The present invention provides methods for determining various biological characteristics of in vitro fertilized embryos, including overall embryo health, implantability, and increased likelihood of developing successfully to term. More specifically, the present invention concerns analyzing media specimens of in vitro fertilization cultures for levels of bioactive lipids in order to determine these characteristics.

The present invention enables the determination of various biological characteristics of in vitro fertilized embryos, including overall embryo health, implantability, viability, and likelihood of successful development to term. More specifically, the present invention concerns analyzing media specimens of in vitro fertilization cultures for levels of bioactive lipids in order to determine the optional characteristics for media formulation to increase embryo viability and reduce the frequency of multiple births.
As many as 30% of couples attempting to conceive suffer from infertility. Due in part to the increasing age of pregnancy, infertility is on the rise. For men, infertility is usually caused by low sperm count or poor motility. For women, common causes include oviduct occlusion, abnormal ovulation cycles, and inhospitable vaginal mucus. Treatment options are available, depending on the source of the problem. However, even with treatment, many couples are not able to conceive in the traditional fashion.
In vitro fertilization (IVF) has been used increasingly to assist infertile couples in becoming pregnant. IVF generally includes the steps of: recovery of mature eggs from the female patient or a donor; incubation of the eggs in culture media; collection of sperm from the male patient or a donor; culture and preparation of the sperm for addition to the egg culture media (including capacitation procedures); fertilization of the eggs by the sperm; monitoring the embryos during early embryogenesis; and, finally, transfer of fertilized oocyte into the uterine cavity.
To harvest the oocytes, gonadotropin is administered to the ovaries of the donor to stimulate follicle growth and to increase the number of oocytes that mature in a single cycle. Just prior to ovulation, oocytes in the late stages of first meiotic division are aspirated from mature ovarian follicles. Generally, harvested oocytes are placed in a culture medium and fertilized by adding semen or cultured sperm. In some cases, sperm are micro-injected directly into the oocyte, bypassing the normal sperm/oocyte fusion process. After fertilization, the zygotes are microscopically monitored to the eight-cell stage, and further into blastocysts. Healthy embryos with normal phenotypic development and morphology are chosen for transfer by visual inspection. The embryos are then transferred directly into the uterine cavity through the cervical opening using a thin catheter.
Only about 20% of the couples utilizing IVF, however, carry an infant successfully to term. To increase the chances for a successful pregnancy, three or four embryos are usually transferred at the same time. Although a single pregnancy is desired, multiple pregnancies often result. Multiple pregnancies pose health risks for the mother during pregnancy, and can cause emotional and economic problems in family planning. The alternative of terminating one of the pregnancies poses a difficult ethical and emotional dilemma for many couples, and successful delivery of the remaining fetus cannot be ensured. Therefore, there is a need for the ability to select single embryos which are likely to develop successfully upon transfer into the uterus. Similarly, there is a need for the ability to de-select embryos that are unlikely to survive to term. As an embryo""s probability of survival generally decreases with culture time, a need also exists for the ability to rapidly assess embryo health as early as possible after fertilization.
In recent years, platelet activating factor (PAF) has been discussed as a possible indicator of embryo viability. PAF (1-alkyl-2-acetoyl-sn-glycero-phosphocholine) seems to be involved in an autocrine hormonal signaling pathway in early embryogenesis. PAF also seems to be involved in paracrine pathway between the mother and the child, and has been detected in the culture medium of embryos that develop successfully to term. However, hormonal signaling in the early stages of development is complex, and a multitude of pathways and signaling mechanisms are involved in embryogenesis. PAF alone has not proven a sufficient indicator of embryo viability, and the role of small hormone molecules both in indicating and maintaining embryo health is not well understood. There is no current adequate molecular marker, or combination of markers, which indicates proper embryonic development at the very early stages of life. The structural formula for PAF is shown below (although the alkyl chain may be of varying lengths):
Thus, in a primary aspect, the present invention provides the ability to determine the implantability, overall viability, and likelihood of successful development of an embryo fertilized in vitro. The present invention comprises analyzing the presence or concentration of one or more bioactive lipids in culture media specimens obtained from IVF cultures containing the embryo. Differences in levels of specific types of bioactive lipids in an IVF culture sample specimen where the bioactive lipid concentrations deviate significantly from the levels of bioactive lipids found in culture specimens from embryos which successfully developed to term, those deviations reveal differences in the biochemical environment in which the embryo develops and are indicative of a difference in probability that the embryo will implant or develop normally to term. The present invention contemplates analyzing bioactive lipids from either single or multiple embryo cultures in media, as well as co-cultures of single or multiple embryos grown on a bed of feeder cells and using this analysis to make informed choices about the likelihood of successful fertilization and full-term development.
Specifically, the invention contemplates measuring the presence or concentration of bioactive lipids, comparing these concentrations to a normal, pre-existing, or established values for the specific embryo or media, and correlating these differences to the underlying biochemical status of the embryo, including the probability that the embryo will accede through viability to normal development, or will result in a multiple-birth pregnancy. Also, The ability to analyze the underlying biochemical status of the embryo provides the ability to analyze the chemical composition of the media and the ability to alter the chemical composition of the media through supplementation with chemical or biochemical agents to affect the viability of the embryo or the probability of a multiple-birth pregnancy. By analyzing one or more lipids, a multi-component lipid profile can be constructed for the culture media such that the lipid profile of the culture media can be correlated to viability, or, where appropriate, the composition of the culture media may be changed according to the concentration of one lipid or the multi-lipid profile. Moreover, an individual embryo, or group of embryos, can be monitored over time to determine how the lipid profile and the culture media is altered through natural development or through chemical or biochemical intervention to titrate the composition of the culture media to reach a desired level indicative of viability or the avoidance of multiple pregnancies. Therefore, the invention enables both the selection of healthy embryos and the deselection of unhealthy embryos to increase the probability of success of in vitro fertilization procedures. The ability to transfer a single healthy embryo with an increased probability of success, instead of several embryos with unknown probabilities of success, will result in fewer undesired multiple birth events.
Suitable bioactive lipids for analysis in the present invention include lysophospholipids, phospholipids, sphingolipids, and combinations and subspecies thereof. In addition to analyses of bioactive lipid species, precursors and metabolites of these bioactive lipids, such as glycerophosphatidyl compounds, may also be analyzed in the method as indicators of embryo health and viability. Various analytical methods of measuring the levels of bioactive lipids in the embryo culture media are contemplated in the present invention. In a preferred embodiment, enzymatic assays are used to measure levels of bioactive lipids in the sample embryo culture media. Generally, in enzymatic methods, lysophospholipids or glycerophosphatidyl compounds in the specimen are selectively converted into glycero-3-phosphate, and measured by a redox coupling reaction with NADH or NADPH. In an alternative embodiment, liquid chromatography-mass spectrometry is used to detect and measure the bioactive lipids present in the culture media sample. In another alternative embodiment, antibodies specific for particular bioactive lipid species or derivatives may be utilized in an immunoassay for the bioactive lipids.
In another aspect, the present invention also provides pre-packaged diagnostic kits for determining embryo biological characteristics by measuring certain bioactive lipids in an embryo culture media specimen. In preferred embodiments, these kits contain enzymes and reagents necessary to assay the sample for bioactive lipid concentrations, as well as standards and instructions for determining whether the assayed level is indicative of an increased or decreased probability for successful development of the embryo.
As discussed above, the ability to identify an embryo that is likely to implant properly in the uterine cavity and to develop normally to term is of great importance to the in vitro fertilization process. By eliminating the necessity to transfer multiple embryos for implantation, the overall success rate of the in vitro fertilization process may be enhanced, avoiding undesired multiple pregnancies. The present invention provides a method for determining the biological characteristics of embryos, including implantability and likelihood of normal development, by analyzing sample media from IVF embryo cultures at the very early stages of embryo development in order to determine which embryos are likely to develop successfully after transfer into a patient.
In the present invention, bioactive lipids, their precursors, and their metabolites in a sample media are analyzed to determine the viability of the embryo, preferably based on predetermined values or a lipid profile for the media, a measured value for the media, or a titrated value based on a series of measurements of bioactive lipids in the media. Small amounts of bioactive lipids in IVF cultures may be assayed accurately and reliably by biochemical means or mass spectrometry. Bioactive lipids analyzed in the present invention comprise one or more lipids selected from the lysophospholipid (LPX) and sphingolipid families. As used herein, the term bioactive lipids also encompasses precursors or metabolites of those lipids, such as glycerophosphitidyl (GPX) compounds, although they are not technically lipids. Bioactive lipid precursors may be measured in the methods of the invention.
LPX species for analysis pursuant to the invention include, among others, lysophosphatidic acid (LPA), lysophosphatidyl choline (LPC), lysophosphatidyl serine (LPS), lysophosphatidyl ethanolamine (LPE), lysophosphatidyl inositol (LPI), and lysophosphatidyl glycerol (LPG). Lysophospholipids have the general structure of a glycerol backbone with a phosphate or a derivatized phosphate such as choline, inositol, ethanolamine, glycerol, or serine at the sn-3 position; a single fatty acid chain located at the sn-1 or sn-2 position, linked to the glycerol backbone by an acyl linkage; and a hydroxyl at the unoccupied sn-1 or sn-2 position. Alternatively, an long-chain alcohol is linked to the glycerol backbone at the sn-1 or the sn-2 position by an alkyl or alkenyl linkage with a hydroxyl at the unoccupied sn-1 or sn-2 position. These compounds have the following general structures:
where X is any fatty acid, alkyl, or alkenyl chain. Such carbon chains include, but are not limited to 18:0, 16:0, 18:1, 18:2, 20:4n-6 and 22:6n-3, attached through an acyl (Gxe2x80x94Oxe2x80x94COOR1), alkyl (Gxe2x80x94Oxe2x80x94CH2xe2x80x94R1) or alkenyl bond (Gxe2x80x94Oxe2x80x94CHxe2x95x90R1),
where xe2x80x9cGxe2x80x9d is the glycerol backbone. X may include such fatty acids as palmitic, palmitoleic, stearic, oleic, linoleic, arachidonic, and docasahexahoic fatty acid linked. R can be any phosphate derivative moiety, including, but not limited to, hydrogen, choline, inositol, ethanolamine, glycerol, and serine. More generally, the lysophospholipids may be represented by the structural formula:
