A transmission apparatus switches to a line of one standby system upon occurrence of a fault on any one of n lines of a working system. The transmission apparatus includes a switching controller that when switching from a line of the working system to a line of the standby-system upon the occurrence of the fault and executing a given command to put the line causing the fault in a given state of line switching according to the given command, causes the working system to maintain the state of line switching according to the given command even after restoration from the fault.

Conventional transmission systems include a system that uses a transmission apparatus having a 1-to-n redundant configuration in which one station is connected to another station through normally used n working-system transmission paths and one standby-system transmission path. When a fault occurs on a working-system path (transmission path, unit, or line), the transmission apparatus switches from the working-system line (working line) to a standby-system line (protection line). An optical signal or electrical signal is transmitted through the transmission path.
1-to-1 or 1-to-n line switching carried out upon the occurrence of a fault is defined in the communication standard provided in GR-253 and ITU-T Standard G. 841. In line switching, a fault countermeasure command is transmitted and received between a monitoring device and the transmission apparatus, and 1-to-1 or 1-to-n line switching is carried out. Switching information is defined as such a command in K1 and K2 bytes included in the header of a transmission frame. When a fault occurs on the working system, the K1 and K2 bytes representing switching information are transmitted and received between one transmission apparatus and another transmission apparatus, using a frame transmitted through the standby-system line.
FIG. 29 is a diagram of a definition of K1 and K2 bytes in conventional line switching. In the K1 byte, four bits (bits 1-4) define a switching request, while the other four bits (bits 5-8) define a switch request channel (switch request channel number). In the K2 byte, four bits (bits 1-4) define information of a channel switched to a standby-system channel (bridging channel number), one bit (bit 5) defines the line switching method (architecture), and three bits (bits 6-8) define the mode.
In the diagram, switching requests defined by the bits 1-4 of the K1 byte are listed in descending order. Line switching is thus carried out according to the switching request highest in priority, i.e., highest on the list. For example, Forced Switch represented by the value of the bits 1-4 "1110" is higher in priority than Manual Switch represented by "1000".
Among conventional techniques for carrying out line switching according to such a definition is a technique in which a standby-system line is allocated dynamically in a standby system in a 1-to-n redundant configuration to prevent instantaneous disconnection that occurs in a switchback process ensuing restoration from a fault to enable long hours of operation using a standby-system transmission path. The technique provides a configuration such that a standby-system line is not established statically upon the occurrence of a fault and such that a transmission path used as a standby-system path is determined dynamically based on the quality and priority of a transmission path at the time of the occurrence of a fault and restoration from the fault (see, e.g., Japanese Patent Application Laid-Open Publication No. 2001-339370).
FIG. 30 is a diagram of state transition that is made in response to a switching request based on a conventional definition. FIG. 30 depicts transition between different states, with attention being focused on switching in slots (units or lines) of the station. For convenience, only some of defined states of FIG. 29 are depicted in FIG. 30. The depicted states include No Request P1, Manual Switch P2, Signal Fail (SF) P3, and Forced Switch P4. Signal Degrade (SD), etc., is also included as a defined state.
In a state of Manual Switch P2, redundant path switching is executed automatically, irrespective of a line switching instruction by a maintenance person when a path error rate exceeds a threshold for the bit error rate specified for a state of SD or when the path enters a state of SF. In contrast, in a state of Forced Switch P4, even when the path enters the state of SD, where the path error rate is higher than the threshold for the bit error rate specified in the state of SD, or enters the state of SF, automatic redundant switching in response to such transition is not executed consequent to the Forced Switch having a higher priority than SD and SF (P4), as depicted in FIG. 29.
A problem arises, however, when removal or a fault of a unit of the transmission apparatus, or a line fault, such as the disconnection of a cable from the unit, occurs after transition to the state of Manual Switch P2 defined in FIG. 30. This is a situation, for example, where such a fault occurs when the maintenance person has set the unit to the state of Manual Switch for a given reason. This situation is equivalent to a condition for a state having a higher priority than the state of Manual Switch P2 (SF (High) P3 in FIG. 30), resulting in transition p2 (depicted in FIG. 30) to automatically clear the state of Manual Switch P2 and make transition to the state of SF (High) P3 (depicted in FIG. 30). This consequently means that the state of Manual Switch set by the maintenance person changes into another state without being noticed by the maintenance person.
After the occurrence of such a fault, when the maintenance person carries out restoration work, such as replacement of the unit, a Small Form Factor Pluggable (SFP), which is equivalent to a port and serves as an optical transmitting/receiving module mounted on the unit, an optical fiber, etc., and mounts or connects a new unit, SFP, cable, etc., the corresponding path abruptly switches back from a standby-system line to a working-system line. At this time, the maintenance person is not able to check the switchback or errors, leading to a problem in that if trouble occurs with a newly mounted unit or cable, signal disconnection occurs.
To prevent such a problem, the maintenance person may set the unit in the state of Forced Switch P4 rather than the state of Manual Switch. Although the state of Forced Switch P4 has a high priority, this state is incapable of relieving a different unit or line from a fault and may cause serious trouble if the maintenance person errantly forgets to clear the state of Forced Switch P4. It is desirable, therefore, for the state of Forced Switch P4 to not be used if possible. In this manner, dealing with a specific event may be difficult if the conventional definition of states alone is applied.


A non-reclosing overpressure protection device such as a rupture disc provides a non-reclosing opening upon forcible contact with a knife blade. A bellows, having an inlet capable of being sealably connected to a source of pressure (the vacuum system) and an outlet containing the rupture disc, transmits the pressure in the system to the disc. The bellows maintains the disc away from the knife when the pressure is below an overpressure amount, and carries the disc to a position when the pressure is above an overpressure amount where the disc is ruptured by the knife.

This invention is directed to the field of overpressure relief devices for vacuum systems, and, more particularly, to overpressure devices that rupture when a vacuum system is pressurized.
A typical vacuum system easily tolerates a higher pressure on the outside than the inside (negative pressure), but is susceptible to damage when there is a higher pressure on the inside than the outside (positive pressure). In many systems, even a small amount of positive pressure may cause damage to expensive equipment, such as manipulators, connected to a vacuum system. Accordingly, where possible vacuum systems often incorporate pressure relief devices to vent the system if the internal pressure exceeds a predetermined amount, which amount may be as little as 1 psig.
Overpressure for vacuum systems is most likely to occur when systems are connected to an external source of gas used for known reasons such as purging, chemical vapor deposition, or sputter cleaning. Some internal gas sources, such as cryopumps and uranium hydride beds, can also release enough gas to overpressurize a vacuum system.
An effective pressure relief device for a vacuum system must meet several criteria. Most importantly, it must be able to resist the negative pressure the system is designed to undergo during normal vacuum operations. On the other hand, without any operator interaction, it must also reliably vent the system when the positive pressure exceeds a predetermined value. The device must also be impervious to corrosive chemicals which may be used within the system.
U. S. Pat. No. 4,512,419 of DeGood et al. discloses a rupture disc, one conventional pressure relief device. The design principle employed in a rupture disc to ensure predicable bursting pressures is simply a tension failure of the single metal membrane of the disc, the overpressure escaping through the ruptured disc. The design problem for a rupture disc is making it strong enough to resist negative pressure, and weak enough to fail at a predetermined positive pressure.
The reverse acting rupture disk is another overpressure device where a diaphragm has a bulging surface pulled by the negative pressure towards the interior of the vacuum system. In the event of overpressure, the bulging surface is pushed outwardly, where it is pierced by a knife blade, causing the diaphragm to rupture.
A problem with rupture discs is that the size of the disc varies inversely with the rupture pressure. For example, one commercially available disc has a diameter of 24" to provide 4 PSIG overpressure protection. The large size and cost of such a disc limits its usefulness in many applications.

Non-reclosing pressure relief device for vacuum systems


Methods and compositions are provided for analyzing differences in the RNA profiles between a plurality of different physiological samples. In the subject methods, a set of a representational number of distinct gene specific primers is used to generate labeled nucleic acids from each of the different physiological samples. The labeled nucleic acids are then compared to each other and differences in the RNA profiles are determined. The subject methods find use in methods of identifying differential gene expression.

