effects of botulinum toxin have been applied to therapeutic and cosmetic facial applications such as treatment of wrinkles, frown lines, and other results of spasms or contractions of facial muscles.
In addition to the type A form of botulinum toxin, there are seven other serologically distinct forms of botulinum toxin that are also produced by the gram-positive bacteria Clostridium botulinum. Of these eight serologically distinct types of botulinum toxin, the seven that can cause paralysis have been designated botulinum toxin serotypes A, B, C, D, E, F and G. Each of these is distinguished by neutralization with type-specific antibodies. The molecular weight of each of the botulinum toxin proteins is about 150 kD. Due to the molecule size and molecular structure of botulinum toxin, it cannot cross stratum corneum and the multiple layers of the underlying skin architecture. The different serotypes of botulinum toxin vary in the effect and in the severity and duration of the paralysis they evoke in different animal species. For example, in rats, it has been determined that botulinum toxin type A is 500 times more potent than botulinum toxin type B, as measured by the rate of paralysis. Additionally, botulinum toxin type B has been determined to be non-toxic in primates at a dose of 480 U/kg, about 12 times the primate LD50 for type A.
As released by Clostridium botulinum bacteria, botulinum toxin is a component of a toxin complex containing the approximately 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. These endogenous non-toxin proteins are believed to include a family of hemagglutinin proteins, as well as non-hemagglutinin protein. The non-toxin proteins have been reported to stabilize the botulinum toxin molecule in the toxin complex and protect it against denaturation by digestive acids when toxin complex is ingested. Thus, the non-toxin proteins of the toxin complex protect the activity of the botulinum toxin and thereby enhance systemic penetration when the toxin complex is administered via the gastrointestinal tract. Additionally, it is believed that some of the non-toxin proteins specifically stabilize the botulinum toxin molecule in blood.
The presence of non-toxin proteins in the toxin complexes typically causes the toxin complexes to have molecular weights that are greater than that of the bare botulinum toxin molecule, which is about 150 kD, as previously stated. For example, Clostridium botulinum bacteria can produce botulinum type A toxin complexes that have molecular weights of about 900 kD, 500 kD or 300 kD. Botulinum toxin types B and C are produced as complexes having a molecular weight of about 700 kD or about 500 kD. Botulinum toxin type D is produced as complexes having molecular weights of about 300 kD or 500 kD. Botulinum toxin types E and F are only produced as complexes having a molecular weight of about 300 kD.
To provide additional stability to botulinum toxin, the toxin complexes are conventionally stabilized by combining the complexes with albumin during manufacturing. For example, BOTOX® (Allergan, Inc., Irvine, Calif.) is a botulinum toxin-containing formulation that contains 100 U of type A botulinum toxin with accessory proteins, 0.5 milligrams of human albumin, and 0.9 milligrams of sodium chloride. The albumin serves to bind and to stabilize toxin complexes in disparate environments, including those associated with manufacturing, transportation, storage, and administration.
Typically, the botulinum toxin is administered to patients by carefully controlled injections of compositions containing botulinum toxin complex and albumin. However, there are several problems associated with this approach. Not only are the injections painful, but typically large subdermal wells of toxin are locally generated around the injection sites, in order to achieve the desired therapeutic or cosmetic effect. The botulinum toxin may migrate from these subdermal wells to cause unwanted paralysis in surrounding areas of the body. This problem is exacerbated when the area to be treated is large and many injections of toxin are required to treat the area. Moreover, because the injected toxin complexes contain non-toxin proteins and albumin that stabilize the botulinum toxin and increase the molecular weight of the toxin complex, the toxin complexes have a long half-life in the body and may cause an undesirable antigenic response in the patient. For example, some patients will, over time, develop an allergy to the albumin used as a stabilizer in current commercial formulations. Also, the toxin complexes may induce the immune system of the patient to form neutralizing antibodies, so that larger amounts of toxin are required in subsequent administrations to achieve the same effect. When this happens, subsequent injections must be carefully placed so that they do not release a large amount of toxin into the bloodstream of the patient, which could lead to fatal systemic poisoning, especially since the non-toxin proteins and albumin stabilize the botulinum toxin in blood.