wherein S1 is either X (as defined above) or hydrogen; S2 is hydrogen if S1 is X, or X if S1 is hydrogen; and R is as defined above. Common lysophospholipids for detection using the invention method include, but are not limited to, LPA, LPC, LPS, LPE, LPI, and LPG, and their specific fatty acid/alcohol side chain forms.
Sphingolipids contemplated for analysis in the present invention comprise, among others, sphinganine-1-phosphate, sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), and sphingomyelin. The structural formulae for these sphingosyl compounds are shown below: Sphingosine-1-Phosphate:
Sphinganine-1-Phosphate:
Sphingomyelin:
Sphingosine-phosphorylcholine (SPC):
Thus, sphingosyl compounds, which are the sphingomyelin analogs of lysophospholipids, have the basic structural formula:
wherein S1 is any alkyl or alkenyl of 15 carbons, and wherein R is any phosphate derivative moiety, including, but not limited to, hydrogen, choline, inositol, ethanolamine, glycerol, and serine. Thus, in addition to the derivative SPC, sphingosylphosphorylinositol (SPI), sphingosylphosphorylethanolarnine (SPE), sphingosylphosphorylglycerol (SPG), and sphingosylphosphorylserine (SPS) are also suitable bioactive lipid analytes for measurement in the methods of the present invention. By convention, 1-unsaturated S1 sphingosyl compounds are called xe2x80x9csphingosinexe2x80x9d compounds, and saturated S1 sphingosyl compounds are called xe2x80x9csphinganinexe2x80x9d compounds.
In addition to the bioactive lipids described above, precursors and metabolites of bioactive lipids may also be assayed. Precursors include, among others, one or more of phospholipids (2 fatty acid chains), glycerophosphatidyl compounds (GPX), monoglyceride (MG), and digycleride (DG). Metabolites comprise, among others, one or more of glycerophospholipids (GPX), and monoglyceride (MG). Glycerophosphatidyl compounds (GPX) suitable for assaying in the methods of the present invention comprise, among others, glycerol-3-phosphate (G3P), glycerophosphatidyl inositol (GPI), glycerophosphatidyl choline (GPC), glycerophosphatidyl serine (GPS), glycerophosphatidyl glycerol (GPG), and glycerophosphatidyl ethanolamine (GPE). These compounds are metabolically related to lysophospholipids, and have the basic structure above, wherein both S1 and S2 are hydrogen. In addition, bioactive lipid xe2x80x9cRxe2x80x9d groups may be measured, such as choline, serine, ethanolamine, inositol, glycerol, and phosphate derivatives of these groups. GPX, MG, DG, and the derivative groups may constitute either a precursor or a metabolite. Additional precursors and metabolites not specifically mentioned herein are well known to one of ordinary skill in the art.
To ascertain standard levels of bioactive lipids which are indicative of a desirable biological characteristic, such as viability, media specimens from successful IVF embryo cultures are analyzed for the levels of bioactive lipids present. xe2x80x9cSuccessfulxe2x80x9d IVF embryos will be defined by the desirable biological characteristic to be measured, including implantation success rate, viability of the embryo in the uterus, length of development to term, successful development to term, and avoidance of multiple pregnancies. Media specimens from unsuccessful cultures may also be analyzed for comparison and validation of the standard level. By comparing the levels of bioactive lipids in the culture media of successful and unsuccessful embryos, one of ordinary skill in the art is able to determine levels of bioactive lipids which are indicative of viability, implantability, and an increased likelihood of development to term.
In general, a standard level or profile of bioactive lipids is developed from this data which correlates with the improved viability, implantability, and likelihood of developing to term for an embryo in IVF culture. Significant deviation from this standard level (meaning a deviation greater that seen in embryos which are successful) is indicative of a decreased extent of the desirable biological characteristic (i.e., a decreased probability of implantation, or a decreased probability of developing to term.) In preferred embodiments, this standard level may be determined empirically from a statistically significant population of successful embryo culture specimens, and expressed as a number with a standard deviation. The standard level may the mean or median level of the bioactive lipid for culture specimens from embryos which exhibit the desirable biological characteristic, or may be derived through some other statistically valid means. This approach will be useful where the detection mechanism is sufficiently accurate to yield reproducible quantitative results. In another embodiment, the standard level for comparison may be a sample of culture media which is spiked with bioactive lipids to the standard level, or a culture media specimen (or mixture of specimens) derived from successful embryos. Based on the foregoing information, the chemical formulation of the cultured media may be designed or altered to reach a specific profile of bioactive lipids to achieve the aims of the in vitro fertilization. Thus, the invention includes specially formulated in vitro fertilization media based on the analysis of preferred bioactive lipid concentrations. Once this data is possessed, the chemical composition of the media may be altered in respect of the combination of one or more bioactive lipids. For example, particular bioactive lipids may be added to supplement the concentration in the media, or chemical agents may be introduced to remove or inactivate elevated levels of undesirable bioactive lipids. When the bioactive lipid profile is monitored over time, the individual concentrations of various lipids can be supplemented or altered as desired.
To determine viability of an embryo in question, a media specimen is collected from the IVF embryo culture, and then analyzed to determine the level of one or more bioactive lipids, precursors, or metabolites present in the specimen. Preferably, the level of the analyte is compared to a control media sample which has not been incubated with an embryo. Utilizing media controls can assist in correcting for natural variations in media compositions which may occur in culture media which are not wholly defined. By comparing the types of bioactive lipids and their concentrations to the standard levels, a determination is able to be made as to the likelihood of whether the embryo will implant and develop successfully to term. In the embodiments of the present invention, the level of a single bioactive lipid may be detected, or two or more bioactive lipids may be detected.
In alternative embodiments, multiple IVF culture specimens may be collected over a time interval, and the change in bioactive lipid levels over the course of the time interval determined. This is compared to a standard change in the level of the bioactive lipids assayed, obtained in a manner similar to that described above for the standard level, wherein multiple samples are collected from the IVF embryo cultures over a similar time interval. Similar to the analysis above, a significant deviation in the change observed in the test culture specimen, as compared to the standard change, would indicate a decreased extent of the desirable biological characteristic. Thus, the invention analyzes one or more bioactive lipids, correlates that measurement to a standard that has been predetermined or is determined according to the practice of the invention, and then determines a biological characteristic of the embryo, such as viability, probability of a certain term length or full term of the chance of a multiple pregnancy. Based on a determination of the selected characteristic, a decision is made whether to use the embryo or select an embryo with more desirable characteristics based on the analysis of the media described herein.
The determination of the desirable biological characteristic may be expressed in terms of a xe2x80x9cbright line testxe2x80x9d or a probability or other gradient measure. For example, in a bright line test, if a specific bioactive lipid is present at a zero or undetectable level in the media specimen, or below a definite threshold, the embryo is deemed not likely to survive, whereas if the bioactive lipid is present at detectable levels or above a certain threshold, the embryo is deemed likely to survive. Alternatively, the determination may be expressed as an embryo""s likelihood or percentage of success. For example, where the analysis involves three bioactive lipids, detecting all three lipids at the standard level would give the strongest indication (i.e., 90% chance) of likelihood of success followed by detecting only two lipids (i.e., 60% chance). Detecting only one of the three lipids would indicate a lower chance of success (i.e., 30% chance). As will be appreciated by those of skill in the art, the analysis or measurement of the bioactive lipids can be conducted by determining an increase or decrease from a pre-existing standard, an increase or decrease from a previous measurement, or an absolute value relative to zero or a standard threshold or variable concentration established through testing or experience.
Various types of in vitro fertilization culture media may be used. To insure that the standard level is correct for any particular culture media type, it is preferred that a standard level be determined empirically for any type of culture media assayed. Moreover, because the chemical composition of various culture media may differ depending on the manufacturer or the specific formulation used by an in vitro fertilization clinic or facility, it may be necessary to determine an individual lipid profile or series of profiles for each type of media or each specific formulation used. Because this analysis necessarily involves a variation of multiple factors, although it is preferred that samples come from a single-embryo culture, which will provide information about that specific embryo, multiple embryo culture specimens may also be assayed utilizing the invention. The information generated from these tests is limited, however, to determinations regarding the embryos as a population, and the bioactive lipids characteristics of one embryo may mask those of another.
IVF culture media specimens include those derived from cultures where the fertilized ovum is cultured alone (as the only cell) in IVF media o for use with co-culture systems, wherein embryos are fertilized and allowed to develop on a bed of feeder cells. The feeder cells are thought to interact and communicate with the embryo in biochemical pathways to facilitate growth and development. Active communication between the feeder cells and the embryo may include important bioactive lipids in paracrine communication pathways. In some IVF culture systems, reproductive tissues such as oviduct cells and endometrial cells are used to produce feeder cell lines. In a more preferred practices, human cell lines are used as feeder cell lines, sometimes derived from the patient receiving the IVF embryo. This reduces the chances that an immunological response will develop upon transfer of the embryo. Further, multiple cell lines may be established from a single donor, and a most suitable cell line may be chosen for use in a co-culture system. In another aspect of the present invention, cell lines derived form a patient for use as feeder cells may be analyzed for the production of one or more bioactive lipids in order to choose the most suitable cell line. Essential bioactive lipids may indicate that a cell line is suitable for co-culturing. In contrast, cell lines that lack essential bioactive lipids may be deemed unfit for use as feeder cells.
In analyzing the media specimen, various sample sizes may be used. A suitable amount of culture media collected for sampling ranges preferably from approximately 1 to approximately 100 xcexcl. Generally, no more than approximately 50 xcexcl is needed for sampling. For the analytical assays described below, a preferred specimen volume ranges from approximately 1 to approximately 50 xcexcl, more preferably from approximately 2 to approximately 10 xcexcl, and most preferably approximately 5 xcexcl. The present invention is advantageous in that only a small amount of sample specimen is required for an accurate analysis.