1. Technical Field
The technical field of this invention is the analysis of differential gene expression.
2. Background
In higher organisms, any given cell expresses only a fraction of the total number of genes present in its genome. The small fraction of the total number of genes that is expressed determine the life processes carried out by the cell, e.g. development and differentiation, homeostasis, response to insults, cell cycle regulation, aging, apoptosis, and the like. Alterations in gene expression decide the course of normal cell development and the appearance of diseased states, such as cancer. Because the choice of which genes are expressed has such a profound effect on the nature of any given cell, methods of analyzing gene expression are of critical import to basic molecular biological research. Identification of differentially-expressed genes can provide a key to diagnosis, prognosis and treatment of a variety of diseases or condition states in animals, including humans, and plants. Additionally, these methods can be used to identify differentially-expressed sequences due to changes in gene expression level associated with predisposition to disease, influence of external treatments, factors or infectious agents. Identification of such genes helps in development of new drugs and diagnostic methods for treating or preventing the occurrence of such diseases.
One way of analyzing gene expression in a particular cell is to perform differential gene expression assays, in which the expression of genes in different cells is compared and any discrepancies in expression are identified, where the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
One method currently employed to identify differentially expressed genes begins with the generation of cDNA xe2x80x9ctargetsxe2x80x9d obtained from analogous cells, tissues or organs of a healthy and diseased organism. The cDNA targets are then hybridized to a set of target nucleic acid xe2x80x9cprobexe2x80x9d fragments immobilized on membrane. Differences between he resultant hybridization patterns are then detected and related to differences in gene expression in the two sources. In this procedure the number of analyzed gene-specific probes can reach several hundred thousand.
Modifications have been made to the above basic method in order to obtain improved results. These modifications include replacement of the traditional radioactive labeling procedure of the target nucleic acid sequences with nonisotopic labels, mainly fluorescent labels. Other modifications have focused on improved methods of immobilization of an array of the probe nucleic acids to surfaces of a variety of solid supports.
Despite the promise of analysis of differential expression using arrays of probes on solid supports, there is a continuing need for improvement of the methods currently employed by researchers. In current methods, hybridization of xe2x80x9ctargetxe2x80x9d to xe2x80x9cprobexe2x80x9d is slow. Furthermore, a number of additional events such as competitive hybridization events between distinct target sequences, nonspecific binding between xe2x80x9ctargetxe2x80x9d and xe2x80x9cprobe,xe2x80x9d and formation of secondary structures in target sequences can occur which adversely effect the results.
Accordingly, there is continued interest in the development of new methods of analyzing differential gene expression, where such methods provide for fast hybridization and high specificity of binding of xe2x80x9ctargetsxe2x80x9d to xe2x80x9cprobes.xe2x80x9d
Patents of interest include: EP 0 328 829 B1 and U.S. Pat. Nos. 5,468,613; 5,580,726; 5,599,672; 5,512,462; 5,162,209 and 5,162,209. Methods of analyzing differential gene expression are also described in Maniatis, et al., Molecular Cloning, A Laboratory Manual, (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)(1989); Nucleic Acid Hybridization, A Practical Approach (Hames, B. D., and Higgins, S. J. eds, IRL Press, Oxford)(1985); WO 95/21944; Chalifour, et al., Anal. Biochem. (1994) 216: 299-304; Nguyen et al., Genomics (1995) 29: 207-216; Pietu et al., Genome Res. (1996) 6: 492-503; and Zhao et al., Gene (1995) 166: 207-213.
Use of non-isotopic labels in methods of differential gene expression analysis are described in: Schena et al. Science (1995) 270: 467-470; Schena et al., Proc. Natl. Acad. Sci. USA (1996) 93: 10614-10619; DeRisi et al., Nature Genet. (1996) 14: 457-460; and Lockhart et al., Nature Biotechnol. (1996) 14: 1675-1680.
Methods of stably associating probes to the surface of substrates are described in: Hermanson, et al. Immobilized Affinity Ligand Techniques, Academic Press, (1992); WO 89/11548; European Patent No. 0 281 390 B1; WO 88/01302; European Patent Application No. 0392546; U.S. Pat. No. 5,436,327; U.S. Pat. No. 5,445,934.
Methods of improving hybridization of target to substrate surface associated probe are described in: EP 0 318 245 B1 (solution hybridization of probe to target followed by binding of hybridization complex to surface of substrate); Lockhart et al., Nature Biotechnol. (1996) 14: 1675-1680, EP 0 328 829 B1 (preamplification of target DNA/RNA); Maniatis et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., (1989), Nucleic Acid Hybridization, A Practical Approach (Hames, B. D., and Higgins, S. J. eds. IRL Press, Oxford) (1985), EP 0 229 442 (addition of an inert polymers such as dextran sulfate); U.S. Pat. No. 5,387,510, EP 0 318 245 B1 (use of xe2x80x9chelperxe2x80x9d oligonucleotides which reorder secondary and tertiary structure of target polynucleotide); WO 89/11548 (attaching probes to surface of substrate through long spacer arms).
Methods of improving specificity of hybridization are described in: U.S. Pat. Nos. 5,449,603 and 5,547,843 (use of single stranded nucleic acid binding protein); U.S. Pat. Nos. 4,888,274 and 5,223,414, EP 0 481 065 B1 (use of RecA protein-coated nucleoprotein target molecules); Khrapko et al., FEBS Lett. (1989) 256: 118-122 and U.S. Pat. No. 5,503,980 (continuous stacking interaction between short oligonucleotides of target and probe molecules, followed by enzymatic ligation step); and U.S. Pat. No. 5,434,047 (use of non-target probe which hybridized with non-target nucleic acid).
Methods and compositions for identifying differences between the nucleic acid profiles of a plurality of biological samples are provided. In the subject methods, a set of a representational number of different gene specific primers is used to generate labeled target nucleic acids from samples of nucleic acids, usually ribonucleic acids, derived from at least two different physiological sources. The labeled target nucleic acids derived from each physiological source are then compared, preferably by hybridization to arrays of probe nucleic acids stably associated with the surface of a substrate. The subject methods find use in differential gene expression analysis.
Methods and compositions are provided for analyzing differences in the ribonucleic acid profiles between two or more physiological sources. In the subject methods, a set of a representational number of gene specific primers is used to generate labeled target nucleic acids from the physiological sources. The labeled target nucleic acids from each of the samples are then compared, preferably by hybridizing the labeled target nucleic acids from each sample to an array of probe nucleic acids stably associated with the surface of a substrate. Also provided are sets of gene specific primers employed in the subject methods, as well as kits comprising the sets of gene specific primers. The subject methods find use in a variety of applications, including differential gene expression assays.
Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
It must be noted that as used in this specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
Critical to the subject invention is the use of a set (i.e. pool, mixture, collection) of a representational number of gene specific primers to generate labeled nucleic acids from a sample of nucleic acids, usually ribonucleic acids (RNAs), where the labeled nucleic acids may act as xe2x80x9ctargetxe2x80x9d in subsequent hybridization assays, described in greater detail below. As used herein, the term nucleic acid is used in the broadest sense to refer to any sized multimer of nucleotide monomeric units, including short multimers such as dimers, trimers and the like, as well as longer multimers such as oligonucleotides and polynucleotides, where oligonucleotides generally denotes single stranded nucleotide multimers of from about 10 to 100 nucleotides and up to 200 nucleotides in length, and polynucleotides typically refers to single or double stranded nucleotide monomers of generally greater than 100 nucleotides in length.
As the subject sets comprise a representational number of primers, the total number of different primers in any given set will be only a fraction of the total number of different or distinct RNAs in the sample, where the total number of primers in the set will generally not exceed 80%, usually will not exceed 50% and more usually will not 20% of the total number of distinct RNAs, usually the total number of distinct messenger RNAs (mRNAs), in the sample. Any two given RNAs in a sample will be considered distinct or different if they comprise a stretch of at least 100 nucleotides in length in which the sequence similarity is less then 98%, as determined using the FASTA program (default settings). As the sets of gene specific primers comprise only a representational number of primers, with physiological sources comprising from 5,000 to 50,000 distinct RNAs, the number of different gene specific primers in the set of gene specific primers will typically range from about 20 to 10,000, usually from 50 to 2,000 and more usually from 75 to 1500.
Each of the gene specific primers of the sets described above will be of sufficient length to specifically hybridize to a distinct nucleic acid member of the sample, e.g. RNA or cDNA, where the length of the gene specific primers will usually be at least 8 nt, more usually at least 20 nt and may be as long as 25 nt or longer, but will usually not exceed 50 nt. The gene specific primers will be sufficiently specific to hybridize to complementary template sequence during the generation of labeled nucleic acids under conditions sufficient for first strand cDNA synthesis, which conditions are known by those of skill in the art. The number of mismatches between the gene specific primer sequences and their complementary template sequences to which they hybridize during the generation of labeled nucleic acids in the subject methods will generally not exceed 20%, usually will not exceed 10% and more usually will not exceed 5%, as determined by FASTA (default settings).
Generally, the sets of gene specific primers will comprise primers that correspond to at least 20, usually at least 50 and more usually at least 75 distinct genes as represented by distinct mRNAs in the sample, where the term xe2x80x9cdistinctxe2x80x9d when used to describe genes is as defined above, where any two genes are considered distinct if they comprise a stretch of at least 100 nt in their RNA coding regions in which the sequence similarity does not exceed 98%, as determined by FASTA (default settings).