In view of the drawbacks associated with current botulinum toxin formulations, it would be highly desirable to have an injectable botulinum toxin formulation that is efficacious and stable, but exhibits reduced antigenicity and a lower tendency to diffuse locally after injection. It would also be desirable to use such a botulinum toxin formulation for various therapeutic, aesthetic and/or cosmetic purposes.


An apparatus and method to detect abnormal combustion conditions for use as a feedback control of a lean burn reciprocating engine using ionization signals is presented. The system receives a succession of ionization signals for successive cycles of a running engine and processes a plurality of related ionization signals for signal stability. The ionization signals are checked to determine if an abnormal combustion condition such as knock or misfire has occurred. The variation of an ionization signal that changes with respect to an engine parameter over a combustion event of the lean burn reciprocating engine operating with an air to fuel ratio corresponding to a lambda (xcex) greater than 1.4 is measured and a floating bounded space is associated with the ionization signal. An indication that the abnormal combustion condition has been detected is provided if a portion of the ionization signal is within the floating bounded space.

The present invention relates generally to ignition systems in spark ignited engines, and more particularly relates to such systems in lean burn engines in which the excess air factor xcex is greater than 1.4.
Industry has developed various techniques using ionization signals for detecting abnormal combustion conditions such as misfire, knock, and approximate air/fuel ratio for stochiometric engines . Free ions present in the combustion gases are electrically conductive and are measurable by applying a voltage across an ionization probe. Alternatively, the voltage is applied across the electrodes of a spark plug after the spark plug has ignited the combustion mixture. The applied voltage induces a current in the ionized gases which is measured to provide the ionization signal. The ionization signal is used as a control parameter in the control of the engine. For example, in U.S. Pat. No. 6,029,627, ionization signals and a single O2 sensor in the exhaust are used to control the air/fuel ratio in engines to achieve stoichiometric operation. This technique uses the O2 sensor to achieve stoichiometry of the overall stoichiometric mixture of the engine and then equalizes the amplitude or location of the first local peak of the ionization signal in each individual cylinder. Another technique disclosed in U.S. Pat. No. 5,992,386 performs a frequency analysis of the ionization signal to detect abnormal combustion conditions such as knock. These systems integrate the ionization signal and compare the magnitude of the integrated signal to the magnitude of the integrated signal of a normal combustion event. The abnormal combustion condition is detected if the magnitude of the integrated signal is above a threshold level, which is set above the magnitude of the integrated signal of a normal combustion event.
One of the drawbacks of stochiometric engines is the emission of pollutants. With fixed engine timing and load, the NOx, emissions level of a typical gas engine is dependent upon the air/fuel ratio. Near a chemically correct (i.e., stoichiometric) ratio, the NOx, emissions peak and then drop significantly as the amount of excess air is increased. Maintaining a stable combustion process with a high air/fuel ratio is difficult to manage. As a result, conventional spark-ignited gas engines typically operate near the stoichiometric air/fuel ratio and depend upon exhaust after treatment with catalytic converters to reduce the NOx emissions.
Government agencies and industry standard setting groups are reducing the amount of allowed emissions in an effort to reduce pollutants. As a result, industry is moving towards using lean burning engines to reduce emissions despite the difficulty of maintaining a stable combustion process in lean burning engines. By using more air during combustion, turbocharged lean-bum engines can enhance fuel efficiency without sacrificing power and produce less nitrous oxide pollutants than conventional stoichiometric engines.
Ionization sensing has not been utilized to any significant extent in these lean burn engines. Because of the lean nature of the mixture, the ionized species concentration, including NOx, is much less than at stoichiometric conditions. As a result, the ionization signal is of very low intensity and has great variability. The techniques developed using ionization signals for stochiometric operation are unsuitable for lean burn operation and do not work. For example, the ionization signals of some lean burn engines are sufficiently variable and are low enough in magnitude that integrating the signal can not be done reliably due to a number of factors. These factors include higher levels of noise relative to the ionization signal magnitude, the variability of the ionization signal, and the low magnitudes of the resultant integrated signal.