In normal IVF culture practices, the culture media is changed approximately two or three days after fertilization. The supernatant is decanted and fresh medium is added. In a preferred embodiment, the decanted media is used advantageously for the IVF culture specimen. The culture media, however, may be collected anytime during IVF. Preferably, the media specimen is collected when the embryo is between one hour to twenty days old, more preferably between one day and ten days old, and more preferably between two and five days old. For embodiments where the change of the detected bioactive lipid is assayed, two or more samples may be collected over a time interval within any of the above periods. Preferably, samples are collected over 1-5 days, more preferably over 1-3 days, and most preferably over 1 day or over 2 days. A small volume of media specimen (10-50 xcexcl) may be carefully extracted without disrupting development of the embryo.
Several analytical techniques may be used for detecting and measuring bioactive lipids, including mass spectroscopy procedures and enzymatic methods to detect or quantitatively measure bioactive lipids in a sample specimen. In one embodiment of the methods of the invention, liquid chromatography and mass spectroscopy (LC-MS) is used to analyze sample specimen for bioactive lipids. Conditioned media samples from cultured embryos are diluted in a suitable solvent (e.g., 1:5 dilution in methanol) and centrifuged to remove any precipitate. In some embodiments, proteln-lipid interactions may be disrupted by adding acid, or other substances, to the sample. The samples are separated on an LC or high pressure (HP) LC column, and the individual bioactive lipids detected and quantified by mass spectroscopy. In a preferred embodiment, electrospray (ESI) MS or atmospheric pressure chemical ionization (APCI) MS is used. The mass spectroscopy technique is very flexible, and may be used to measure lysophospholipids, sphingolipids, and glycerophosphatidyl compounds, as well as to distinguish between various fatty acid or long-chain alcohol side chains and phosphatidyl substituents within the general species.
In a preferred embodiment, the bioactive lipids are assayed enzymatically. The enzymatic assay measures the total concentration of lysophospholipids or glycerophosphatidyl compounds present in a sample, and may also be used to measure the concentrations of specific bioactive lipids. The enzymatic assay preferably measures total GPX and/or LPX in the IVF culture specimen. Preferred embodiments for determining levels of LPX, LPA, and LPC in sample specimen are discussed in detail below. The specification of U.S. application Ser. No. 09/314,780, ENZYME METHOD FOR DETECTING LYSOPHOSPHOLIPIDS AND PHOSPHOLIPIDS AND FOR DETECTING AND CORRELATING CONDITIONS ASSOCIATED WITH ALTERED LEVELS OF LYSOPHOSPHOLIPIDS, filed May 15, 1999, and PCT Publication No. WO 00/23612, which is instructive for teaching methods of measuring LPX, LPA and LPC levels:, are fully incorporated herein by reference. In addition, the specification of U.S. application Ser. No. 09/558,880, METHOD OF DETECTING CARCINOMAS, filed Apr. 26, 2000, which is instructive for teaching methods of measuring GPX, G3P, and GPC levels, is also fully incorporated herein by reference.
A preferred method for enzymatically measuring bioactive lipids comprises measuring GPX and LPX in the specimen. The analytic method generally comprises converting GPX or LPX into G3P and assaying for the concentration of G3P produced in the sample. To convert LPX into G3P, lysophospholipase is used in the enzymatic reaction to cleave the fatty acid group from the G3P and other glycerophosphatidyl compound (GPX) backbones. GPX is preferably digested using glycerophosphatidyl compound phosphodiesterase (GPX-PDE) to cleave the substituent from the phosphate of the G3P backbone. Thus, the amount of G3P produced by both of these enzymatic cleavage reactions is directly proportional to the total amount of LPX in the media specimen. Similarly, to determine the amount of GPX in a sample, only the glycerophosphatidyl compound phosphodiesterase is used to cleave the phosphatidyl substituents from the GPX species.
The amount of G3P in the cleaved and uncleaved portions of the sample specimen is then quantified using conventional or enzymatic techniques. If the size of the media specimen is 2 ml or less, a quantification technique capable of detecting picomole amounts of the glycero compound is used. Suitable conventional techniques for detecting picomole amounts include mass spectrometry.
Another preferred technique for determining the amount of G3P in the samples is an enzymatic cycling reaction. Specifically, an enzyme cycling reaction using glycerol-3-phosphate dehydrogenase (GDH), glycerol-3-phosphate oxidase (GPO) and NADH is used to accumulate H2O2 and NAD. In the reaction, G3P is converted into dihydroxyacetone phosphate (DAP) and H2O2 using GPO in the presence of oxygen and water. In the presence of DAP, G3P dehydrogenase converts dihydroxyacetone phosphate back to G3P and oxidizes NADH to NAD. Alternatively, depending on the sensitivity of the NADH or H2O2 detection, a non-cycling reaction may be used.
The disappearance of NADH is monitored spectrophotometrically preferably at OD340. In alternative embodiments, H2O2 production may be measured by colorimetry, fluorometry, or by chemiluminescence. For the colorimetric assay, any of a number of chromogenic substrates, such as 4-aminoantipyrine (AAP), pyrogallol, 2-(21-Azinobis (3-ethylbenzthiazoline-sulfonic acid)(ABTS) and 3,31,5,51-tetramethylbenzidine) (TMB), may be used with a peroxidase to generate detectable signal (e.g., OD505 for AAP). Numerical values are obtained from a standard curve consisting of known concentrations of G3P, and assays are preferably performed in duplicate with both positive and negative controls. The difference between the detectable signal (e.g., OD340 or OD505) before and after the enzyme cycling reaction is directly proportional to the amount of G3P present.
Additional bioactive lipids, such as glycerol-3-phosphate (G3P) and lysophosphatidic acid (LPA), may also be determined as above by first separating LPA and G3P from the total lysophospholipid in the sample. In a preferred embodiment for determining the concentration of LPA and G3P, G3P is measured after being liberated from LPA in the absence of GPX-PDE. In this embodiment, G3P is detected using the enzymatic cycling reaction described above, but the remaining GPX compounds other than G3P are not detected. LPA is first cleaved into glycerol-3-phosphate and fatty acid using phospholipase B or lysophospholipase. The level of G3P is then measured using G3P dehydrogenase and oxidase in the cycling reaction as described above.
Additional bioactive lipids, such as glycerophosphatidylcholine and lysophosphatidylcholine, may also be determined as above by first separating LPC and GPC from the total lysophospholipid in the sample. In a preferred embodiment for determining the concentration of LPC and GPC, choline is measured after being liberated from GPC and LPC. LPC is first cleaved into glycerophosphatidyl choline and fatty acid using phospholipase B or lysophospholipase. The level of LPC is then determined by liberating choline and glycero-3-phosphate (G3P) from glycerophosphorylcholine using glycerophosphorylcholine phosphodiesterase (GPC-PDE), followed by a calorimetric enzymatic determination of choline using choline oxidase, 4-aminoantipyrine (AAP), 3,5 Dichloro-2-hydroxybenzenesulfonic acid sodium salt (HDCBS) and peroxidase. Choline is preferably detected by oxidizing to H2O2 and betaine and using peroxidase to form quinoneimine dye. Alternatively, G3P is measured using G3P dehydrogenase and oxidase in the cycling reaction as described above.
In other embodiments of the invention, immunoassay techniques may be utilized to measure the levels of bioactive lipids in the IVF media specimen. Antibodies to various biological lipids suitable for analysis have been described in the literature. For example, Chen, et al., xe2x80x9cProduction and Application of LPA Polyclonal Antibody,xe2x80x9d Bioorg. and Med. Chem. Letters 10:1691-1693 (2000), describes the production and use of an antibody to LPA, utilizing colloidal gold as an antigen carrier. Similar antibodies may be raised to other lysophospholipid species and utilized in immunoassay formats (e.g., sandwich or competitive immunoassays.) In addition, antibodies to bioactive lipid precursors or metabolites have been reported. For example, Echelon Labs (Salt Lake City, Utah) offers an anti-phosphoinositol antibodies, and immunoassay kits for the detection of phosphoinositol species. One of ordinary skill in the art would be able to readily adapt these antibodies for analysis of bioactive lipids in media to determine embryo characteristics pursuant to the invention.
To optimize detection of lysophospholipids, inhibitors may be used to prevent degradation of the glycerophosphatidyl compounds and lysophospholipids in the sample. Such inhibitors include phosphodiesterase inhibitors such as IBMX (3-Isobutyl-1-methylxanthine, CalBiochem, La Jolla, Calif.); Ro-20-1724 (CalBiochem); Zaprinast (CalBiochem) and Pentoxifylline (CalBiochem); general protease inhibitors such as E-64 (trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane, Sigma); leupeptin (Sigma); pepstatin A (Sigma); TPCK (N-tosyl-L-phenylalanine chloromethyl ketone, Sigma); PMSF (Phenylmethanesulfonyl fluoride, Sigma); benzamidine (Sigma) and 1, 10-phenanthroline (Sigma); organic solvents including chloroform and methanol; detergents such as SDS or Trident X100; proteases that would degrade phospholipases such as trypsin (Sigma) and thermostable protease (Boehringer Mannheim Biochemicals, Indianapolis, Ind.); and metal chelators such as EDTA (Ethylenediaminetetracetic acid, Sigma) and EGTA (Ethylene glycol-bis-(beta-aminoethyl ether), Sigma). In some embodiments, MgCl2 and/or EDTA are included in the assay buffers to optimally determine levels of each analyte.
In a preferred embodiment, microtiter plates may be used for small volumes of samples and reagents. An ELISA reader may also be used to monitor and help automate the assay, and the reduced processing times may in turn reduce variability between results. Also, micro-scale automated assay equipment, such as the Immuno I system available from Bayer, the Access system available from Beckman Coulter, or the Dimension R-L HM system available from Dade Behring may be used.
The present invention also contemplates convenient pre-packaged diagnostic kits for enzymatically detecting levels of bioactive lipids such as GPX, GPC, LPX and/or LPC in IVF culture media specimens. Preferably, these kits contain enzymes and reagents necessary for determining the level of GPX, GPC, LPX and/or LPC as described above, as well as instructions to correlate the bioactive lipid levels assayed using the kits to embryo characteristics as described herein. For example, diagnostic kits preferably include enzymes and buffers for the cleavage of GPX, GPC, LPX and LPC. Exemplary enzymes for inclusion in such kits are phospholipase B, lysophospholipase, glycerophosphitidyl compound phosphodiesterase, and glycerophosphatidylcholine phosphodiesterase. In addition, the kits of the present invention preferably include reagents for determining concentrations of G3P, including enzymatic reaction reagents such as glycerol-3-phosphase dehydrogenase, glycerol-3-phosphate oxidase, NADH and other ancillary agents such as buffering agents, colorimetric reagents for the detection of peroxide generation, and EDTA for inhibiting degradation of G3P.
Optionally, the kits may include reagents necessary to separate GPC or LPC from the other lysophospholipids in the sample. Also optionally, the kits of the present invention include reagents for measuring choline liberated from GPC or LPC in the specimen. Such reagents may include, for example, choline oxidase, peroxidase, 4-aminoantipyrine (AAP), 3,5 Dichloro-2-hydroxybenzenesulfonic acid sodium salt (HDCBS), and other ancillary agents such as buffering agents.
The kits of the invention also comprise containers and measuring apparatus for carrying out the appropriate measurements. The kits of the invention may also include standards for comparison, which are preferably IVF culture media spiked with the appropriate level of the assayed bioactive lipids, in order to assure that the clinician has properly performed the analysis described herein when using the kit. Less preferably, such standards may also be derived from successful embryo culture media. Variations of specific container and combination embodiments of the kits of the invention may readily be devised by those of ordinary skill in the art utilizing the guidance herein provided.