The gene specific oligonucleotide primers may be synthesized by conventional oligonucleotide chemistry methods, where the nucleotide units may be: (a) solely nucleotides comprising the heterocyclic nitrogenous bases found in naturally occurring DNA and RNA, e.g. adenine, cytosine, guanine, thymine and uracil; (b) solely nucleotide analogs which are capable of base pairing under hybridization conditions in the course of DNA synthesis such that they function as the above nucleotides found in naturally occurring DNA and RNA, where illustrative nucleotide analogs include inosine, xanthine, hypoxanthine, 1,2-diaminopurine and the like; or (c) from combinations of the nucleotides of (a) and nucleotide analogs of (b), where with primers comprising a combination of nucleotides and analogues thereof, the number of nucleotide analogues in the primers will typically be less than 25 and more typically less than 5. The gene specific primers may comprise reporter or hapten groups, usually 1 to 2, which serve to improve hybridization properties and simplify detection procedure.
Depending on the particular point at which the gene specific primers are employed in the generation of the labeled nucleic acids, e.g during first strand eDNA synthesis or following one or more distinct amplification steps, each gene specific primer may correspond to a particular RNA by being complementary or similar, where similar usually means identical, to the particular RNA. For example, where the gene specific primers are employed in the synthesis of first strand cDNA, the gene specific primers will be complementary to regions of the RNAs to which they correspond.
Each gene specific primer can be complementary to a sequence of nucleotides which is unique in the population of nucleic acids, e.g. mRNAs, with which the primers are contacted, or one or more of the gene specific primers in the set may be complementary to several nucleic acids in a given population, e.g. multiple mRNAs, such that the gene specific primer generates labeled nucleic acid when one or more of set of related nucleic acid species, e.g. species having a conserved region to which the primer corresponds, are present in the sample. Examples of such related nucleic acid species include those comprising: repetitive sequences, such as Alu repeats, Al repeats and the like; homologous sequences in related members of a gene-family; polyadenylation signals; splicing signals; or arbitrary but conversed sequences.
The gene specific primers of the sets of primers according to the subject invention are typically chosen according to a number of different criteria. In some embodiments of the invention, primers of interest for inclusion in the set include primers corresponding to genes which are typically differentially expressed in different cell types, in disease states, in response to the influence of external agents, factors or infectious agents, and the like. In other embodiments, primers of interest are primers corresponding to genes which are expected to be, or already identified as being, differentially expressed in different cell, tissue or organism types. Preferably, at least 2 different gene functional classes will be represented in the sets of gene specific primers, where the number of different functional classes of genes represented in the primer sets will generally be at least 3, and will usually be at least 5. In other words, the sets of gene specific primers comprise nucleotide sequences complementary to RNA transcripts of at least 2 gene functional classes, usually at least 3 gene functional classes, and more usually at least 5 gene functional classes. Gene functional classes of interest include oncogenes; genes encoding tumor suppressors; genes encoding cell cycle regulators; stress response genes; genes encoding ion channel proteins; genes encoding transport proteins; genes encoding intracellular signal transduction modulator and effector factors; apoptosis related genes; DNA synthesis/recombination/repair genes; genes encoding transcription factors; genes encoding DNA-binding proteins; genes encoding receptors, including receptors for growth factors, chemokines, interleukins, interferons, hormones, neurotransmitters, cell surface antigens, cell adhesion molecules etc.; genes encoding cell-cell communication proteins, such as growth factors, cytokines, chemokines, interleukins, interferons, hormones etc.; and the like. Less preferred are gene specific primers that are subject to formation of strong secondary structures with less than xe2x88x9210 kcal/mol; comprise stretches of homopolymeric regions, usually more than 5 identical nucleotides; comprise more than 3 repetitive sequences; have high, e.g. more than 80%, or low, e.g. less than 30%, GC content etc.
The particular genes represented in the set of gene specific primers will necessarily depend on the nature of physiological source from which the RNAs to be analyzed are derived. For analysis of RNA profiles of eukaryotic physiological sources, the genes to which the gene specific primers correspond will usually be Class II genes which are transcribed into RNAs having 5xe2x80x2 caps, e.g. 7-methyl guanosine or 2,2,7-trimethylguanosine, where Class II genes of particular interest are those transcribed into cytoplasmic mRNA comprising a 7-methyl guanosine 5xe2x80x2 cap and a polyA tail.
For analysis of RNA profiles of mammalian physiological sources, of particular interest are gene specific primers corresponding to the functional gene classes listed above. For analysis of RNA profiles of human physiological sources, the gene specific primers of particular interest are the gene specific primers identified in Table 1 of application Ser. No. 08/859,008 filed May 21, 1997 (the disclosure of which is herein incorporated by reference) as SEQ ID NO:01 to SEQ ID NO:1372, where sets of these primers will usually include at least 20 and more usually at least 50 of these specific sequences. Also of interest are those gene specific primers corresponding to those genes (and specific capable of producing target capable of hybridizing to those specific regions of the genes) as listed in the following copending patent applications, the disclosures of which are herein incorporated by reference: U.S. application Ser. No. 09/221,480 entitled Apoptosis Array; U.S. application Ser. No. 09/222,432 entitled Cancer Array; U.S. application Ser. No. 09/222,436 entitled Cell Cycle Array; U.S. application Ser. No. 09/222,437 entitled Cell Interaction Array; U.S. application Ser. No. 09/222,251 entitled Cytokine Receptor Array; U.S. application Ser. No. 09/221,481 entitled Human Array; U.S. application Ser. No. 09/222,256 entitled Human Stress Array; U.S. application Ser. No. 09/222,248 entitled Mouse Array; and U.S. application Ser. No. 09/222,253 entitled Oncogene/Tumor Suppressor Array.
Depending on the particular nature of the labeled nucleic acid generation step of the subject methods, the gene specific primers may be modified in a variety of ways. One way the gene specific primers may be modified is to include an anchor sequence of nucleotides, where the anchor is usually located 5xe2x80x2 of the gene specific portion of the primer and ranges in length from 10 to 50 nt in length, usually 15 to 40 nt in length. The anchor sequence may comprise a sequence of bases which serves a variety of functions, such as a sequence of bases which correspond to the sequence found in promoters for bacteriophage RNA polymerase, e.g. T7 polymerase, T3 polymerase, SP6 polymerase, and the like; arbitrary sequences which can serve as subsequent primer binding sites; and the like.
Turning now to the methods employing the above sets of gene specific primers, the first step in the subject methods is to obtain a sample of nucleic acids, usually RNAs or nucleic acid derivatives thereof, from a physiological source, usually a plurality of physiological sources, where the term plurality is used to refer to 2 or more distinct physiological sources. The physiological source of RNAs will typically be eukaryotic, with physiological sources of interest including sources derived single celled organisms such as yeast and multicellular organisms, including plants and animals, particularly mammals, where the physiological sources from multicellular organisms may be derived from particular organs or tissues of the multicellular organism, or from isolated cells derived therefrom. Thus, the physiological sources may be different cells from different organisms of the same species, e.g. cells derived from different humans, or cells derived from the same human (or identical twins) such that the cells share a common genome, where such cells will usually be from different tissue types, including normal and diseased tissue types, e.g. neoplastic, cell types. In obtaining the sample of RNAs to be analyzed from the physiological source from which it is derived, the physiological source may be subjected to a number of different processing steps, where such processing steps might include tissue homogenation, nucleic acid extraction and the like, where such processing steps are known to the those of skill in the art. Methods of isolating RNA from cells, tissues, organs or whole organisms are known to those of skill in the art and are described in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press)(1989).
The next step in the subject methods is the generation of labeled nucleic acids representative of the nucleic acid, usually RNA, profile of the physiological source. As mentioned above, a set or pool of gene specific primers is used to generate the labeled nucleic acids from the sample of RNAs, where the labeled nucleic acids generated in this step may serve as xe2x80x9ctargetxe2x80x9d in subsequent assays in which the differences in the RNA profiles of at least two sources are analyzed. Since the subject sets or pools of primers are employed, a sub-population of nucleic acids is generated from the initial source, where the sub-population corresponds to only a portion or fraction of the initial nucleic acid source. As used herein, the term xe2x80x9ctargetxe2x80x9d refers to single stranded RNA, single stranded DNA and double stranded DNA, where the target is generally greater than 50 nt in length.
The set of primers may be used either in first strand cDNA synthesis or following one or more amplification steps. Furthermore, the actual synthesis of the labeled nucleic acids may be at the same step during which the sets of gene specific primers are employed, or the synthesis of the labeled nucleic acids may be one more steps subsequent to the step in which the sets of gene specific primers are employed.
In a first embodiment of the invention, the set of gene specific primers is used to generate labeled first strand cDNA, where the labeled first strand cDNA is representative of the RNA profile of the physiological source being assayed. The labeled first strand cDNA is prepared by contacting the RNA sample with the primer set and requisite reagents under conditions sufficient for hybrid duplexes (i.e. double stranded primer complexes) to be produced followed by reverse transcription of the RNA template in the sample. Requisite reagents contacted with the primers and RNAs are known to those of skill in the art and will generally include at least an enzyme having reverse transcriptase activity and dNTPs in an appropriate buffer medium.
A variety of enzymes, usually DNA polymerases, possessing reverse transcriptase activity can be used for the first strand cDNA synthesis step. Examples of suitable DNA polymerases include the DNA polymerases derived from organisms selected from the group consisting of a thermophilic bacteria and archaebacteria, retroviruses, yeasts, Neurosporas, Drosophilas, primates and rodents. Preferably, the DNA polymerase will be selected from the group consisting of Moloney murine leukemia virus (M-MLV) as described in U.S. Pat. No. 4,943,531 and M-MLV reverse transciptase lacking RNaseH activity as described in U.S. Pat. No. 5,405,776 (the disclosures of which patents are herein incorporated by reference), human T-cell leukemia virus type I (HTLV-I), bovine leukemia virus (BLV), Rous sarcoma virus (RSV), human immunodeficiency virus (HIV) and Thermus aquaticus (Taq) or Thermus thermophilus (Tth) as described in U.S. Pat. No. 5,322,770, the disclosure of which is herein incorporated by reference. Suitable DNA polymerases possessing reverse transcriptase activity may be isolated from an organism, obtained commercially or obtained from cells which express high levels of cloned genes encoding the polymerases by methods known to those of skill in the art, where the particular manner of obtaining the polymerase will be chosen based primarily on factors such as convenience, cost, availability and the like.
