According to one example of the present disclosure, a system includes a computing element configured to provide requests for memory access operations and a memory module comprising a plurality of memories, a plurality of independent data channels, each of the independent data channels coupled to one of the plurality of memories, a plurality of internal address/control channels, each of the independent address/control channels coupled to one of the plurality of memories, and control logic coupled to the plurality of internal address/control channels and configured to receive and decode address and control information for a memory access operation, the control logic further configured to selectively provide the decoded address and control information to a selected internal address/control channel for a selected independent data channel of the plurality of independent data channels based on the received address and control information for the memory access operation.

The popularity of computing systems continues to grow and the demand for improved processing architectures thus likewise continues to grow. Ever-increasing desires for improved computing performance/efficiency has led to various improved processor architectures. For example, multi-core processors are becoming more prevalent in the computing industry and are being used in various computing devices, such as servers, personal computers (PCs), laptop computers, personal digital assistants (PDAs), wireless telephones, and so on.
In the past, processors such as CPUs (central processing units) featured a single execution unit to process instructions of a program. More recently, computer systems are being developed with multiple processors in an attempt to improve the computing performance of the system. In some instances, multiple independent processors may be implemented in a system. In other instances, a multi-core architecture may be employed, in which multiple processor cores are amassed on a single integrated silicon die. Each of the multiple processors (e.g., processor cores) can simultaneously execute program instructions. This parallel operation of the multiple processors can improve performance of a variety of applications.
A multi-core CPU combines two or more independent cores into a single package comprised of a single piece silicon integrated circuit (IC), called a die. In some instances, a multi-core CPU may comprise two or more dies packaged together. A dual-core device contains two independent microprocessors and a quad-core device contains four microprocessors. Cores in a multi-core device may share a single coherent cache at the highest on-device cache level (e.g., L2 for the Intel® Core 2) or may have separate caches (e.g. current AMD® dual-core processors). The processors also share the same interconnect to the rest of the system. Each "core" may independently implement optimizations such as superscalar execution, pipelining, and multithreading. A system with N cores is typically most effective when it is presented with N or more threads concurrently.
One processor architecture that has been developed utilizes multiple processors (e.g., multiple cores), which are homogeneous. As discussed hereafter, the processors are homogeneous in that they are all implemented with the same fixed instruction sets (e.g., Intel's x86 instruction set, AMD's Opteron instruction set, etc.). Further, the homogeneous processors access memory in a common way, such as all of the processors being cache-line oriented such that they access a cache block (or "cache line") of memory at a time, as discussed further below.
In general, a processor's instruction set refers to a list of all instructions, and all their variations, that the processor can execute. Such instructions may include, as examples, arithmetic instructions, such as ADD and SUBTRACT; logic instructions, such as AND, OR, and NOT; data instructions, such as MOVE, INPUT, OUTPUT, LOAD, and STORE; and control flow instructions, such as GOTO, if X then GOTO, CALL, and RETURN. Examples of well-known instruction sets include x86 (also known as IA-32), x86-64 (also known as AMD64 and Intel® 64), AMD's Opteron, VAX (Digital Equipment Corporation), IA-64 (Itanium), and PA-RISC (HP Precision Architecture).
Generally, the instruction set architecture is distinguished from the microarchitecture, which is the set of processor design techniques used to implement the instruction set. Computers with different microarchitectures can share a common instruction set. For example, the Intel® Pentium and the AMD® Athlon implement nearly identical versions of the x86 instruction set, but have radically different internal microarchitecture designs. In all these cases the instruction set (e.g., x86) is fixed by the manufacturer and directly hardware implemented, in a semiconductor technology, by the microarchitecture. Consequently, the instruction set is traditionally fixed for the lifetime of this implementation.