In view of the foregoing, an object of the present invention is to reliably detect abnormal combustion conditions such as misfire and knock of lean burn engines based on ionization signals.
The foregoing objects are among those attained by the invention, which provides a method of detecting an abnormal combustion condition in a combustion chamber of a lean burn reciprocating engine. The abnormal combustion condition includes misfire and knock. The method measures the variation of an ionization signal that changes with respect to an engine parameter over a combustion event of the lean burn reciprocating engine operating with an air to fuel ratio corresponding to a lambda (xcex) greater than 1.4, associates a floating bounded space with the ionization signal, determines if a portion of the ionization signal is within the floating bounded space, and provides an indication that the abnormal combustion condition has been detected if the portion of the ionization signal is within the floating bounded space.
A method to determine the floating bounded space and a starting point for the floating bounded space is also disclosed. The method includes receiving a set of ionization signals that change with respect to an engine parameter over a combustion event. The set of ionization signals has ionization signals corresponding to normal combustion conditions and ionization signals corresponding to at least one abnormal combustion condition for an engine operating with an air to fuel ratio corresponding to a xcex greater than 1.4. The method further includes the step of adjusting the starting point and size of the floating bounded space such that selected portions of the ionization signals corresponding to the at least one abnormal combustion condition reliably fall within the floating bounded space and the ionization signals corresponding to normal combustion conditions reliably fall outside the floating bounded space.

Method and apparatus for detecting abnormal combustion conditions in lean burn reciprocating engines


A reference pattern renewing method includes inputting an input pattern of an utterance of a word, comparing the input pattern with reference patterns stored in a dictionary memory to thereby generate at least a first candidate having the highest degree of similarity and a second candidate having the second highest degree of similarity, and calculating a ratio of the highest degree of similarity to the second highest degree of similarity. The method further includes determining whether or not the ratio is equal to or less than a predetermined threshold value, calculating, from the input pattern, a renewed reference pattern to be substituted for a corresponding one of the reference patterns when it is determined that the ratio of the highest degree of similarity to the second highest degree of similarity is neither equal to nor less than the predetermined threshold value, and registering the renewed reference pattern in the dictionary memory instead of the corresponding one of the reference patterns.

The present invention generally relates to a speech recognition, and more particularly to a method of renewing a reference pattern stored in a dictionary.
Recently, there has been considerable activity in the research and development of a speech recognition system. A dictionary which stores reference patterns used for identifying an unknown input speech is provided in a speech recognition system. Reference patterns are created beforehand and registered in the dictionary. It is frequently required to renew the reference patterns registered in the dictionary. Conventionally, various methods of renewing a reference pattern have been proposed. For example, the user creates and registers a modified reference pattern instead of the corresponding old reference pattern.
Alternatively, an unknown input pattern is recognized by using a modified reference pattern prepared by the user before it is registered in the dictionary. When the user decides that a candidate having the highest degree of similarity is correct, the old reference pattern is renewed by the modified reference pattern. This process is complicated because the renewing process needs the user's decision. Further, when a modified reference pattern that is decided to be substituted for the old reference pattern is created under a noisy condition which the user is not aware of, the quality of the dictionary is degraded

Method of renewing reference pattern stored in dictionary


A split sprocket assembly, related apparatus, and a method of mounting a sprocket to a rotatable structure, such as a drive or idler drum, are disclosed. In one embodiment, the sprocket assembly is comprised of at least three sections or segments, each of which is secured to the adjacent section or segment by a fastener, such as a tangentially-extending bolt and nut combination. The sprocket assembly includes a plurality of receivers, each for receiving a key that corresponds to a keyway formed in the rotatable structure. In one embodiment, at least one of the keyways is truncated and, thus, extends only partially across the rotatable structure in the axial direction. A key substantially identical to the truncated keyway in length and width is placed therein, such that the sprocket is prevented from traveling or xe2x80x9cwalkingxe2x80x9d along the rotatable structure.