Determination of biological characteristics of embryos fertilized in vitro by assaying for bioactive lipids in culture media


A radial bearing may include a bearing body, which includes a receiving space for a shaft to be arranged therein. The bearing body may have an inner wall facing the receiving space, in which transverse grooves running in an axial direction and separated from one another in a circumferential direction by separating portions are formed. The radial bearing may include a supply channel penetrating the bearing body provided in at least one separating portion. The at least one separating portion may have at least one oil pocket spaced from the associated supply channel.

A radial bearing serves for mounting a rotating shaft and is employed for example in an exhaust gas turbocharger for an internal combustion engine. Such a generic radial bearing is known from DE 10 2008 000 853 A1. Here, the radial bearing comprises a cylindrically formed bearing body, which forms a receiving space for the shaft to be arranged therein. Within the bearing body, supply channels running in radial direction are provided, which feed a lubricant, for example lubricating oil to the receiving space, which lubricating oil upon rotation of the shaft disposes itself between the shaft and an inner wall of the bearing body facing the receiving space or the shaft in order to avoid a direct mechanical contact between the shaft and the inner wall. To avoid or reduce undesirable vibration effects, for example the so-called oil whirl and/or oil whip, multiple transverse grooves are formed in the inner wall, which run in the axial direction and are separated in the circumferential direction by separating portions, said supply channels being arranged in the separating portions. Here, a lubricant shortage can occur during the rotation of the shaft in the bearing body, in particular at high rotational speeds of the shaft, so that shaft and bearing body directly contact one another. This lubricant shortage results in an increased wear of the radial bearing and can at times result in a failure of the radial bearing.


Supported catalysts of the following formula (I) for preparing ethylene by conversion of methane or purified natural gas and preparation thereof, and process for preparation of ethylene by direct conversion of methane or purified natural gas using said catalysts: EQU Ma'/S formula (I). (wherein, M is a compound selected from the group of RuCl.sub.2 (PPh.sub.3).sub.3, RuCl.sub.2 (CO).sub.2 (PPh.sub.3).sub.2, Ru.sub.3 (CO).sub.12, RhCl(CO)(PPh.sub.3).sub.2, IrCl(CO)(PPh.sub.3).sub.2, Pd(PPh.sub.3).sub.4, Pt(PPh.sub.3).sub.4, RuCl.sub.3 .multidot.xH.sub.2 O, RhCl.sub.3 .multidot.xH.sub.2 O, IrCl.sub.3 .multidot.xH.sub.2 O, PdCl.sub.2 .multidot.xH.sub.2 O, H.sub.2 PtCl.sub.6 .multidot.xH.sub.2 O, S is an inorganic carrier selected from .alpha.-Al.sub.2 O.sub.3, .gamma.-Al.sub.2 O.sub.3, SiO.sub.2, SiO.sub.2 --Al.sub.2 O.sub.3, Y-zeolite, MgO and TiO.sub.2, a' is weight percentage of metal in catalyst, ranging from 0.25 to 5 wt %.)

Few of literatures and patents about the catalyst which can be used to produce ethylene by direct conversion of methane is disclosed; moreover, it is different in technical field from present invention. By conventional processes, large amounts of by-product such as carbon dioxide are produced, separation and removal thereof is difficult and environmental pollution is likely to be caused. Also, synthesis of hydrocarbon(s) by conventional dehydrogenation is conducted at relatively high temperature of about 1500.degree. to 1550.degree. C. through thermal or electric cracking reaction to cause some problems such as necessity of supply of high energy, expense of high temperature equipment (plan) as well as enormous loss of thermal energy expense, particulary severe corrosion of reactor.
As prior art describing synthesis of hydrocarbon by oxidative coupling or dehydrogenation reaction, there are U.S. Pat. Nos. 5,066,629, 5,068,486 and 5,118,654, Canadian patent no. 2016675 and Japanese patent nos. 04352730, 04368342.

Supported catalysts for converting methane or purified natural gas, preparation thereof, and process for preparation of ethylene using said catalysts

Korea Research Institute of Chemical Technology


A system, machine readable medium and method for utilizing protocol conversions in policy changing enforcement is disclosed. A message, in a first protocol, is received from a network gateway device including identifying information unique to a client attempting to access a resource from a server. The message is processed using one or more portions of the client identifying information as a unique key identifier. A policy access request is generated, in a second protocol, and includes at least the unique key identifier. The policy access request is sent to a policy server, wherein the policy server is configured to provide policy enforcement information of the client associated with the policy access request. The policy enforcement information is received and one or more policies from the policy enforcement information are enforced to network traffic between the client and the server.

In existing systems, client devices, such as mobile devices, will attempt to access a service or resource from one or more servers via a cellular based network. During initiation of the connection, the client device will start a data context with a gateway node in which the gateway node will send an Authorization, Authentication and Accounting (AAA) message to a policy server in a cellular network based protocol. In the case that a virtual policy enforcement proxy device is positioned between the gateway node and the server, and the policy enforcement device must retrieve policy information of the client device from a policy server in which messages sent between the proxy device and policy server may be in a protocol different than the protocol of the AAA message. This can be costly and burdensome using current technologies.
What is needed is a network traffic management device which is able to utilize unique information of the user in generating a policy access request to a policy server, wherein the network traffic management device is able to apply policy enforcement functions from the policy server to the network traffic between the client device and the server(s).

System and method for on the fly protocol conversion in obtaining policy enforcement information


A method of transferring a decorative stenciled image to an irregular object utilizes flexible plastic static cling sheet material as the stencil medium. The cutout stencil is shaped approximately to the size of the surface of the object that will receive the image and conforming the stencil medium to the surface smoothly, adhering the sheet material such that the static cling attribute of the material will bond to the object's surface without lifting or breaking that would cause gaps or bubbles. The method is completed by well-known steps of applying stencil ink or paint by brush or other suitable applicator, removing the stencil medium and optionally curing the image by baking or other fixation step.

Decorative stenciling by applying paint or ink through patterned cutouts in sheet or screen material has long been popular both as a home craft method and as a limited or custom production method for small articles. Properly done, stenciling can accurately and repetitively transfer simple and complex artistic and graphic patterns and lettering to a variety of small objects, enhancing the attractiveness of the objects. Many such objects may be the subject of application of decorative designs by transfer of liquid or semi-solid artistic media to the surface of the object and fixing the media to the object by curing, drying or baling. In particular, ceramic, metallic and glass objects are suitable for application of decorative stencil patterns by reason of the relatively smooth exterior surfaces they offer to receive and display the designs as the object is in use.
A critical factor in proper application of a stencil design is adherence of the sheet stencil medium to the surface of the object to receive the design. Flat surfaces on objects present little problem, as the flat stencil sheet can be closely pressed to the flat object and, if necessary, secured by tape or other clamping-type pressure during the application of the stencil design, typically by brush or other flexible applicator that will carry the fluid art media to the surface through the stencil cutout. However, curved or irregular surfaces are far more difficult to achieve the necessary close adherence of stencil sheet medium to object surface. Even more difficult is an object with an outside corner that the stencil must wrap around. Since the stencil sheet medium is provided in flat format, bending the medium to a curved surface deforms the sheet medium which will then tend to lift away from the object surface. If the stencil design is not tightly fitted to the object surface, ink or paint will leak and spread under the stencil when applied, resulting in an unacceptably poor image due to smudging, blurring and inexact lines.
The present invention is expressed herein as a method of applying a stencil design utilizing a novel stencil sheet material that enhances the exact adherence of the stencil pattern to the surface of the object to be decorated.