FIG. 1 shows a block-diagram representation of an exemplary prior art system 100 in which multiple homogeneous processors (or cores) are implemented. System 100 comprises two subsystems: 1) a main memory (physical memory) subsystem 101 and 2) a processing subsystem 102 (e.g., a multi-core die). System 100 includes a first microprocessor core 104A and a second microprocessor core 104B. In this example, microprocessor cores 104A and 104B are homogeneous in that they are each implemented to have the same, fixed instruction set, such as x86. In addition, each of the homogeneous microprocessor cores 104A and 104B access main memory 101 in a common way, such as via cache block accesses, as discussed hereafter. Further, in this example, cores 104A and 104B are implemented on a common die 102. Main memory 101 is communicatively connected to processing subsystem 102. Main memory 101 comprises a common physical address space that microprocessor cores 104A and 104B can each reference.
As shown further in FIG. 1, a cache 103 is also implemented on die 102. Cores 104A and 104B are each communicatively coupled to cache 103. As is well known, a cache generally is memory for storing a collection of data duplicating original values stored elsewhere (e.g., to main memory 101) or computed earlier, where the original data is expensive to fetch (due to longer access time) or to compute, compared to the cost of reading the cache. In other words, a cache 103 generally provides a temporary storage area where frequently accessed data can be stored for rapid access. Once the data is stored in cache 103, future use can be made by accessing the cached copy rather than re-fetching the original data from main memory 101, so that the average access time is shorter. In many systems, cache access times are approximately 50 times faster than similar accesses to main memory 101. Cache 103, therefore, helps expedite data access that the micro-cores 104A and 104B would otherwise have to fetch from main memory 101.
In many system architectures, each core 104A and 104B will have its own cache also, commonly called the "L1" cache, and cache 103 is commonly referred to as the "L2" cache. Unless expressly stated herein, cache 103 generally refers to any level of cache that may be implemented, and thus may encompass L1, L2, etc. Accordingly, while shown for ease of illustration as a single block that is accessed by both of cores 104A and 104B, cache 103 may include L1 cache that is implemented for each core.
In many system architectures, virtual addresses are utilized. In general, a virtual address is an address identifying a virtual (non-physical) entity. As is well-known in the art, virtual addresses may be utilized for accessing memory. Virtual memory is a mechanism that permits data that is located on a persistent storage medium (e.g., disk) to be referenced as if the data was located in physical memory. Translation tables, maintained by the operating system, are used to determine the location of the reference data (e.g., disk or main memory). Program instructions being executed by a processor may refer to a virtual memory address, which is translated into a physical address. To minimize the performance penalty of address translation, most modern CPUs include an on-chip Memory Management Unit (MMU), and maintain a table of recently used virtual-to-physical translations, called a Translation Look-aside Buffer (TLB). Addresses with entries in the TLB require no additional memory references (and therefore time) to translate. However, the TLB can only maintain a fixed number of mappings between virtual and physical addresses; when the needed translation is not resident in the TLB, action will have to be taken to load it in.
As an example, suppose a program's instruction stream that is being executed by a processor, say processor core 104A of FIG. 1, desires to load data from an address "Foo" into a first general-purpose register, GPR1. Such instruction may appear similar to "LD<Foo>, GPR1". Foo, in this example, is a virtual address that the processor translates to a physical address, such as address "123456". Thus, the actual physical address, which may be formatted according to a global physical memory address format, is used to access cache 103 and/or memory 101.
In operation, each of cores 104A and 104B reference main memory 101 by providing a physical memory address. The physical memory address (of data or "an operand" that is desired to be retrieved) is first presented to cache 103. If the addressed data is not encached (i.e., not present in cache 103), the same physical address is presented to main memory 101 to retrieve the desired data. Main memory 101 may be implemented in whole or in part via memory module(s), such as dual in-line memory modules (DIMMs), which may employ dynamic random access memory (DRAM) or other memory storage.