The various dNTPs and buffer medium necessary for first strand cDNA synthesis through reverse transcription of the primed RNAs may be purchased commercially from various sources, where such sources include Clontech, Sigma, Life Technologies, Amersham, Boehringer-Mannheim. Buffer mediums suitable for first strand synthesis will usually comprise buffering agents, usually in a concentration ranging from 10 to 100 xcexcM which typically support a pH in the range 6 to 9, such as Tris-HCl, HEPES-KOH, etc.; salts containing monovalent ions, such as KCl, NaCl, etc., at concentrations ranging from 0-200 mM; salts containing divalent cations like MgCl2, Mg(OAc) etc, at concentrations usually ranging from 1 to 10 mM; and additional reagents such as reducing agents, e.g. DDT, detergents, albumin and the like. The conditions of the reagent mixture will be selected to promote efficient first strand synthesis. Typically the set of primers will first be combined with the RNA sample at an elevated temperature, usually ranging from 50 to 95xc2x0 C., followed by a reduction in temperature to a range between about 0 to 60xc2x0 C., to ensure specific annealing of the primers to their corresponding RNAs in the sample. Following this annealing step, the primed RNAs are then combined with dNTPs and reverse transcriptase under conditions sufficient to promote reverse transcription and first strand cDNA synthesis of the primed RNAs. By using appropriate types of reagents, all of the reagents can be combined at once if the activity of the polymerase can be postponed or timed to start after annealing of the primer to the RNA.
In this embodiment, one of either the gene specific primers or dNTPs, preferably the dNTPs, will be labeled such that the synthesized cDNAs are labeled. By labeled is meant that the entities comprise a member of a signal producing system and are thus detectable, either directly or through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include isotopic and fluorescent moieties incorporated into, usually covalently bonded to, a nucleotide monomeric unit, e.g dNTP or monomeric unit of the primer. Isotopic moieties or labels of interest include 32p, 33p, 35S, 125I, and the like. Fluorescent moieties or labels of interest include coumarin and its derivatives, e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such as Bodipy FL, cascade blue, fluorescein and its derivatives, e.g. fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. texas red, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g. Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye(trademark), fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, TOTAB, etc. Labels may also be members of a signal producing system that act in concert with one or more additional members of the same system to provide a detectable signal. Illustrative of such labels are members of a specific binding pair, such as ligands, e.g. biotin, fluorescein, digoxigenin, antigen, polyvalent cations, chelator groups and the like, where the members specifically bind to additional members of the signal producing system, where the additional members provide a detectable signal either directly or indirectly, e.g. antibody conjugated to a fluorescent moiety or an enzymatic moiety capable of converting a substrate to a chromogenic product, e.g. alkaline phosphatase conjugate antibody; and the like. For each sample of RNA, one can generate labeled oligos with the same labels. Alternatively, one can use different labels for each physiological source, which provides for additional assay configuration possibilities, as described in greater detail below.
In a variation of the above embodiment, where desired one can generate labeled RNA instead of labeled first strand cDNA. In this embodiment, first strand cDNA synthesis is carried out in the presence of unlabeled dNTPs and unlabeled gene specific primers. However, the primers are optionally modified to comprise a promotor for an RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and the like. In this embodiment, following first strand cDNA synthesis, the resultant single stranded cDNA is then converted to double stranded CDNA, where the resultant double stranded CDNA comprises the anchor sequence comprising the promoter region. Conversion of the mRNA:cDNA hybrid following first strand synthesis can be carried out as described in Okayama and Berg, Mol. Cell. Biol. (1982) 2:161-170, and Gubler and Hoffman, Gene (1983) 25: 253-269, where briefly the RNA is digested with a ribonuclease, such as E.coli RNase H, followed by repair synthesis using a DNA polymerase like DNA polymerase I, etc., and E.coli DNA ligase. One may also employ the modification of this basic method described in Wu, R, ed., Methods in Enzymology (1987), vol. 153 (Academic Press). Next, the double stranded cDNA is contacted with RNA polymerase and dNTPs, including labeled dNTPs, to produce linearly amplified labeled ribonucleic acids. For cDNA lacking the anchor sequence comprising a promoter region, a polymerase that does not need a promoter region but instead can initiate RNA strand synthesis randomly from cDNA, such as core fragment of E.Coli RNA polymerase, may be employed.
In another embodiment of the subject invention, the labeled nucleic acid generation step comprises one or more enzymatic amplification steps in which multiple DNA copies of the initial RNAs present in the sample are produced, from which multiple copies of the initial RNA or multiple copies of antisense RNA (aRNA) may be produced, using the polymerase chain reaction, as described in U.S. Pat. No. 4,683,195, the disclosure of which is herein incorporated by reference, in which repeated cycles of double stranded DNA denaturation, oligonucleotide primer annealing and DNA polymerase primer extension are performed, where the PCR conditions may be modified as described in U.S. Pat No. 5,436,149, the disclosure of which is herein incorporated by reference.
In one embodiment involving enzymatic amplification, the set of gene-specific primers are employed in the generation of the first strand cDNA, followed by amplification of the first strand cDNA to produce amplified numbers of labeled cDNA. In this embodiment, as a set of gene-specific primers is employed in the first strand synthesis step, only a representative proportion of the total RNA in the sample is amplified during the subsequent amplification steps.
Amplification of the first strand cDNA can be conveniently achieved by using a CAPswitch(trademark) oligonucleotide as described in U.S. patent application Ser. No. 08/582,562, the disclosure of which is herein incorporated by reference. Briefly, the CAPswitch(trademark) technology uses a unique CAPswitch(trademark) oligonucleotide in the first strand cDNA synthesis followed by PCR amplification in the second step to generate a high yield of ds cDNA. When included in the first-strand cDNA synthesis reaction mixture, the CAPswitch(trademark) oligonucleotide serves as a short extended template. When reverse transcriptase stops at the 5xe2x80x2 end of the mRNA template in the course of first strand cDNA synthesis it switches templates and continues DNA synthesis to the end of the CAPswitch(trademark) oligonucleotide. The resulting ss cDNA incorporates at the 3xe2x80x2 end, sequence which is complimentary to complete 5xe2x80x2 end of the mRNA and the CAPswitch(trademark) oligonucleotide sequence.
Of particular interest as the CAPswitch(trademark) oligonucleotide are oligonucleotides having the following formula:
5xe2x80x2-dN1-dN2- . . . dNm-rN1-rN2 . . . rNn-3xe2x80x2
wherein:
dN represents a deoxyribonucleotide selected from among dAMP, dCMP, dGMP and dTMP;
m represents an integer 0 and above, preferably from 10 to 50;
rN represents a ribonucleotide selected from the group consisting of AMP, CMP, GMP and UMP, preferably GMP; and
n represents an integer 0 and above, preferably from 3 to 7.
The structure of the CAPswitch(trademark) oligonucleotide may be modified in a number of ways, such as by replacement of 1 to 10 nucleotides with nucleotide analogs, incorporation of terminator nucleotides, such as 3xe2x80x2-amino NMP, 3xe2x80x2-phosphate NMP and the like, or non-natural nucleotides which can improve efficiency of the template switching reaction but still retain the main function of the CAPswitch(trademark) oligonucleotide i.e. CAP-depended extension of full-length cDNA by reverse transcriptase using CAPswitch(trademark) oligonucleotide as a template.
In using the CAPswitch(trademark) oligonucleotide, first strand cDNA synthesis is carried out in the presence of a set of gene specific primers and a CAPswitch(trademark) oligonucleotide, where the gene specific primers have been modified to comprise an arbitrary anchor sequence at their 5xe2x80x2 ends. The first strand cDNA is then combined with primer sequences complementary to: (a) all or a portion of the CAPswitch(trademark) oligonucleotide and (b) the arbitrary anchor sequence of the gene specific primers and additional PCR reagents, such as dNTPs, DNA polymerase, and the like, under conditions sufficient to amplify the first strand cDNA. Conveniently, PCR is carried out in the presence of labeled dNTPs such that the resultant, amplified cDNA is labeled and serves as the labeled or target nucleic acid. Labeled nucleic acid can also be produced by carrying out PCR in the presence of labeled primers, where either or both the CAPswitch(trademark) oligonucleotide complementary primer and anchor sequence complementary primer may be labeled. In yet an alternative embodiment, instead of producing labeled amplified cDNA, one may generate labeled RNA from the amplified ds cDNA, e.g by using an RNA polymerase such as E.coli RNA polymerase, or other RNA polymerases requiring promoter sequences, where such sequences may be incorporated into the arbitrary anchor sequence.
Instead of using the set of gene specific primers in the first strand cDNA synthesis step followed by subsequent amplification of only a representative fraction of the total number of distinct RNA species in the sample, one may also amplify all of the RNAs in the sample and use the set of gene specific primers to generate labeled nucleic acid following amplification. This embodiment may find use in situations where the RNA of interest to be amplified is known or postulated to be in small amounts in the sample.
In this embodiment, first strand synthesis is carried out using: (a) an oligo dT primer that usually comprises an arbitrary anchor sequence at its 5xe2x80x2 end and (b) a CAPswitch(trademark) oligonucleotide. During first strand synthesis the oligo(dT) anneals to the polyA tail of the mRNA in the sample and synthesis extends beyond the 3xe2x80x2 end of the RNA to include the CAPswitch(trademark) oligonucleotide, yielding a first strand cDNA comprising an arbitrary sequence at its 5xe2x80x2 end and a region complementary to the CAPswitch(trademark) oligonucleotide at its 3xe2x80x2 end. The length of the dT primer will typically range from 15 to 30 nts, while the arbitrary anchor sequence or portion of the primer will typically range from 15 to 25 nt in length.