A gutter protection system is adapted for covering a gutter and preventing debris from falling into the gutter. The gutter protection system comprises an elongate sheet metal member or panel adapted to engage a portion of the gutter and to cover the gutter. The sheet metal member has a longitudinal axis and a generally smooth top surface interrupted by a plurality of canals and perforations formed therein. Each canal extends down and away from the smooth top surface. The canals are formed from the material of the sheet metal member, each canal formed so that water entering the canal exits through one of the perforations and flows into the gutter below. The canals enable water to move laterally within the canal, thereby increasing the water volume handled by the system without necessarily increasing the number of holes or perforations.

Gutter protection systems such as gutter screens attempt to direct water into the gutter while preventing debris from entering the gutter. The screen should be designed so that it does not itself become clogged with debris and is securely attached to the gutter. However, some conventional gutter screens have holes that often trap debris instead of allowing the debris to slide over the screen. Debris may build up in the holes and cause rainwater to bypass the holes and flow over the gutter instead of into the gutter, or may cause rainwater to build up above the gutter. The debris must thereafter be removed from the screens by hand. Thus, many conventional gutter screens are not maintenance free. Moreover, the pattern of holes in prior art gutter screens is such that, even without debris clogging, water may flow along a path that avoids all the holes in the screen and thereby flows over the screen and gutter. In other words, water is not satisfactorily directed by the gutter screen into the gutter. Accordingly, a cost-effective, maintenance-free gutter screen that directs ample rainwater to flow into the gutter is needed.

Perforated gutter protection system having canals


The present invention provides an improved Chua's circuit providing current mode operation, access to all state variables, minimum use of grounded passive elements, and freedom from passive component matching comprising a dual output current conveyer based inductor having one grounded terminal, a capacitor connected across the second terminal of said inductor, a resistor having one terminal connected to the second terminal of said inductor, the second terminal of said resistor connected to one terminal of a second capacitor the other end of which is grounded, and a pair of dual output current conveyers connected together to form a 2-terminal negative resistance having one terminal connected to ground and the second terminal connected to the second terminal of said resistance.

FIG. 1 shows a generalized Chua's circuit that has a parallel combination of a capacitor C2, a Chua's diode NR1 connected to an L-C tank circuit that includes a second capacitor C1 connected in parallel with a serially connected inductor L1. In addition, a first voltage V1 is defined across capacitor C1 and a second voltage V2 is defined across capacitor C2 with the positive orientation of voltages V1 and V2 both being at the positive end of the corresponding capacitor. Finally, there is a linear resistor R1 connected between the positive terminals of each capacitor.
In a typical configuration, the non-linear curve is piece-wise linear with symmetrical slope discontinuities around the current axis. It satisfies the following equation: IR=GaVR+(½) (Ga−Gb) {|vR+BP|−|vR−Bp|}, where Ga and Gb are the slopes of respective linear portions of the piece-wise linear current/voltage curve characterizing the nonlinear resistor, and Bp is the absolute value of the two voltage points at which discontinuities in the current/voltage curve lie, as shown in FIG. 2. The circuit has a circuit driving subsystem, an L-C tank circuit, and a response subsystem with a parallel combination of a capacitor and a non-linear resistor interconnected through a resistor.
By choosing values of R, L, C1 and C2, the circuit can operate in different operating regions, for example, the double scroll region. Herein, a Chua's circuit can be made that will oscillate chaotically or quasi-periodically. Given a specified physical configuration, a specified initial state specified by V1, V2 and IL, the voltage across the capacitors C1 and C2, and the current through the inductor L, the evolution of the Chua's circuit is deterministic, but chaotic. That is, any Chua's circuit with the same physical parameters and initial conditions will follow the same course of states over time, and this course will repeat itself over a very long period. However, to an observer it looks mainly like noise. Also, such a system's trajectory is sensitive to initial conditions. In addition, the power spectral density function is spread over a wide range of frequencies, with the peak frequency of the fundamental being governed by the L-C tank circuit.
Due to its simple circuitry and ability to demonstrate most well known routes to chaos, a Chua's Circuit and Chua's Oscillator (CC/CO) are an active topic of research in the study of non-linear dynamic circuits and systems. Recently, there has been an increasing interest in designing inductor-less Chua's circuits and Chua's oscillators (CC/CO). Moreover, due to its advantages, the attainability of all the three state variables of a CC/CO is also attracting the designers. Simultaneously, literature is also witnessing the shift of analog integrated circuit designs from voltage mode processing to current-mode processing (CMP). A number of chaotic circuits have been implemented using current-mode active building blocks. The Dual-Output Current Conveyor (DO-CCII) is also emerging as a versatile block to implement current-mode circuits.
To improve the performance of the circuit, different researchers have done extensive research. A paper by Kennedy M. P. (Kennedy M. P., 'Robust op-amp realization of Chua's circuit', Frequenz, 1992, 46, pp. 66–80) suggests a Chua's circuit using off the shelf components. Further, in another research paper by Torres, L. A. B. and Aguirre, L. A. (Torres, L. A. B. and Aguirre, L. A., Inductorless Chua's circuit, Electron. Lett., 2000, 36, (23), pp. 1915–1916) report a Chua's circuit using an operational amplifier to generate a Chua's oscillation at a very low frequency for bio-medical operations.
In a design proposed by Morgul, 0. (Morgul, O., 'Inductorless realization of Chua's oscillator', Electron. Lett., 1995, 31, pp. 1403–1404) synthetic inductors using op-amps were used along with the operational amplifier, thereby making the design suitable for monolithic implementation.
Senani R. and Gupta S. S. (Senani R. and Gupta S. S., 'Implementation of Chua's chaotic circuit using current feedback op-amps', Electron. Lett., 1998, 34, (9), pp. 829–830) proposed a Chua's circuit using a Current Feedback Operational Amplifier (CFOA), thus making available the third state variable through the inductor (iL).
In another architecture designed by Elwakil A. S. and Kennedy M. P. (Elwakil A. S. and Kennedy M. P., 'Improved implementation of Chua's chaotic oscillator using current-feedback op-amp.', IEEE Trans. CAS-I, 2000, 47, (1), pp. 76–79) the CFOA was efficiently used in the Chua's circuit to provide a higher bandwidth of the chaotic signal with a buffered output of one state variable.
All the above architectures individually provide advantages of a Chua's circuit, but so far, there is no circuit that simultaneously provides: current mode operation; use of minimum grounded passive elements; availability of all the state variables; availability of two state variables in the form of current which can be used to further generate other complex chaotic circuits; a circuit free from passive component matching; use of lesser active components as compared to that represented by FIG. 2; and generation of a reduced hardware higher order chaotic circuit (also called a hyper-chaotic circuit) using one of the available currents mentioned above. Thus, there is a need to develop a circuit that can provide all of these simultaneously.
A Chua's circuit, apart from being a device for demonstrating, studying and modeling a chaotic real world system, has been proposed to generate a hyper-chaotic circuit (T. Kapitaniak, L. O. Chua and G. Zhong, 'Experimental Hyperchaos in Coupled Chua's Circuits', IEEE Trans. CAS-I, Vol 41, No.7, July 1994). Referring to FIG. 3, it has been shown in (T. Kapitaniak, L. O. Chua and G. Zhong, 'Experimental Hyperchaos in Coupled Chua's Circuits', IEEE Trans. CAS-I, Vol 41, No.7, July 1994) that a Chua's circuit (100) may be coupled to a similar Chua's circuit (101) such that the non-grounded terminal of the inductor is connected to the input of a voltage buffer (10) whose output is connected to one of the terminals of a controlling resistor (11), and another end of the controlling resistor is connected to the non-grounded terminal of another Chua's circuit (101). The system coupling is achieved by interconnecting n Chua's circuit in the fashion described above, and the last Chua's circuit of the ring is either connected to the first Chua's circuit (100) or is left open. This type of coupling results in the following cases:
For the value of the controlling resistor greater than a specific value (called a threshold value), all the Chua's circuits will synchronize with each other. This value of the controlling resistor depends on the number of Chua's circuits used in the chain. During this state, the curve between the state variable of one Chua's circuit to the corresponding state variable in another Chua's circuit of the chain will be a straight line.
If the value of the controlling resistor is less than the specific value (called a threshold value), the Chua's circuits will loose synchronization with each other and the system will undergo a state of hyper-chaos, which is more sensitive to the initial condition. This value of the controlling resistor depends on the number of Chua's circuits used in the chain. During this state the curve between the state variable of one Chua's circuit to the corresponding state variable in another Chua's circuit of the chain will not be a straight line.
With such a proposal, a monolithic implementation of the hyper-chaotic circuit can be achieved by using any of the above-mentioned variations of the Chua's circuit (i.e., prior art Chua's circuits can be used to generate the hyper-chaotic circuit using the above described scheme). However, the use of any of these circuits will not only add to the disadvantage of those proposed Chua's circuits, but also one voltage buffer and one floating resistor per coupling is required, which in turn, makes the final circuit bulky and inefficient in terms of power consumption.