The present invention relates to the conveyor art and, more particularly, to an improved split sprocket assembly, a related apparatus, and a related method of mounting a sprocket to a rotatable structure, such as a drum or shaft.
Today, in many modern manufacturing facilities, modular conveyor systems are extensively utilized to transport articles to and from various work stations during all stages of production. In recent years, manufacturers using production lines with conveyors as an integral component of the material handling system have realized reasonably significant gains in productivity and resource utilization. As a result, modular conveyor systems have become even more widely implemented and have been adapted to meet an even wider scope of the material handling needs of producers of a multitude of consumer and industrial goods. Therefore, the continual development of improved modular conveyors is necessary in order to keep pace with the demands and expectations of the users of such conveyors.
Conventional conveyor systems employing endless, modular link or roller chain belts are typically driven at one end of an elongated guide structure, such as a rail or the like supported above the ground by a frame. The driving force is transmitted from a motive device, such as a variable speed electric motor, to a plurality of gang-driven sprockets coupled to a rotating drive structure, such as a shaft or drum. At the opposite end of the guide structure, idler sprockets are coupled to a freely-rotating idler shaft or drum. As should be appreciated, the drive and idler sprockets assist in supporting and guiding the endless belt as it makes the transition from the forward run to the return run, or vice versa, at each respective end of the guide structure.
Of course, both the drive and idler sprockets are subject to wear as a result of the substantially continuous engagement maintained with the conveyor belt. If not kept in check, this wear may affect the overall performance of the conveyor system. For example, worn sprockets may allow the belt to slip, which creates both timing and efficiency problems. Hence, it is necessary to perform an inspection of the sprocket(s) from time to time to determine whether some form of ameliorative action, such as a repair operation or perhaps even a complete replacement, is required.
While inspection may only require removing the belt to gain visual access to the sprocket, repairing or completely replacing a unitary sprocket requires removing it from the corresponding support shaft or drum altogether. In the case where a single drive or idler sprocket is carried on the shaft or drum, removing the belt and the sprocket is a relatively uncomplicated undertaking. However, in the situation where a plurality of sprockets are gang driven or carried on a single support shaft or drum, removing any of the inner sprockets becomes a time and labor intensive process, since all adjacent outer sprockets and any other corresponding structures (side plates, chain guides/strippers, conveyor frame components or other support structures, etc.) must first be removed. As should be appreciated, removing even one of the sprockets in a gang-driven arrangement significantly increases the time and labor involved. Additionally, the wholesale removal and replacement of multiple sprockets carried on a shaft may create alignment and/or timing problems that must be corrected to ensure efficient operation. Of course, the time and labor required to remedy these problems further exacerbates the situation.
Others in the past have proposed a number of different types of arrangements to solve these problems. One popular proposal is to use a split sprocket consisting of two mating halves that are coupled together over a shaft. With this arrangement, removal of the sprocket can be accomplished without dismantling the entire conveyor assembly. Modem examples of various types of split sprockets are found in U.S. Pat. No. 6,202,834 to Layne et al, U.S. Pat. No. 6,146,299 to Harvey, and U.S. Pat. No. 6,086,495 to Stebnicki et al., the disclosures of which are hereby incorporated by reference.
In the case of a rotatable drive or idler drum, it is known to couple a single-piece, unitary sprocket to the drum using a plurality of cylindrical keys or pins. For example, U.S. Pat. No. 5,934,447 to Kanaris discloses an arrangement in which the drum includes a plurality of shallow grooves extending axially along its entire surface. Keys received in the sprocket fit in the corresponding shallow grooves and, as a result, keep it spaced from the outer surface of the drum. A radially extending set screw is used to press each key into the corresponding groove to rotatably couple the sprocket to the drum.