Following first strand synthesis, the cDNA is amplified by combining the first strand CDNA with primers that correspond at least partially to the anchor sequence and the CAPswitch(trademark) oligonucleotide under conditions sufficient to produce an amplified amount of the cDNA. Labeled nucleic acid is then produced by contacting the resultant amplified cDNA with a set of gene specific primers, a polymerase and dNTPs, where at least one of the gene specific primers and dNTPs are labeled.
The labeled nucleic acids produced above provide a representation of the total RNA profile of the particular source from which the labeled nucleic acids are generated. Accordingly, the labeled nucleic acids find use in comparing the characteristic RNA profiles of different physiological sources and identifying differences in the RNA profiles between different physiological source. Comparison of the RNA profiles of two or more physiological sources finds particular use in methods of identifying differential gene expression in two physiological samples, such as cells or tissues derived from the same or different individual organisms, where the tissues may represent different diseased or normal states, different organ or tissue types, etc.
The labeled nucleic acids of the plurality of physiological sources may be compared in a number of different ways. Thus, one may compare the labeled nucleic acids from each source by separately resolving the labeled nucleic acids from each source under substantially identical electrophoretic conditions to yield an electrophoretic pattern of resolved bands for each of the different populations of labeled nucleic acids. The resultant electrophoretic patterns can then be resolved to identify differences between the labeled nucleic acid populations, which differences can then be attributed to differences in the RNA profiles of the each of the physiological sources, where such differences can, in turn, be attributed to difference in gene expression. See Liang and Pardee, Science (1992) 257: 967. Conveniently, electrophoretic separation under identical electrophoretic conditions can be achieved by running the labeled nucleic acids derived from each physiological source of interest in separate, side by side lanes on a slab gel. Automated electrophoretic machines as described in U.S. Pat. Nos. 5,410,412; 5,275,710; 5,217,591; and 5,104,512, the disclosures of which are herein incorporated by references, may be employed to resolve the labeled nucleic acids. In a modification of the above, where each set of labeled nucleic acids or targets of each physiological source has been labeled with a distinct and distinguishable label, the opportunity arises to resolve the nucleic acids in the same electrophoretic medium, e.g. the same column or in the same lane of a slab, thereby ensuring that the nucleic acids are resolved under identical electrophoretic conditions.
Alternatively, one may hybridize the labeled nucleic acids to predefined arrays of probe polymeric molecules stably associated with the surface of a substrate, where the probe polymeric molecules are capable of sequence specific base pair hybridization with complementary labeled target nucleic acids. A variety of different arrays which may be used are known in the art. The polymeric or probe molecules of the arrays may be oligonucleotides or hybridizing analogues or mimetics thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phophorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, e.g. hexose phosphodiester; peptide nucleic acids; and the like. The length of the probes will generally range from 10 to 1000 nts, where oligonucleotide probes usually range from 15 to 150 nts and more usually from 15 to 100 nts in length, and polynucleotide probes usually range in length from 150 to 1000 nts, where the polynucleotide probes may be single or double stranded, usually single stranded, and may be PCR fragments amplified from cDNA.
The probe molecules on the surface of the substrates will preferably correspond to known genes of the physiological source being analyzed so that positive hybridization events may be correlated to expression of a particular gene in the physiological source. Of particular interest are arrays of probes which correspond to a particular subset of the total genes expressed by a particular physiological source. For example, for analysis of human physiological sources, preferably the arrays of probes will correspond to a particular subset of all the expressed human genes, such as those genes associated with cell-cell communication, cancer related genes, etc. The arrays of probes may have sequences that are complementary to the template and/or non-template strands of the gene to which they correspond, depending on the nature of the labeled target nucleic acid to which they are to hybridize.
The substrates with which the probe molecules are stably associated may be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like.
A variety of different methodologies have been developed for producing arrays of probes stably associated to the surface of a substrate. Representative methodologies include spotting methods, in which probes are immobilized or spotted on the surface of substrates as described in WO 95/35505 the disclosure of which is herein incorporated by reference, and methods in which the probes are synthesized or grown on the surface of the substrates, such as EP 0 373 203 B1 and U.S. Pat. No. 5,445,934, the disclosures of which are herein incorporated by reference. Arrays of probes spotted onto nylon membranes are described in Lennon and Lerach, Trends in Genetics (1991) 7:314-317; Gress et al., Mammalian Genome (1992) 3:609-619; Meier-Ewert et al., Nature (1993) 361:375-376; Nguyen et al., Genomics (1995) 29:207-216; Zhao et al., Gene (1995) 156:207-213; Takahashi et al., Gene (1995) 164:219-217; Milosavlijevic et al., Genome Research (1996) 6:132-141; Pietu et al., Genome Research (1996) 6:492-503; and Drmanac, Science (1993) 260:1649-1652. Arrays of probes spotted onto the surface of modified microscope glass slides are described in Shena et al., Science (1995) 270: 467-470 and Shalon et al., Genome Research (1996) 6: 639-645. Arrays in which the probes have been grown on the surface of a substrate are described in Lockhart et al., Nature Biotechnology (1996) 14:1675.
Of particular interest for use in the analysis of differential gene expression in human physiological sources are the arrays of subsets human cDNAs sold under the trademark Atlas(trademark) by Clontech and described in CLONTECHniques (April, 1997) 12:4-7.
In analyzing the differences in the population of labeled nucleic acids generated from two or more physiological sources using the arrays described above, each population of labeled nucleic acids are separately contacted to identical probe arrays under conditions of hybridization, preferably under stringent hybridization conditions, such that labeled nucleic acids hybridize to their complementary probes on the substrate surface. Suitable hybridization conditions are well known to those of skill in the art and reviewed in Maniatis et al, supra and WO 95/21944.
As with the electrophoretic analysis, where all of the target sequences comprise the same label, different arrays will be employed for each physiological source (where different could include using the same array at different times). Alternatively, where the labels of the targets are different and distinguishable for each of the different physiological sources being assayed, the opportunity arises to use the same array at the same time for each of the different target populations.
In one preferred embodiment of assays using arrays, the number of gene specific primers used to generate the target will be chosen in view of the number of distinct probes present on the surface of the substrate of the array. In such instances, the number of gene specific primers in the set will not vary by more than 10 fold from the number of distinct probes, usually by not more than 5 fold and more usually by not more than 2 fold from the number of distinct probes in the array.
Following hybridization, non-hybridized labeled nucleic acid is removed from the support surface, conveniently by washing, generating a pattern of hybridized nucleic acid on the substrate surface. A variety of wash solutions are known to those of skill in the art and may be used.
The resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement and the like.
Following detection or visualization, the hybridization patterns may be compared to identify differences between the patterns. Where arrays in which each of the different probes corresponds to a known gene are employed, any discrepancies can be related to a differential expression of a particular gene in the physiological sources being compared.
The subject methods find use in, among other applications, differential gene expression assays. Thus, one may use the subject methods in the differential expression analysis of: (a) diseased and normal tissue, e.g. neoplastic and normal tissue, (b) different tissue or subtissue types; and the like.
Also provided are kits for use in carrying out the subject methods, e.g. generating populations of labeled nucleic acids, performing differential gene expression analysis and the like. The kits according to the subject invention include at least the set of gene specific primers that are employed to generate the labeled oligonucleotides. Of particular interest are kits comprising a set of primers selected from the primers identified as SEQ ID NO: 01-1372 in application Ser. No. 08/859,008 filed May 21, 1997, the disclosure of which is herein incorporated by reference, where in these kits of particular interest, at least twenty, usually at least 50 and more usually at least 100 of the gene specific primers in the kit will be selected from this group of primers identified as SEQ ID NO: 01-1372. Also of interest are kits comprising those gene specific primers corresponding to those genes (and specifilc capable of producing target capable of hybridizin to those specific regions of the genes) as listed in the following copending patent applications, the disclosures of which are herein incorporated by reference: U.S. application Ser. No. 09/221,480 entitled Apoptosis Array; U.S. application Ser. No. 09/222,432 entitled Cancer Array; U.S. application Ser. No. 09/222,437 entitled Cell Cycle Array; U.S. application Ser. No. 09/222,437 entitled Cell Interaction Array; U.S. application Ser. No. 09/222,251 entitled Cytokine Receptor Array; U.S. application Ser. No. 09/221,481 entitled Human Array; U.S. application Ser. No. 09/222,256 entitled Human Stress Array; U.S. application Ser. No. 09/222,248 entitled Mouse Array; and U.S. application Ser. No. 09/222,253 entitled Oncogene/Tumor Suppressor Array. The kits may further comprise one or more additional reagents employed in the various methods, such as additional non-gene specific primers sequences, such as SEQ ID NO: 1373 to 1375 of application Ser. No. 08/859,008 filed May 21, 1997, the disclosure of which is herein incorporated by reference, dNTPs and/or rNTPs, which may be either premixed or separate, one or more uniquely labeled dNTPs and/or rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, enzymes, such as reverse transcriptases, DNA polymerases, and the like, various buffer mediums, e.g. hybridization and washing buffers, prefabricated probe arrays, labeled probe purification reagents and components, like spin columns, etc., signal generation and detection reagents, e.g. streptavidin-alkaline phosphatase conjugate, chemifluorescent or chemiluminescent substrate, and the like.