Chua's circuit and it's use in a hyperchaotic circuit


A method of converting a control set from an identifiable first control pattern configuration to an identifiable second control pattern configuration. The method provides a pair of control assemblies that may be easily rotated from a first position to a second position to accomplish the conversion. Each of the control assemblies include a respective control lever, rocker assembly, and linkage rods. After rotation of the control assemblies, the control levers are interchanged and connected to the opposite control assembly to establish an ergonomic position for the control levers. The method of converting the control set is completed without the need for additional components or complex modifications.

This invention relates generally to a control set for operating a work machine, and more particularly, to a method of converting the control set to obtain two distinct control pattern configurations that are easily identifiable.
It is well known that a control set for operating a backhoe loader may be generally constructed for use in a variety of pattern configurations to operate various control valves via a control lever. Generally, the control set may be removed and modified in order to accommodate the preference of an operator who may be accustomed to a particular control pattern configuration. The removal and modification of the control set, however, may necessitate the use of complex or additional components that increase the cost and time necessary for the modification. Additionally, although some control sets may be easily modified into various control pattern configurations, such modifications are not within the manufacturer specifications and do not include identification of the modified control pattern configuration being utilized. Accordingly, it is desirable to provide a simple method of converting the control set so that additional or complex components are not required and the control pattern configuration being utilized may be easily identified.
A control structure is disclosed in U.S. Pat. No. 4,464,778 issued on Mar. 3, 1987 by Kazuhiko Tsuji and Youji Tsutsui. This patent utilizes a valve control structure for a working vehicle that operates two control valves by means of a control lever rockable crosswise. One of the valves is operable by a rocking movement in a first direction of the control lever and the other valve is operable by a rocking movement in a second direction of the control lever. A connection switching mechanism is provided between the control lever and the valves to switch interlocking relations of the two control valves with respect to the rocking directions of the control lever. The switching is readily carried out by operating a single switch lever to permit an operator to use the vehicle after selecting the preferred interlocking relations. In this patent, switching may be readily obtained; however, complex components must be included to provide the capability. Such components increase costs of the control set and the time needed for assembly.
Another such control structure is disclosed in U.S. Pat. No. 4,736,647 issued on Apr. 12, 1988 by Shizuo Shimoie, Kazuhiko Tsuji, and Masahiko Kobayashi. This patent utilizes a valve control structure for a working vehicle that comprises a control lever rockable crosswise, a first input rod pushed and pulled by rocking movements in a first direction of the control lever, a second input rod pushed and pulled by rocking movements in a second direction of the control lever, a first output rod connected to a first valve, a second output rod connected to a second valve, and an interlock switching device including bellcranks provided between the input rods and the output rods. The interlock switching device is operable to switch interlocking relationship between the input and output rods through the bellcranks. In one interlocking relationship, the first valve is controlled by rocking movements in the first direction of the control lever. In the other interlocking relationship, the second valve is controlled by the rocking movements in the first direction of the control lever and the first valve by the rocking movements in the second direction of the control lever. In this patent, the addition of the necessary components (such as the bellcranks) increases the complexity of the control set with cooperative switching mechanisms. Also, manufacturing costs and the time necessary for assembly of the control structure is increased. Further, the control set relates to the operation of different valves without changing their functionality.
The present invention is directed to overcoming the problems as set forth above.
In one aspect of the present invention, a method of converting a control set from a first control pattern configuration to a second control pattern configuration is disclosed. The method of converting the control set comprises the steps of providing a pair of spaced control levers with each of the control levers having a mounting plate connected thereto. Next, connecting each of the mounting plates to a bracket that has spaced first and second tab portions. Then, connecting a pair of linkage rods at a first end portion to the respective first and second tab portions of each of the brackets and connecting the pair of linkage rods at a second end portion to a respective pair of control valves. Next, connecting a rocker joint to each of the pair of brackets to define a pair of control assemblies with the respective control levers and linkage rods. Then, connecting each of the control assemblies to a base plate at a first predetermined position to define the first control pattern configuration wherein movement of either of the control levers about the respective rocker joint actuates the control valves in a predetermined manner via the pair of linkage rods. Next, substantially disconnecting each of the control assemblies from the base plate. Then, reconnecting the control assemblies at a second predetermined position angled from the first predetermine position to define the second control pattern configuration wherein movement of either of the control levers about the respective rocker joints actuates the control valves in a predetermined manner different from the predetermined manner of the first control pattern configuration. Finally, identifying whether the first control assembly is in either of the first or second control pattern configurations.
In another aspect of the present invention, a method of converting a control set from a first control pattern configuration to a second control pattern configuration is disclosed. The method of converting the control set comprises the steps of providing a first control assembly for actuating a first control valve. Connecting the first control assembly at a first predetermined location on a mounting member to establish a first predetermined position of the first control assembly that defines the first control pattern configuration. Movement of the first control assembly actuates the first control valve in a predetermined manner. Substantially disconnecting the first control assembly from the mounting member. Reconnecting the first control assembly to the mounting member at a second predetermined location to establish a second predetermined position of the first control assembly that defines the second control pattern configuration. Movement of the first control assembly actuates the first control valve in a predetermined manner different from the predetermined manner of the first control pattern configuration. Limiting connection of the first control assembly to either of the first or second locations on the mounting member.
The present invention is able to easily convert a control set from a first control pattern configuration to a second control pattern configuration without the need for additional components or complex modification.

Method of converting a control set to obtain various control pattern configurations


A magnetic recording medium and a method for manufacturing the same are provided, which provide high surface recording density and high recording/reading performance. The magnetic recording medium is manufactured by forming an intermediate protective layer between a continuous recording layer and a first mask layer, dividing the continuous recording layer to form divided recording elements, and then removing the first mask layer while leaving the intermediate protective layer on the top of the divided recording elements. Further, gaps between the divided recording elements are filled with a non-magnetic material before removing the first mask layer.

1. Field of the Invention
The present invention relates to a method for manufacturing a magnetic recording medium and to a magnetic recording medium.
2. Description of the Related Arts
In the field of magnetic recording media such as hard discs, the surface recording density has been increased remarkably by various technical improvements, for example, by making magnetic particles, which constitute the recording layer, finer, changing materials to more effective ones, sophisticating the head processing, and so on, and further improvement of the surface recording density is still expected.
However, the improvement of the surface recording density by such conventional methods as making magnetic particles finer has already reached the limit. A discrete type magnetic recording medium is now proposed as a novel magnetic recording medium expected to be capable of further improving the surface recording density. The discrete type magnetic recording medium is produced by dividing a continuous recording layer into a number of divided recording elements, and filling the gaps between these divided recording elements with a non-magnetic material (see Japanese Patent Laid-Open Publication No. Hei 9-97419, for example).
An example of processing techniques capable of achieving minute division of a continuous recording layer is a dry etching process, such as a reactive ion etching process in which CO (carbon monoxide) gas with a nitrogen based compound gas such as NH3 (ammonia) gas added is used as the reactive gas (see Japanese Patent Laid-Open Publication No. H 12-322710, for example). More specifically, a mask layer is formed in a predetermined pattern on the surface of a continuous recording layer, removing the part of the continuous recording layer exposed from the mask layer by the aforementioned dry etching process using CO gas and so on as a reactive gas, and thereby dividing the continuous recording layer into a number of divided recording elements.
The mask layer left on the divided recording elements can be removed by a reactive ion etching process or the like using a chemically active gas as a reactive gas. The chemically active gas may be a gas commonly used in the field of semiconductor manufacturing, for example, fluorine-based gas such as SF6 (sulfur hexafluoride), CF4 (carbon tetrafluoride), NF3 (nitrogen trifluoride), or CHF3 (fluoroform), and chlorine-based gas such as Cl2 (chlorine), BCl3 (boron trichloride), or CHCl3 (chloroform).
In order to achieve stable flying of head, it is preferable to process and flatten the surfaces of a non-magnetic material and divided recording elements so that the surface roughness is limited to a certain degree. Processing techniques used in the field of semiconductor manufacturing, such as wet-process CMP (Chemical Mechanical Polishing) may be used for such flattening processing.
However, when the mask layer is removed from the divided recording elements using a reactive gas with strong chemical activity such as SF6 or CF4, a region around the top face and side face of the divided recording elements is apt to be subjected to deterioration such as oxidation, corrosion, or the like.
Also when a CMP process or the like is used for processing the surface of the divided recording elements and non-magnetic material, regions around the top face and side face of the divided recording elements are apt to be chemically and physically influenced by slurry or the like, and are often subjected to deterioration such as corrosion.
Further, such deterioration of the divided recording elements may occur with time after the processing.
Such deterioration of the divided recording elements may deteriorate the magnetic properties and hence decrease the recording/reading performance of the magnetic recording medium.
In other words, since magnetic recording media have peculiar problems such as their magnetic material being prone to oxidize and so on, it has been difficult to manufacture a discrete type magnetic recording media affording high recording/reading performance while reliably preventing the deterioration of divided recording elements, by directly applying a conventional processing method, that might be very effective in the field of semiconductor manufacturing.

Method for manufacturing magnetic recording medium and magnetic recording medium


A jaw horse apparatus and method for using the same to support materials. The apparatus includes an adjustable bi-leg assembly which has a set of jaws which engage with a main elongated portion of a uni-leg assembly to form a three leg support. The main portion has an aperture which receives a rod holding an elongated head piece that has an adjustable hinge. An extension arm attaches to the leg of either the uni-leg or bi-leg assembly. The head piece and extension arm may be combined with head pieces on other jaw horse apparatuses or may be used separately to support various materials. The head piece may be a roller type, ball head type or a flat drilled head type, depending on the use of the apparatus. The apparatus when not in use may be folded up into a flat package.