While this arrangement does provide the limited advantage of allowing for the adjustment of the position of the sprocket along the drum, there are countervailing disadvantages. For example, during extended periods of use, the set-screws may loosen and allow the keys and, hence, the sprocket to xe2x80x9cwalkxe2x80x9d or travel axially along the surface of the drum. This is deleterious, since it not only increases the wear on the sprocket, but also causes chattering and wear on the belt. If the set-screws loosen to a sufficient degree to allow the key to move out of the shallow groove in the drum, the sprocket may slip and, in the case of a driven drum, transmit no drive power or torque to the belt. Of course, the set-screws may be frequently inspected or tightened down in an effort to prevent these deleterious occurrences, but this inevitably increases the maintenance effort and overall operational cost.
Accordingly, the foregoing discussion identifies a particular need for an improved manner of mounting a sprocket to a rotatable structure, such as a drive or idler drum in a conveyor system. It is envisioned that by splitting the sprocket into two or more sections or segments, it would be possible to provide a key for each section or segment that corresponds to a separate keyway formed in the outer surface of the rotatable structure. The sections or segments could also be coupled together and held in tension, such as by tangentially-extending fasteners. The tension would create a sufficient radially-directed holding force to ensure that the keys remain securely held in the keyways during rotation while eliminating the need for radially-extending set-screws. One or more of the keyways could also be truncated and the keys sized for fitting in the truncated keyway(s) in relatively tight seated engagement. This would prevent the sprocket from traveling or xe2x80x9cwalkingxe2x80x9d along the surface of the drum in use. Overall, this combination of features would result in a sprocket assembly that is a significant improvement over those proposed in the past, especially in terms of maintenance cost, operational efficiency, and reliability.
In accordance with a first aspect of the invention, a sprocket assembly intended for mounting on a rotatable structure, such as a drum or shaft, for engaging an endless conveyor belt making the transition between a forward and a return run or vice-versa is disclosed. The assembly comprises at least three unitary sprocket sections or segments, with each of the sections or segments including an arcuate inner surface for engaging an outer surface of the rotatable structure. A plurality of fasteners are also provided, each for fastening one of the plurality of sprocket sections to an adjacent sprocket section to form the sprocket assembly.
Each of the arcuate sprocket sections or segments includes a first end and a second end. In one embodiment, the first end includes a tangentially-extending bore and the second end includes a corresponding aperture for receiving a threaded bolt forming a part of one of the fasteners. The engagement of the bolt extending through the aperture in the first end of one sprocket section or segment into the bore in a second end of the adjacent sprocket section or segment assists in securing the two structures together. In another embodiment, each of the ends includes a mating face having a matching aperture formed therein for receiving a threaded bolt forming a part of one of the fasteners. When the ends of two adjacent sprocket sections or segments are abutting in the mated position, the apertures are positioned such that a centerline extending through them is generally parallel to a line drawn tangent to a point located at an interface between the adjacent ends. To complete the fastener and secure the adjacent sections or segments together, a nut is then placed on the bolt and tightened.
Each of the sections or segments may include a receiver, or alternatively two adjacent sections or segments may together form a receiver. Each receiver is capable of receiving and holding a key that is adapted to fit into a keyway formed in or on the rotatable structure, such as a shaft or drum. The key is preferably cylindrical, in which case, the receiver is C-shaped. However, using keys having other shapes is entirely possible, with the corresponding receiver and keyway generally having a matching shape.
Instead of the three sections or segments described above, the sprocket assembly may comprise at least four sprocket sections or segments. As with the three piece embodiment, each of the four sprocket sections or segments may include a first end and a second end. Preferably, each end includes an aperture for receiving a threaded bolt forming a part of one of the plurality of fasteners. When the ends of two sprocket sections or segments are positioned adjacent to each other in abutment, a centerline extending through the matching apertures is generally parallel to a line drawn tangent to a point at an interface between the adjacent ends. To complete the assembly of the two adjacent sections or segments, a nut may be placed on the bolt and tightened.