Methods of assaying differential expression


An encoding apparatus comprises a frame processor (105) which receives a multi channel audio signal comprising at least a first audio signal from a first microphone (101) and a second audio signal from a second microphone (103). An ITD processor 107 then determines an inter time difference between the first audio signal and the second audio signal and a set of delays (109, 111) generates a compensated multi channel audio signal from the multi channel audio signal by delaying at least one of the first and second audio signals in response to the inter time difference signal. A combiner (113) then generates a mono signal by combining channels of the compensated multi channel audio signal and a mono signal encoder (115) encodes the mono signal. The inter time difference may specifically be determined by an algorithm based on determining cross correlations between the first and second audio signals.

Efficient encoding of audio signals is critical for an increasing number of applications and systems. For example, mobile communications use efficient speech encoders to reduce the amount of data that needs to be transmitted over the air interface.
For example, the International Telecommunication Union (ITU) is standardizing a speech encoder known as the Embedded Variable Bit Rate Codec (EV-VBR) which can encode a speech signal at high quality with data rates ranging from 8 to 64 kbps. This encoder, as well as many other efficient speech encoders, uses Code Excited Linear Prediction (CELP) techniques to achieve the high compression ratio of the encoding process at the lower bit rates of operation.
In some applications, more than one audio signal may be captured and in particular a stereo signal may be recorded in audio systems using two microphones. For example, stereo recording may typically be used in audio and video conferencing as well as broadcasting applications.
In many multi channel encoding systems, and in particular in many multi channel speech encoding systems, the low level encoding is based on encoding of a single channel. In such systems, the multi channel signal may be converted to a mono signal for the lower layers of the coder to encode. The generation of this mono signal is referred to as down-mixing. Such down-mixing may be associated with parameters that describe aspects of the stereo signal relative to the mono signal. Specifically, the down mixing may generate inter-channel time difference (ITD) information which characterises the timing difference between the left and right channels. For example, if the two microphones are located at a distance from each other, the signal from a speaker located closer to one microphone than the other will reach the latter microphone with a delay relative to the first one. This ITD may be determined and may in the decoder be used to recreate the stereo signal from the mono signal. The ITD may significantly improve the quality of the recreated stereo perspective since ITD has been found to be the dominant perceptual influence on stereo location for frequencies below approximately 1 kHz. Thus, it is critical that ITD is also estimated.
Conventionally, the mono signal is generated by summing the stereo signals together. The mono signal is then encoded and transmitted to the decoder together with the ITD.
For example, the European Telecommunication Standards Institute has in their Technical Specification ETSI TS126290 "Extended Adaptive Multi-Rate—Wideband (AMR-WB+) Codec; Transcoding Functions" defined a stereo signal down-mixing where the mono signal is simply determined as the average of the left and right channels as follows.xML(n)=0.5(xLL(n)+xRL(n))where xML(n) represents the nth sample of the mono signal, xLL(n) represents the nth sample of the left channel signal and xRL(n) represents the the nth sample of the right channel signal.
Another example of a downmix is provided in H. Purnhagen, "Low Complexity Parametric Stereo Coding in MPEG-4", Proceedings 7th International Conference on Digital Audio Effects (DAFx'04), Naples, Italy, Oct. 5-8, 2004, pp 163-168. In this document, a down-mixing method is described which obtains an output mono signal as a weighted sum of the incoming channels on a band-by-band frequency basis using information obtained about the inter-channel intensity difference (IID).
Specifically:M[k,i]=glL[k,i]+grR[k,i]where M[k,i] represents the ith sample of the kth frequency bin of mono signal, L[k,i] represents the ith sample of the kth frequency bin of the left channel signal and R[k,i] represents the ith sample of the kth frequency bin of the right channel signal, gl is the left channel weight and gr is the right channel weight.
A characteristic of such approaches is that they either result in mono signals having a high reverberation time or else have high complexity and/or delay. For example, the AMR-WB+ method of down-mixing provides an output whose reverberation time is approximately that of the room plus the flight time between the two microphones. The downmix provided in Purnhagen is of high complexity and imposes a delay due to the frequency analysis and reconstruction.
However, many mono encoders provide the best results for signals with low reverberation times. For example, low bit rate CELP speech coders, and other encoders which employ pulse-based excitation to represent speech and audio signals, perform best when presented with signals having short reverberation times. Accordingly, the performance of the encoder and the quality of the resulting encoded signal tend to be suboptimal.
Hence, an improved system would be advantageous and in particular a system allowing increased flexibility, facilitated implementation, improved encoding quality, improved encoding efficiency, reduced delay and/or improved performance would be advantageous.

This patent is subject to a terminal disclaimer.

Apparatus and method for encoding a multi-channel audio signal


The present invention provides a tubular surgical cutter fabrication method comprising forming one or more tabs which extend beyond an end of a tube. Each of the tabs is then folded across at least a portion of the end of the tube, and affixed in the folded position to form an end structure extending across the end of the tube. A cutting edge is imposed on the tube adjacent to the end structure, thereby providing a tubular surgical cutter having the structural reinforcement of a closed end without resorting to welding or otherwise attaching a closed end structure about the perimeter of the tube.

1. Field of the Invention
This invention relates generally to surgical cutting devices, and in particular, provides a method for forming a tubular surgical cutter for use in endoscopy, arthroscopy, internal tissue resection, and other minimally-invasive surgical procedures, the method including forming the end of the cutter by folding tabs of tubal wall material.
Arthroscopic surgery techniques often involve manipulating a cutting probe through a small incision. For example, arthroscopic knee surgery often involves manually positioning the distal end of the probe against a tissue to be cut, typically against a meniscus in the knee joint. The piece of meniscus that is to be trimmed protrudes into an aperture formed in an outer tubular structure of the probe. An inner tubular structure rotates within this outer tubular structure, the inner tube including a chopping edge which sweeps by the aperture. Hence, the meniscus (or any other hard or soft tissues which protrude into the aperture) is sheared between the chopping edge of the inner tube and the edge of the aperture on the outer tubular structure.
U.S. patent application Ser. No. 08/136,426, and co-pending U.S. patent application Ser. Nos. 08/322,680, and 08/542,289 (Attorney Docket No. 16944-000130), the full disclosures of which are incorporated herein by reference, describe exemplary tissue resection devices having chopping mechanisms formed from cooperating tubular cutters. These exemplary tissue resectors include strip-cutting members which remove axial strips of tissue from an internal body cavity, the axial strips being chopped into tissue fragments by the chopping mechanism to facilitate evacuation of the tissue through the inner tube. These patent applications further describe a preferred method of use of the exemplary resection device for trans-cervical fibroid removal from the uterus and the like.
Known tubular cutters have typically been fabricated by drawing a cup of suitable material into the shape of a closed tube. This closed tube is then welded to the end of standard tubing of the appropriate size. The drawn cups are manufactured to sizes that allow the inner tubular structure to rotate smoothly inside the outer cup of the outer tubular structure, and generally provide small clearances for a good shearing action. After welding, the tube-cup assemblies are ground to create the desired opening, and also to impose the cutting edges in the tubal wall.
Although the above welded cup fabrication method has proven effective at producing tubular surgical cutters, the process does have certain drawbacks. The process requires a number of steps which must be very accurately performed to ensure cooperation between the inner and outer tubular structures. The cups must be drawn to sizes of very tight tolerance, and each must be precisely and welded to allow rotation. Achieving the required straightness in the welded structure typically requires fixturing, greatly increasing tooling costs. Furthermore, the fabrication process requires drawability, which limits the number of suitable materials and may also increase material costs.
For the above reasons, it is desirable to provide improved tubular surgical cutters and methods for their production. It would be particularly desirable if such tubular cutter production methods did not require welding of a separate cup onto the cutter tube. It would be especially beneficial if such cutter fabrication methods could be used for both the inner and outer cutter tubes, and if such fabrication methods relied on economical tube-forming procedures to provide a disposable tubular cutter assembly for use in a wide variety of minimally-invasive surgical procedures.
2. Description of the Background Art
U.S. Pat. No. 5,112,299 describes an arthroscopic surgical apparatus and method in which an elongate outer sheath member and an inner cylindrical cutting blade member are each provided with longitudinally-extending diametrically-opposed tabs having cutting edges. The tabs extend distally so that tissue positioned between the tabs is severed as the cutting blade member passes the sheath member. There is no suggestion that these tabs be folded to structurally reinforce the open ends of the cutting tubes.
U.S. Pat. No. 4,850,354 describes a surgical cutting instrument having inner and outer tubes. An opening having a corner is cut into at least one of the inner and outer tube, while the tubes themselves preferably include hemispherical ends.
U.S. Pat. No. 5,269,798, and European Patent Application Publication No. 0 499 465 A1 describe a surgical cutting instrument formed from cooperating inner and outer elongate tubular members, the distal end of each tubular member again including a hemispherical surface.
U.S. Pat. No. 4,819,635 describes a tubular microsurgery cutting apparatus having a hemispherically-ended outer tubular member in which a tubular inner sleeve reciprocates axially.
Australian Patent No. AU-B-85855/91 and German Patent No. 2848314 each describe surgical cutting instruments having rotary cutting tubes. U.S. Pat. No. 5,133,360 is generally relevant.

Folded-end surgical tubular cutter and method for fabrication


An optical pickup includes first and second objective lenses; a first raising optical element that reflects an optical beam from a first direction within a plane substantially orthogonal to an optical axis of the first objective lens, thereby the optical beam being incident on the first objective lens; and a second raising optical element that reflects an optical beam from a second direction at an angle to the first direction within a plane substantially orthogonal to the optical axis of the second objective lens, thereby the optical beam being incident on the second objective lens. At least one lateral surface of at least one of the first and second raising optical elements is at an angle of 90 degrees or smaller to a reflection surface thereof. The lateral surface of the one raising optical element is arranged to face a lateral surface of the other raising optical element.