This invention relates to a method and apparatus for supporting materials and more particularly to a tri-leg jaw horse which supports objects during construction.
Saw horses have been used in construction sites for resting material thereon so that the material can be cut and drilled. Conventional saw horses have two pairs of downward extending legs that attach to a main member extending across between the pairs. Horses are placed adjacent each other with flat boards resting on the main members. These flat boards create a working surface on which material is then laid.
A drawback to these horses is that they are bulky and take up a lot of room. Consequently horses cannot easily fold up to be stored. Further these horses typically have four legs. Thus when the horses are placed on uneven surfaces the working surface tends to rock. Another drawback to horses is that their maximum height is limited as their legs are fixed in length. Consequently the horses cannot be quickly and easily adapted to positioning material thereon at different height levels.
The angle of prior horses main member is fixed with respect to horizontal. It is often desirable to change the angle of the working surface to accommodate drilling or other construction tasks. A requirement in construction is to be able to use these horses to position materials at different levels so that they can be easily worked on. Prior horses have a fixed height and thus cannot accommodate working with materials placed at various levels.
Another requirement is that once the materials are placed on the saw horse and the construction on the materials completed, these materials are removed and replaced with new material. Prior horses have a rigid main member that restricts the movement of the material when laid thereon. Consequently it is often difficult to remove the completed material from the horse itself. Another objective in construction is to place material at different levels simultaneously so that different tasks can be accomplished simultaneously. Prior horses restrict the material to working at one level at a time unless many different sized horses must be used. Adding a large number of different size horses increases space requirement which is often limited on construction sites.


A CCD area sensor comprising two horizontal transfer registers and a charge discharging section comprising a sweep-out electrode adjacent to the side of a horizontal register opposite to an image section and drain section, wherein the horizontal transfer register has a multi-channel structure comprising two transfer channels and a distribution electrode.

1. Field of the Invention
The present invention relates generally to the field of solid-state image sensing devices, and more particularly, the present invention relates to an improved solid-state image sensing device and method of processing image information.
2. Description of the Related Art
Some solid-state image sensing devices, such as, for example CCD area sensors, have a structure that includes a drain section 103 for discharging signal charge and a sweep-out electrode 104 as shown in FIG. 13. These structures are provided on the side opposite an image section 101 of a horizontal transfer register 102. Application of a sweep out clock .phi.HOG to the sweep-out electrode 104 for a given period, sweeps out the signal charges in the horizontal transfer register 102 to the drain section 103.
On the other hand, some CCD area sensors generally have a horizontal transfer register 102 with a wide channel width for increasing the capacity for involved charges because the pitch in the horizontal direction of the horizontal transfer register 102 is inevitably determined by the number of pixels and the optical system. In particular, the charge capacity of the horizontal transfer register 102 is proportional to the area per 1 bit under the condition of constant amplitude of the transfer clock, and therefore the charge capacity can be increased by increasing the length and width for one bit. However, the length is limited by the number of pixels of the image section 101 and only the channel width can therefore be increased.
In a CCD area sensor provided with a horizontal transfer register 102 with a wide channel width, if the structure for selectively sweeping out the signal charge in the horizontal transfer register 102 as described herein above is applied, it has been difficult to completely sweep out the signal charge within a predetermined time because the transfer electric field from the horizontal transfer register 102 to the drain section 103 is weak, particularly the transfer electric field at the middle portion in the width of the channel is weak when the signal charge in the horizontal transfer register 102 is intended to be swept out to the drain section 103 through the sweep-out electrode 104. In order to sweep out the signal charge in the horizontal transfer register 102, it takes a long time for sweeping out charges, and high speed sweeping out has been difficult to achieve.
This is true not only for a CCD area sensor having one horizontal transfer register 102 but also for a CCD area sensor having a structure which includes two or more horizontal transfer registers such as registers 102a and 102b shown in FIG. 14. This structure is provided with a transfer electrode 105 between both registers 102a and 102b in order to independently horizontally transfer and output the signal charge of two vertical lines for one horizontal period. A drain section 103 and sweep-out electrode 104 are provided adjacent to the outside horizontal transfer register 102b in order to selectively sweep out the signal charge in the horizontal transfer registers 102a and 102b.
As described herein above, the CCD area sensor having two horizontal transfer registers 102a and 102b can independently read the signal charge of all pixels of the image section (so-called all pixel reading) by independently reading signal charge of two vertical lines for one horizontal period. Therefore, the vertical resolution is improved in comparison with a device that reads signal charges for two vertical lines which are mixed. For a CCD area sensor of this sort, it is inevitable that the channel width of each is widened in order to increase the charge capacity of the horizontal transfer registers 102a and 102b. Thus the residual transfer of signal charge due to the decrease of the transfer electric field is a problem, as is true for a CCD area sensor having one horizontal transfer register 102.
This invention is accomplished in view of the above-mentioned problem. One object of the present invention to provide a solid-state image sensing device capable of completely sweeping out the signal charge in a horizontal transfer register and for sweeping out the signal charge to a drain section. Another object of the present invention is to provide a device which is capable of high speed sweeping out operation and a method for driving thereof. Yet another object of the present invention is to provide a device and method of processing CCD video information wherein only a portion of the complete image is initially processed at a higher speed and the remaining portion of the image is stored and later processed or processed initially at a slower speed. Other objects and advantages of the present invention will be apparent from the following summary and detailed description of the invention when viewed in light of the drawings.

Solid-State image sensing device, method for driving thereof and camera employing the same


The present invention is directed to preparations of birdseed treated with capsaicin or capsaicin derivatives or analogues thereof in an amount sufficient to be unpalatable to animals having capsaicin sensitive receptors, and more specifically to mammals such as rodents. These "hot" compounds, extracts or whole plant material containing these compounds may be coated on, impregnated in or combined (e.g., mixed) with birdseed to repel troublesome mammals which recognize these compounds as "hot". These "hot" compounds, in contrast, will not repel birds because birds do not recognize these compounds as "hot" since they do not have capsaicin sensitive receptors. The invention is further directed to a method of selectively repelling animals having capsaicin sensitive receptors, which comprises feeding the treated birdseed of the invention to birds, in an amount effective for repelling animals having capsaicin sensitive receptors, thereby discouraging said animals from eating the treated birdseed.

1. Field of the Invention
The present invention relates to preparations of bird seed treated with a material containing capsaicin or capsaicin derivatives or analogues thereof (including resiniferatoxin (RTX) or RTX analogues) in an amount sufficient to be unpalatable to mammals (e.g., rodents), which have capsaicin receptors.
2. Description of Related Art
U.S. Pat. No. 779,634 by Allen discloses a poison composition for the extermination of rodents which are destructive to crops. The poison comprises one pound of cayenne pepper, three (3) gallons of bisulfid of carbon, and one gallon of carbolic acid. A piece of absorbent material is saturated with the preparation and placed in locations frequented by unwanted rodents.
U.S. Pat. No. 631,738 by Dowie discloses a powdered composition for expelling rats consisting of twenty percent chili pepper, five percent hellebore, eight percent sulphate of lime, eight percent phosphate of lime, fifty four percent carbonate of lime and five percent oxide of iron. The powdered composition is intended to be sprinkled on the premises where rats are troublesome, and as a result of its intense irritating and burning qualities effectually prevents the rats from again invading places where the composition has been used. The hellebore and pepper are mixed with a medium to form a very light powder so as to float on the air and permeate the areas where the rats may be. It is said to be impossible to use hellebore without the pepper as it would be unbearable to human beings distributing it.
U.S. Pat. No. 4,455,304 by Yaralian discloses a method and composition for repelling birds from crops by applying the composition in the vicinity of the crop to be treated. The composition comprises finely divided garlic and finely divided dried cayenne pepper, the garlic being present in at least about one-fifth to at least five times the weight of the pepper, dry weight, and the total amount of garlic and pepper applied being from at least about 0.37 to 3.7 kilograms per hectare.
Szolcsanyi et al, Pain, 27 (1986) 247-260, discloses the sensitivity of birds to the nociceptive action of capsaicin differs from that of mammals. This was investigated by local instillation of capsaicin into the eye and by close arterial injection in pigeons and guinea-pigs. More specifically, instillation of capsaicin into the eyes of the pigeons did not evoke more reactions than the solvent alone. Thus, pigeons were found to reveal selective insensitivity to capsaicin and the peripheral chemoreceptive nerve endings in pigeons are almost totally insensitive to capsaicin.
Capsaicin and its analogues have been commercially used as repellents, such as in HALT, an aerosol preparation sold to repel attacking dogs.
Monsereenvsorn et al (1982) CRC Crit. Rev. Toxicol. 10, 321-339, discloses the physical and chemical properties of capsaicin.
Rozin, P. and Schiller, D., "The nature and acquisition of a preference for chili pepper by humans", Motiv. Emot. 4, 77-101, 1980, teaches that the maximum tolerated dose of chili pepper for humans is between 1,300 Scoville units (1 part in 1.25.times.10.sup.4 of capsaicin) and 10,400 Scoville units (1 part in 1.5.times.10.sup.3 of capsaicin).
Rozin, P. and Gruss, L., and Berk, G., "Reversal of innate aversions: attempts to induce a preference for chili peppers in rats", J. Comp. Physio. Psychol. 93, 1001-1014, 1979, teaches that incorporation of chili pepper in the feed of rats at a level of 400 Scoville units (corresponding to 1 part capsaicin in 40,000) causes 95% rejection of the treated food relative to untreated food. The report further teaches that the geometric mean for the avoidance threshold is 1 part capsaicin in 1.5.times.10.sup.6 and that all animals (out of 12 tested) avoided the treated feed at an avoidance threshold of 1 part capsaicin in 25,000 in the feed.
Sann, H., Harti, G., Pierau, F. K., and Simon, E., "Effect of capsaicin upon afferent and efferent mechanism of nociception and temperature regulation in birds", Can. J. Physiol. Pharmacol. 65: 1347-1354, 1987, teaches that the acute capsaicin effects on nociception in chickens upon close arterial injection were only observed at a dose two to three orders of magnitude higher than that in mammals.
Kare, M. R., "The special senses" in Avian Physiology, P. D. Sturkie, Ed., Cornell University Press, Ithaca, N.Y., 1965, pp. 406-446, teaches that parrots willingly eat hot peppers.