In accordance with a second aspect of the invention, a sprocket assembly intended for mounting on the outer surface of a rotatable structure, such as a drum or shaft, for engaging an endless conveyor belt making the transition between a forward and a return run is provided. The assembly comprises a plurality of sprocket sections or segments (preferably two, three, or four), with each section or segment having an arcuate inner surface and at least two ends. Each of the ends includes an aperture and a plurality of fasteners for fastening a first end of one of the plurality of sprocket sections to a second, corresponding end of an adjacent sprocket section are provided. Specifically, the fasteners include a threaded bolt for positioning through a matching pair of the apertures in the first and second ends and a nut for placement on a portion of the bolt projecting through one of the apertures. A centerline extending through the matching pair of the apertures is generally parallel to a line drawn tangent to a point at an interface between the ends of two adjacent sprocket sections or segments when positioned in an abutting relationship. As a result of this fastening arrangement, tightening of the nut against a surface of the sprocket section or segment adjacent to the aperture through which the bolt projects creates a tension between the adjacent sprocket sections. Each sprocket section or segment may further include a C-shaped receiver for receiving a substantially cylindrical key, whereby a portion of the key projects from the inner surface of the section or segment for engaging a keyway formed in the rotatable structure and is held in place by the tension mounting.
In accordance with a third aspect of the invention, an apparatus intended for use in engaging and guiding an endless conveyor belt making the transition between a forward and a return run or vice-versa is disclosed. The apparatus comprises a structure, such as a drum or shaft, mounted for rotation about an axis. The rotatable structure includes an outer surface having a first plurality of keyways, with at least one of the first keyways being truncated and thus extending only partially across the outer surface in an axial direction. A first split sprocket comprised of at least three sections or segments is also provided. The first sprocket includes an inner surface and a plurality of receivers. A first plurality of keys are also provided, with each key being positioned at least partially in one of the receivers such that a portion thereof projects inwardly and is adapted for seating engagement in one of the first keyways. A fastener for fastening each sprocket section or segment to an adjacent sprocket section or segment is also provided.
In one embodiment, the rotatable drum is cylindrical and the inner surface of each the sprocket section or segment is arcuate. Additionally, each receiver is substantially C-shaped, in which case each key is cylindrical and is slidably received in the corresponding receiver. The corresponding keyway is also semi-circular. Instead of being slidably received, each key may be received in the corresponding receiver in snap-fit engagement. Preferably, the width of each keyway in the axial direction is substantially identical to the width of the key and the length of each keyway at the surface of the rotatable structure is substantially equal to of less than the diameter of the key.
In addition, the split sprocket may be formed of four sections or segments. The fourth sprocket section or segment is secured to one of the at least two sprocket sections or segments and the third section or segment using fasteners. Also, the fourth sprocket section or segment may include a receiver for slidably receiving a key for engaging one of the first keyways.
In addition to the first keyways, a second plurality of keyways may be provided, with at least one of the second keyways extending only partially across the outer surface of the rotatable structure in the axial direction. The apparatus may further comprise a second split sprocket comprised of at least two sections or segments having an inner surface and a plurality of receivers; a second plurality of keys, each key positioned at least partially in one of the receivers such that a portion thereof projects inwardly from the second split sprocket, the portion being adapted for seating engagement in one of the second plurality of keyways; and a second plurality of fasteners for fastening the at least two sections or segments of the second split sprocket together. A third plurality of keyways, including at least one third keyway extending only partially across the outer surface of the drum or shaft in the axial direction, may also be provided. The first or second split sprockets may be alternately fastened to the rotatable structure using the third keyways.
In accordance with a fourth aspect of the invention, a method of mounting a split sprocket over a rotatable structure, such as a drum or shaft, is provided. The method comprises fastening at least three unitary toothed sections or segments together over the rotatable structure to form a first sprocket, while simultaneously securing the sprocket to the rotatable structure so as to prevent axial movement along the outer surface thereof. The step of fastening may include securing a tangentially-extending fastener between adjacent sprocket sections or segments. The rotatable structure may include a plurality of circumferentially-aligned, truncated keyways, and the step of securing the sprocket to the rotatable structure may include positioning a key in each one of a plurality of receivers formed in the sprocket such that at least a portion of the key projects inwardly therefrom and is received in one of the keyways.