1. Field of the Invention
The present invention relates to an optical pickup that records and/or reproduces information signals on and/or from an optical disc, and an optical disc apparatus using the optical pickup.
2. Description of the Related Art
Hitherto, an optical disc, such as a compact disc (CD) or a digital versatile disc (DVD); or to achieve further high-density recording, an optical disc for recording/reproducing signals using an optical beam with a wavelength of about 405 nm emitted from, for example, a blue-violet semiconductor laser, this type of optical disc being hereinafter referred to as "high-density recording optical disc", has been used as a recording medium for information signals. An optical disc apparatus has been used for recording information signals on such an optical disc, or reproducing information signals recorded on such an optical disc.
Such an optical pickup is being desired to be compatible with a plurality of types of optical discs. Hence, to be compatible with optical discs in different formats with different usable wavelengths and different thicknesses of protection substrates, an optical pickup is being provided, which uses a plurality of optical beams with different wavelengths and has a plurality of objective lenses (for example, see Japanese Unexamined Patent Application Publication No. 2006-4547).
The optical pickup having the plurality of objective lenses includes an objective lens drive unit having a fixed portion and a movable portion. For example, two objective lenses are aligned in a tangential direction or a radial direction on a lens holder functioning as the movable portion of the objective lens drive unit. The optical pickup selects one of the objective lenses depending on the format and type of an optical disc.
The optical pickup also includes optical systems for respectively guiding optical beams with wavelengths corresponding to the objective lenses. In general, an optical beam guided in a plane orthogonal to an optical axis of an objective lens is reflected and raised with a raising mirror toward the objective lens, thereby collecting the optical beam onto the optical disc with the objective lens.
Meanwhile, such an optical pickup is demanded to reduce a distance between the objective lenses for reduction in size and weight of the movable portion as well as the entire optical pickup. To reduce the distance between the objective lenses, it is necessary to reduce a distance between the raising mirrors corresponding to the objective lenses. For example, if two objective lenses and two raising mirrors are provided and optical paths to the raising mirrors do not define an angle to each other within a plane, that is, if optical beams are incident on the raising mirrors in the same direction, or opposite directions, the sizes of the raising mirrors are reduced to a limit of an effective area corresponding to apertures of the objective lenses. In addition, the raising mirrors are adjacently arranged, thereby reducing the distance between the raising mirrors, and the distance between the objective lenses.
However, in view of the optical systems, and reduction in size of the entire optical pickup, an optical path of an optical beam being incident on one of the raising mirrors may define an angle to an optical path of an optical beam incident on the other of the raising mirrors. In such a case, the distance between the raising mirrors may be increased by thicknesses of the raising mirrors. Owing to this, the distance between the objective lenses may be increased, and the size of the lens holder functioning as the movable portion may be increased.

Optical pickup and optical disc apparatus using the same


A bicycle and bicycle parking and lock system wherein a hub locking device that is secured to a bicycle cooperates with a parking rack. The bicycle includes an engagement member that is secured to an axle of the bicycle. The engagement member selectively removably cooperates with a recess formed in the parking and lock system. The parking and lock system includes a number of upstanding members that are positioned to generally flank the front wheel of a bicycle to maintain the bicycle in a generally upright orientation. Each or select number of the upstanding members include a lock system that cooperates with the locking engagement member so as to secure the respective bicycle with respect to the parking system.

The present invention relates generally to bicycles and, more particularly, to a bicycle hub having an integral locking mechanism that cooperates with a bicycle parking stand or rack.
Automated bicycle rental systems have become increasingly popular. Such rental systems typically include a number of rental stations located across a city or other location. The rental stations typically include one or more bicycle stands for supporting a number of rentable bicycles. The bicycles are secured to the bicycle stands by a locking mechanism such that the bicycles are incapable of being removed until payment or identification for the rental of one of the bicycles is recognized.
The bicycle stands are in electronic communication with a rental kiosk or the like. User's of the rental system use the rental kiosk or other means to provide payment and/or identification information for renting of one of the bicycles for temporary use. Once the electronic means of the kiosk verifies the payment and/or identification, the kiosk enables the user the remove one of the bicycles secured to the bicycle stand by unlocking the locking mechanism. Thus, the user is free to take the bicycle and ride it around the city to go from place to place. When the user is finished riding the bicycle, he or she returns the bicycle to one of the bicycle stations and secures the bicycle to the bicycle stand for subsequent use by another user.
Known bicycle rental systems suffer from a number of disadvantages. For instance, such bicycle rental systems are susceptible to a great deal of wear and tear associated with the public use of the bicycles and bicycle stands of the bicycle rental systems. In addition, such publically located systems are susceptible to vandalism and theft. Further, such systems often require the users of the bicycles to lift or otherwise awkwardly manipulate the bicycles of the bicycle rental systems in order to engage or disengage the bicycle with the bicycle stand for storage or removal of the bicycle, respectively. Further, known bicycle rental systems often require relatively precise guidance of the bicycle with respect to the bicycle stand so as to engage the bicycle with the bicycle stand.
Thus, it is desired to provide an apparatus that overcomes at least one of the foregoing disadvantages.

Bicycle hub locking mechanism and parking system


In an active matrix for a liquid crystal display, including a plurality of pixel electrodes in an active zone, each of which can be addressed using one selection line among N selection lines and one data line among M data lines, a storage capacitor is connected to each pixel electrode in the matrix, and a line stabilization capacitor is provided for each selection line in the matrix. A first electrode of the stabilization capacitor is connected to the selection line and a second electrode is connected to a reference voltage. Such a structure may find application to active matrix type liquid crystal displays, particularly with integrated drivers.

According to the state of the art of amorphous silicon technology, integrated line drivers are of the high output impedance type so as to limit the transistor stress and therefore the drift in the threshold voltage of these transistors. With this type of driver, when lines are not selected (which is the case during more than 99% of the display addressing time) they are in a so-called floating state. In this state, the lines are sensitive to all parasite variations and particularly variations related to capacitive couplings due to intersection of lines and columns. More precisely, for each new selected line, the data to be displayed is applied on all columns of the display: as a result of capacitive couplings due to intersection between lines and columns, the voltage levels of unselected lines vary on these columns. This has an effect on the display quality of the display.
In particular, these couplings can lead to unwanted selection of a line on the display and as a result data on this line may be modified. This is the so-called multi-line selection problem.
Other unwanted effects can occur depending on the method used to make the storage capacitor associated with each pixel electrode of the active matrix. In particular, when this storage capacitor is made using the pixel row selection line, these couplings will also cause variations in voltage levels stored on pixels through the storage capacitor. The display quality is affected.
Moreover, in such a structure, the total capacitance of each line depends on the video information displayed on the pixels in this line through the storage capacitor. This total capacitance depends on the sum of all coupling capacitances between this line and each column in the matrix, plus the sum of all storage capacitances in series with the associated liquid crystal capacitances made with this line, as a first approximation. This dependence may introduce display nonuniformities.

Active-matrix liquid crystal matrix display


An electronic device includes an authentication section that authenticates a user of the electronic device, an operation section that includes a display section and operates the electronic device, a storage section that stores programs that each cause the electronic device to perform a job in accordance with a preliminarily set content, and a program processing section that generates a program associated with the user authenticated in the authentication section. Using the operation section, a first user specifies a program to be shared and a second user permitted to use the shared program. As such, the first user who permits to share the program shares the program with the second user.

The present disclosure relates to a technology capable of omitting a setting operation for an electronic device such as an image forming apparatus.
In an electronic device, such as an image forming apparatus, various functions that improve user convenience are added. A function called a program function is one of such functions. The program function provides a capability to omit a setting operation for the image forming apparatus. Using this function, a user performs a setting by operating an operation section, and preliminarily registers the setting as a program. Accordingly, the user is able to cause the image forming apparatus to execute a job by performing an operation for calling the program, without a need to perform an operation relating to that setting.
As an example, for a copying job, the user may perform a setting of two in one, a setting of a paper size of A4, a setting of duplex copying, and a setting of monochrome copying, by operating the operation section, and preliminarily register the settings as a program. By performing an operation for calling the program, the user is able to cause the image forming apparatus to execute the copying job in accordance with those settings.
If, in such an image forming apparatus, users are able to share the program, it is possible to reduce memory capacity necessary to store the program. Therefore, in a case where one user (e.g., a user A) intends to register a program having the same registration content as that of a program that has already been registered by another user (e.g., a user B), a terminal device of the user B is requested to permit sharing of the program with the user A. In some cases, the program is shared with the user A and used.
However, in the above-mentioned image forming apparatus, the degree of freedom of the user B has been restricted with respect to the availability of sharing of the program.

Storage medium recording display control program for function setting, method for operating display control program, and electronic device including the same


An upper insulating layer is formed on an upper surface of a gate electrode formed on an insulating substrate. A gate insulating layer is formed on sidewalls of the gate electrode and the surfaces of the upper insulating layer. A semiconductor layer is formed on the surfaces of the gate insulating layer. Three source/drain regions are formed in the semiconductor layer. Two independent channel regions are formed in the semiconductor layer along both side surfaces of the gate electrode. Source/drain regions are arranged on both ends of two channel regions. Each source/drain region has an LDD structure formed in a self alignment manner by an oblique ion implantation method and a vertical ion implantation method using sidewall insulating layers formed on the channel regions as masks.