Treated bird seed preferentially palatable to birds but not to animals

The United States of America as represented by the Department of Health and Human Services


A composite marine piling is provided comprising (a) a core element comprising filler and a thermoplastic resin and (b) a sheath comprising thermoplastic resin and a light absorbing/emitting material. The filled thermoplastic composition of the core element is melt processed (for example injection or compression molded) to produce the filled thermoplastic composite marine piling core element. The sheath covers top of the core element to provide the top portion of the piling with a light responsive sheath to enhance the visibility of the piling to ship operators. The piling is a useful replacement for wood pilings and has several physical property advantages and preferably being suitable made from recycled plastic and waste byproduct filler.

1. Field of the Invention
The present invention relates to protective pilings and processes for making thereof, and more particularly relates to pilings and processes for making thereof.
2. Description of the Related Art
In the shipping industry, the ships (barges) traveling on narrow waterways such as rivers and bays have need to pass under bridges and adjacent to bridge supports and other structures. Historically, these bridge supports have been damaged by accidental ramming and rubbing by heavily loaded ships and barges resulting in undesired damage to the bridges thereby reducing the useful lives of such bridges and structure, and such ships have also been damaged as a result of such undesired contact with the structures. Prior efforts to protect bridges and structure have included the use of protective outer wood pilings which are placed around the structure and banded or bolted together to provide a protective outer layer. The use of marine fenders has been disclosed in various patents, see for example Young U.S. Pat. No. 4,281,610 issued Aug. 4, 1981 which discloses a marine fender comprising a sheet of resilient material and an elastomeric core; Nill U.S. Pat. No. 5,037,242 which discloses a dock fender made of PVC pipe; Matthews U.S. Pat. No. 3,950,953 issued Apr. 20, 1976 which discloses a piling fender; Smath U.S. Pat. No. 4,804,296 issued Feb. 14, 1989 which discloses a floatable marine fender device; James U.S. Pat. No. 5,007,363 issued Apr. 16, 1991 which discloses a buoyant marine fender; Watkins U.S. Pat. No. 5,013,272 issued May 7, 1991 which discloses a boat fender made of flexible plastic and having a hollow interior; Stevens U.S. Pat. No. 5,018,471 issued May 28, 1991 which discloses a marine fender for pilings of marine structures, all of which are incorporated herein by reference. These various prior fenders exhibit one or more of the following problems: (a) they are not easily visible at night (low light conditions) resulting in them not providing warning to the ship (barge) approaching the structure and/or (b) they exhibit floating characteristics, and other characteristics, which make it difficult to use them as pilings or as mostly submerge fenders.
Consequently, there is a need and a desire to provide pilings which exhibit certain improved characteristics such as improved visibility and improved density characteristics.

Thermoplastic photoluminescent pilings and process for making thereof


A film article release machine has two drive wheels, a conveyer chain surrounds the drive wheels and is driven by the drive wheels, and a plurality of release devices disposed on the conveyer chain. A guide plate is disposed above the conveyer chain. A main chain is disposed above the guide plate. A plurality of hand-shaped molds are disposed on the main chain. Each hand-shaped mold is dipped into a raw material solution such as a PVC solution. After the hand-shaped mold is dried, a film article is formed. A rolling wheel is connected to each hand-shaped mold. A brush wheel brushes the film article to roll up a portion of the film article to form a roll-up portion. Then the film article is blown by a blowing pipe to blow the roll-up portion of the film article to the original position. The release devices clamp the film articles and release the film articles from the hand-shaped molds.

The present invention relates to a film article release machine. More particularly, the present invention relates to a film article release machine which can release a plurality of film articles easily.
A conventional film article release machine comprises a very complex release device in order to release a plurality of film articles, a very complex clamp device to be driven by a screw rod, and a dirve mechanism to drive the screw rod. Since the dirve mechanism is controlled by an electric circuit, the whole structure should be very complex. It will consume a lot of electricity to rotate the heavy and complex release device. Furthermore, the film articles are released at random. Therefore, the film articles cannot be arranged in order automatically.


A depilatory cream composition comprising a depilatory active, and one or more volatile, essential oils selected from the group consisting of orange oil, cedarwood oil, patchouli oil, pine oil, cabreuva oil, guaiacwood oil, lavandin oil, bergamot oil, petitgrain paraguay oil, lavender oil, mint oil, Virginia cedarwood oil, Citrus Aurantium dulcis oil and Citrus grandis oil.

Depilatory creams for removing unwanted hair are known. Such creams typically include a depilatory active that degrades hair keratin.
Typically, the active agent in depilatory creams is based on a sulphur-containing compound such as potassium thioglycolate. An unpleasant odour is commonly associated with this type of compound. The depilatory active achieves its effect by weakening the hair. In doing so the depilatory active acts as reducing which results in the production of volatile sulphur-containing compounds. These sulphur-containing compounds also have an unpleasant odour.
Furthermore, In use the depilation cream is applied to a user's skin and left for a period of up to 10 mins while the active achieves its effect.
It would, therefore, be desirable to use a depilatory cream which avoids the unpleasant odours of creams currently available.


A method comprising encoding a plurality of signals according to a predetermined negation scheme and transmitting the plurality of signals, wherein each signal is transmitted by way of a wireless channel. The method further comprises receiving a signal, wherein the received signal is a combination of the plurality of transmitted signals, and interpolating between data in the received signal to generate a plurality of systems of equations. The method further comprises solving the plurality of systems of equations to determine a gain and phase shift applied to each of the plurality of transmitted signals by a corresponding wireless channel.

Wireless local area networks ("WLAN") allow electronic devices, such as computers, to have network connectivity without the use of wires. Network connections may be established via, for example, radio signals. A wireless access point ("AP") may comprise a wired Internet or Ethernet connection and radio communication circuitry capable of transmitting data to and receiving data from any compatible wireless device. The AP may provide Internet and/or network connectivity to such wireless devices (e.g., portable computers) called receiver stations ("STA") by transmitting and receiving data via radio signals.
Architects of WLAN systems and devices must take various factors into account. One such factor is multipath interference. In multipath interference, a signal transmitted from a source (e.g., an AP) may take multiple paths through a wireless medium and thus reach the intended destination as more than one version of the same signal. FIG. 1a illustrates this phenomenon, in which a signal from an AP is transmitted directly to a STA and also bounces off the walls 10, 12 before reaching the STA. The lengths of the different paths may vary, thereby causing a phase/time difference in the received signals. Accordingly, multipath interference may cause distortion in the signal. Thus, the signal received by the STA may be a distorted version of the signal that was originally transmitted by the AP. The technique of "channel estimation" may be implemented in a STA or AP receiver to eliminate such distortion and generate a version of the signal which is nearly identical to the signal that was originally transmitted by the AP.
Channel estimation comprises transmitting a predetermined signal (described below) from a transmitter to a receiver, where the transmitted predetermined signal is known to both the transmitter and the receiver prior to transmission. Due to multipath interference, the predetermined signal received by the receiver will generally be different from the predetermined signal transmitted by the transmitter. Upon receiving the signal, the receiver may compare the received signal to the transmitted signal to determine how multipath interference has distorted the signal. The receiver may use such information to synchronize the receiver to the transmitter(s) and eliminate distortion present in future received signals.
A signal used specifically for channel estimation comprises a preamble. A preamble comprises, among other things, a short sequence of data and a long sequence of data. The short sequence may be used to perform basic synchronization, including determining whether a packet is en route to the receiver, estimating frequency offset, and other various synchronization operations. The long sequence is the sequence actually used in channel estimation. Standard IEEE 802.11 protocols, such as 802.11a, comprise long sequence designs that enable channel estimation for standard single input, single output ("SISO") systems. Thus, in a system comprising a single transmitter and a single receiver, the receiver is able to successfully estimate the channel between the transmitter and the receiver, thereby eliminating distortions present in a received signal. However, multiple-input, multiple-output ("MIMO") signaling systems comprising a plurality of transmitters and receivers present unique problems for existing channel estimation techniques.
In a MIMO system, the rate at which data is transferred ("data rate") between a transmitter and a receiver may be raised by increasing the number of antennas associated with each wireless device in the system. For instance, a system comprising a transmitter with multiple antennas and a receiver with multiple antennas may have a higher data rate than a system comprising a transmitter with a single antenna and a receiver with a single antenna. The MIMO antennas are part of a design that attempts to achieve a linear increase in data rate as the number of transmitting and receiving antennas linearly increases.
MIMO systems present unique problems for existing channel estimation techniques due to difficulties introduced by signal overlapping, wherein a receiver receives a mixture of signals instead of a single signal. For example, in a system comprising two transmitters and two receivers, each receiver receives a signal that is a combination of the signals transmitted by each of the transmitters. In order to estimate the four channels (i.e., one channel from a first transmitter to a first receiver, a second channel from a first transmitter to a second receiver, a third channel from a second transmitter to a first receiver, a fourth channel from a second transmitter to a second receiver), a receiver must mathematically analyze the received signal to determine a plurality of equations describing distortion imparted by each channel on a signal transmitted through the channel. Each receiver then may successfully estimate all four channels. Channel estimation information subsequently may be used by a receiver to eliminate distortion present in future signals. Thus, a technique to separate mixed signals and eliminate signal distortion in MIMO systems is desirable.

Scalable and backwards compatible preamble for OFDM systems