Split sprocket assembly, related apparatus, and related mounting method


The present invention provides methods of reducing the background signal of a nucleic acid sequencing reaction. In particular, the invention provides methods of specifically degrading unwanted chain termination reaction products generated by the extension of primers carried over from the amplification step of the sequencing reaction. These methods are amenable for use with both one step and two-step amplification/chain termination reaction sequencing protocols.

1. Field of the Invention
The present invention is drawn to the general field of DNA amplification and sequence determination. More specifically, the present invention provides methods for lowering the background signal generated during DNA sequencing due to the carry over of primers from the amplification step. These methods are generally applicable to all known methods of DNA sequencing that rely on the amplification of the target sequence prior to delineation of the nucleotide sequence. Also included in the present invention are kits for the production of nucleic acid sequencing data with reduced background noise due to amplification primer carry over.
2. Background
The Sanger method for determining the sequence of a DNA molecule (PNAS: 74, 5463-5467 (1977)) requires a relatively large amount of purified, single-stranded DNA template. Alternatively, the cycle sequencing method (Murray, V., Nucleic Acids Res.: 17, 8889 (1989)) does not require a single-stranded template and allows the sequence reaction to be initiated with relatively small amounts of template. However, this method does require that the template DNA be purified to almost complete homogeneity. Due to this limitation, template DNA is routinely prepared by means of PCR amplification (Mullis, K. B. and Faloona, F. A., Methods Enzymol.: 155, 335-350 (1987)) when the target sequence is contained in a large mixture of DNA sequences.
In both of the above methods, DNA sequencing is accomplished by first performing an enzymatic chain termination reaction, as described by Sanger, followed by the subsequent delineation of the DNA sequence from the chain termination reaction products. First, a nucleic acid template is used to generate a plurality of labeled nucleic acid fragments of various lengths by the action of a suitable DNA polymerase. This is accomplished by hybridizing to the DNA molecule an oligonucleotide complimentary to the target sequence and extending that oligonucleotide by incorporation of deoxynucleotide triphosphates (dNTPs) in the presence of dideoxynucleotide triphosphates (ddNTPs). Commonly, these ddNTPs are labeled with, for example, a radioactive isotope, such as phosphorus 32, or a fluorescent moiety. These chain termination reaction products are then used to determine the sequence of the target, for example after polyacrylamide gel electrophoresis or capillary electrophoresis.
An important step between the amplification and chain termination reaction steps of a sequencing procedure is the removal of unused primers at the end of the amplification reaction. Incomplete removal of primers can lead to noisy and unreliable sequencing data. While enzymatic, chromatographic and ultrafiltrational methods for removing excess primers are well known in the art (Dugan et al., J. Forensic Sci., 47:811-818 (2002)) (Smith and Ballantyne, J. Forensic Sci., 52:820-829 (2007)), these methods are not always adequate to reduce the noise caused by the extension products from primers carried over from the amplification step. Laboratories that rely on the success of high-throughput DNA sequencing often encounter sequencing data that are unusable due to such noise. Recently, a method called the AmpliSeq (Murphy et al., Clin. Chem., 51:35-39 (2005)) that combines both amplification and sequencing in a single step has been described. Since the AmpliSeq method does not allow the removal of excess primers between the amplification and sequencing step, the final data can be sometimes ambiguous. Therefore, there is a need in the art for a method of reducing the background noise, post chain termination reaction, of a sequencing reaction created by the extension of primers carried over from the amplification step of the procedure. The current invention satisfies this need by providing enzymatic methods of degrading unwanted chain termination reaction products. The methods of the present invention are effective in improving sequencing data for both regular amplification followed by cycle sequencing and one-step amplification/cycle sequencing.

Sequence data by reduction of noise due to carry-over primer