1. Field of the Invention
The present invention relates to a structure in which electric field through a thin film field effect transistor using a polycrystalline silicon layer of a thin film as a channel layer can be decreased and to a manufacturing method of the thin film field effect transistor.
2. Description of the Background Art
A so-called thin film transistor (TFT) is one of types of insulated field effect transistors structured to have semiconductor thin films formed on insulating substrates and channel regions within the thin films.
FIG. 7 is a sectional structural view of a conventional thin film transistor. In the conventional thin film transistor, a gate electrode 5 of a polycrystalline silicon layer is formed on a surface of an insulating substrate or insulating layer 1. A gate insulating layer 7 of an oxide film or the like is formed on the surfaces of insulating substrate 1 and gate electrode 5. A semiconductor layer 8 of polycrystalline silicon or single crystalline silicon is formed on a surface of gate insulating layer 7. A pair of source/drain regions 14, 14 are formed in the semiconductor layer 8, and a channel region 12 is formed between these source/drain regions 14, 14. The surface of semiconductor layer 8 is covered with an interlayer insulating layer 15. Interconnection layers 16 are connected to source/drain regions 14, 14, respectively, through contact holes formed in interlayer insulating layer 15. Such a type of a gate electrode 5 being formed under channel region 12 is called a bottom gate type thin film transistor. FIG. 16 (a) is an equivalent circuit diagram of the bottom gate thin film transistor.
Manufacturing steps of the thin film transistor shown in FIG. 7 will be described. FIGS. 8 through 13 are sectional structural views sequentially showing respective manufacturing steps of the thin film transistor (a first step through a sixth step).
First, as shown in FIG. 8, a polycrystal silicon layer 2 is formed on the surface of insulating layer substrate 1 using a CVD (Chemical Vapor Deposition) method. A resist pattern 4 is formed on the surface of polycrystalline silicon layer 2 using a photolithography method and an etching method. Then, using a resist pattern 4 as a mask, polycrystalline silicon layer 2 is etched and a gate electrode 5 is formed.
Next, as shown in FIG. 9, a gate insulating film 7 and a semiconductor layer 8 of polycrystalline silicon or single crystalline silicon are formed on the whole wafer. A resist pattern 24 to define shapes of active regions is then formed on the surface of semiconductor layer 8 using a photolithography method. Using the resist pattern 24 as a mask, semiconductor layer 8 is patterned. After the resist pattern 24 is removed, impurities of a first type conductivity are ion-implanted into semiconductor layer 8 in a dose of 0-5.times.10.sup.13 /cm.sup.2.
As shown in FIG. 10, a resist pattern 25 is formed on the surface of a region which is to be a channel region of semiconductor layer 8 using a photolithography method. Using resist pattern 25 as a mask, impurity ions 33 of a second type conductivity are ion-implanted into semiconductor layer 8 in a dose of 5.times.10.sup.14 -1.times.10.sup.16 /cm.sup.2, so that a pair of source/drain regions 14, 14 are formed in semiconductor layer 8.
As shown in FIG. 11, an interlayer insulating film 15 is formed on the whole surface. On the surface of interlayer insulating layer 15, resist pattern 26 is formed for forming contact holes. Using resist pattern 26 as a mask, interlayer insulating layer 15 is etched to form contact holes reaching source/drain regions 14, 14.
As shown in FIG. 12, after resist pattern 26 is removed, interconnection layers 16 are formed within contact holes and on the surface of interlayer insulating layer 15 and is patterned to a prescribed interconnection pattern. By following the above steps, wafer process of a thin film transistor shown in FIG. 12 (corresponding to FIG. 7) is finished.
A bottom gate type thin film transistor manufactured by the above stated method has source/drain regions formed by ion implantation using resist 25 as a mask, as shown in FIG. 10. A problem sometimes arises that gate electrode 5 and resist pattern 25 for forming source/drain regions are not in register because of alignment errors of a mask for forming a resist pattern. FIG. 13 is a sectional structural view showing positions of resist pattern 25 and source/drain regions 14, 14 when an alignment error occurs. When such an alignment error shown in the figure occurs, one of source/drain regions 14 is separated from gate electrode 5 and a so-called offset structure is formed. When such a thin film transistor of an offset structure is formed, a problem arises that transistor characteristics deteriorate. In particular, as a device is miniaturized and a channel length becomes 1 .mu.m or shorter than 1 .mu.m, the problem of an alignment error of a mask has been more significant than ever.
In order to avoid the alignment error of a mask, a thin film transistor formed by a method in which an alignment of gate electrode with source/drain regions is carried out using a self-alignment technology has been conceived. FIG. 14 is a sectional structural view of a bottom gate type thin film transistor having source/drain regions formed by the self-alignment technology. The thin film transistor shown is described, for example, in Symposium on VLSI Technology p.8, Sep. 10-12, 1984. FIG. 16 (b) is an equivalent circuit diagram of the thin film transistor shown in FIG. 14. As shown in FIGS. 14 and 16 (b), the thin film transistor includes a pair of source/drain regions 14, 14 formed in semiconductor layer 8 located on the surface of insulating substrate 1 and a source/drain region 14 formed in semiconductor layer 8 located above gate electrode 5. Channel regions 12, 12 are formed in semiconductor layer 8 located on the sidewalls of gate electrode 2. Hence, this thin film transistor has two transistors formed on the sidewalls of the gate and connected in series.
A method of manufacturing a thin film transistor shown in FIG. 14 will be described. Manufacturing step of a first conventional example shown in FIGS. 8 and 9 can be also used in manufacturing steps of the film transistor shown in FIG. 14. Therefore, the description of the steps shown in FIGS. 8 and 9 will not be repeated. Following the steps shown in FIG. 9, as shown in FIG. 15, impurity ions 34 are ion-implanted vertically into semiconductor layer 8. By this ion implantation, semiconductor layer 8 has impurities implanted only in a region extending on the surface of insulating substrate 1 and a region above gate electrode 2 to form source/drain regions 14, 14, but not in a region on the sidewalls of gate electrode 2 which are to be channel regions 12, 12.
Thereafter interlayer insulating layer 15 and interconnection layer 16 are formed as in the first conventional example.
However, in the conventional bottom gate type thin film transistor shown in FIG. 14, a problem exists that large electric field strength is generated in a region of semiconductor layer 8 where a source/drain region 14 is superimposed on gate electrode 2 resulting in an increase in leakage current. Since channel regions 12, 12 are formed on the sidewalls of gate electrode 2, a channel length is short and therefore a problem of a decrease in a breakdown voltage between source/drain regions 14, 14 arises.

Thin film field effect element having an LDD structure


A lightweight, flexible ski pole which is virtually indestructible comprises a filament-reinforced, resin-matrix composite shaft having a diameter of about 0.05 in or less and a tensile strength of about 140,000 or higher. The shaft may be severely bent without damage or deformation. A surface coating of acrylic paint is applied by dip coating. A metal tip is adhesively applied, as are hand grip and basket. The shaft may be solid or hollow, straight or tapered near the lower end. If hollow and tapered, the tapered end is reinforced with a short reinforcing rod.

The standard state-of-the-art ski pole for the past two or three decades comprises a hollow, tapered aluminum shaft, painted with enamel and having a basket and tip mounted on one end and a hand grip mounted on the other end. Such a pole weighs about 6.5 ounces and has a tensile strength of about 50,000 psi.
The principal disadvantage of the traditional aluminum ski pole is the fact that it is relatively easily bent; i.e., the aluminum shaft is soft and tends to permanently deform or even collapse under the bending loads which are commonly encountered during skiing. A partially collapsed shaft exhibits greatly reduced bending resistance and cannot be restored to its original shape and strength. Moreover, the paint is relatively easily chipped off and the resulting exposure of bare aluminum is unsightly.
Another disadvantage of the aluminum shaft is its axial rigidity and inability to absorb shock loads. To compensate for this, one recently introduced pole includes an expensive axial shock absorber near the hand grip.
U.S. Pat. No. 4,301,201 issued in 1981 to Stout discloses a filament/resin composite ski pole comprising an annular array of continuous reinforcing filaments or fibers embedded in a synthetic resin matrix and formed into a hollow tubular shaft by the process known as pultrusion. The filaments extend rectilinearly along the length of the shaft.


An automatic transmission includes a transmission case, a parking gear fixed to the transmission case, and a meshing mechanism, such as a synchromesh mechanism. The meshing mechanism includes a coupling sleeve to engage with the parking gear and hold a transmission output member unrotatable in a park lock state when the coupling sleeve is shifted axially toward the parking gear.

The present invention relates to a parking mechanism of a transmission for a vehicle, and specifically to a parking mechanism of an automated manual transmission.
A meshing type automatic transmission constructed to automate driver's manual operations for an ordinary manual transmission is in practical use. One example is a twin-clutch type automatic transmission including first and second input shafts to be connected with an engine through respective automatic clutches.
A U.S. Pat. No. 5,711,409 (specifically column 12, lines 32-50, FIG. 10)(corresponding to P8004788) shows a parking system for such a twin-clutch automatic transmission in which a Park function for parking lock is attained by a pressing force of an elastic member applied to a pressure plate of one of the automatic clutches, and by selecting one of the gear speed associated with that automatic clutch.


A piston for an internal combustion engine includes a piston main body for the internal combustion engine, and a crosshatched groove being formed on a surface of the piston main body. The crosshatched groove includes a crosshatch angle that corresponds to a crossing angle of lines that configure a crosshatch, the crosshatch angle that is set at a range from equal to or greater than 15 degrees to equal to or less than 35 degrees.

A known piston for an internal combustion engine is disclosed in JP2008-297928A (hereinafter referred to as Patent reference 1). According to Patent reference 1, the piston of the internal combustion engine includes a skirt portion being provided at a piston main body, the skirt portion having plural streaks (stripe-shaped grooves) on a surface of the skirt portion formed by a cutting process using a cutting instrument, or by a form rolling process using a roller. According to the piston of the internal combustion engine, the plural streaks extend in parallel to each other in a sliding direction of the piston main body in a state where the plural streaks are inclined in only one direction by a predetermined angle.
However, according to the piston of the internal combustion engine disclosed in Patent reference 1, because the plural streaks are inclined and extend in the only one direction with respect to the sliding direction of the piston main body, a lubricant oil (oil) being positioned in the grooves is considered to be easily discharged from an edge of the groove by moving within the groove in accordance with a reciprocating motion of the piston. Thus, because the oil retaining performance at a sliding portion cannot be sufficiently ensured, the seizure resistance of the piston main body cannot be secured.
A need thus exists for a piston for an internal combustion engine and a manufacturing method thereof which is not susceptible to the drawback mentioned above.

Piston for internal combustion engine and manufacturing method thereof