In contemporary architectures, the processor cores 104A and 104B are cache-line (or "cache-block") oriented, wherein a "cache block" is fetched from main memory 101 and loaded into cache 103. The terms cache line and cache block are used interchangeably herein. Rather than retrieving only the addressed data from main memory 101 for storage to cache 103, such cache-block oriented processors may retrieve a larger block of data for storage to cache 103. A cache block typically comprises a fixed-size amount of data that is independent of the actual size of the requested data. For example, in most implementations a cache block comprises 64 bytes of data that is fetched from main memory 101 and loaded into cache 103 independent of the actual size of the operand referenced by the requesting micro-core 104A/104B. Furthermore, the physical address of the cache block referenced and loaded is a block address. This means that all the cache block data is in sequentially contiguous physical memory. Table 1 below shows an example of a cache block.
TABLE 1Physical AddressOperandXXX(7)Operand 7XXX(6)Operand 6. . .. . .XXX(1)Operand 1XXX(0)Operand 0
In the above example of table 1, the "XXX" portion of the physical address is intended to refer generically to the corresponding identifier (e.g., numbers and/or letters) for identifying a cache line address. For instance, XXX(0) corresponds to the physical address for an Operand 0, while XXX(1) corresponds to the physical address for an Operand 1, and so on. In the example of table 1, in response to a micro-core 104A/104B requesting Operand 0 via its corresponding physical address XXX(0), a 64-byte block of data may be fetched from main memory 101 and loaded into cache 103, wherein such cache block of data includes not only Operand 0 but also Operands 1-7. Thus, depending on the fixed size of the cache block employed on a given system, whenever a core 104A/104B references one operand (e.g., a simple load), the memory system will bring in 4 to 8 to 16 (or more) operands into cache 103.
There are both advantages and disadvantages of this traditional cache-block oriented approach to memory access. One advantage is that if there is temporal (over time) and spatial (data locality) references to operands (e.g., operands 0-7 in the example of Table 1), then cache 103 reduces the memory access time. Typically, cache access times (and data bandwidth) are 50 times faster than similar access to main memory 101. For many applications, this is the memory access pattern.
However, if the memory access pattern of an application is not sequential and/or does not re-use data, inefficiencies arise which result in decreased performance. Consider the following FORTRAN loop that may be executed for a given application:                DO I=1, N, 4        A(i)=B(i)+C(i)        END DO
In this loop, every fourth element is used. If a cache block maintains 8 operands, then only 2 of the 8 operands are used. Thus, 6/8 of the data loaded into cache 103 and 6/8 of the memory bandwidth is "wasted" in this example.
In multi-processor systems, such as exemplary system 100 of FIG. 1, main memory 101 can be configured to improve performance. FIG. 2 shows a block diagram illustrating a traditional implementation of main memory 101. As shown, memory module 202, which comprises memory (e.g., DRAMs) 203, is accessible via memory controller 201. That is, memory controller 201 controls access to memory module 202. Memory module 202 is commonly implemented as a DIMM (dual in-line memory module) that includes one or more DRAMs (dynamic random access memory) as memory 203. In general, a DIMM is a double SIMM (single in-line memory module). Like a SIMM, a DIMM contains one or several random access memory (RAM) chips on a small circuit board with pins that connect it to the computer motherboard.
Traditional DIMMs provide one data channel 205 and one address/control channel 204 per DIMM. In general, the address/control channel 204 specifies an address and a desired type of access (e.g., read or write), and the data channel 205 carries the corresponding data to/from the specified address for performing the desired type of access. Typically, a memory access operation requires several clock cycles to perform. For instance, address and control information may be provided on the address/control channel 204 over one or more clock cycles, and then the data is provided on the data channel 205 over later clock cycles. In a typical DIMM access scenario, a row select command is sent from memory controller 201 on the address/control channel 204 to the memory module 202, which indicates that an associated address is a row address in the memory cell matrix of the DRAM memory 203. In general, a data bit in DRAM is stored in a memory cell located by the intersection of a column address and a row address. A column access command (e.g., a column read or column write command) is sent from the memory controller 201 over the address/control channel 204 to validate the column address and indicate a type of access desired (e.g., either a read or write operation).
The row select command may be sent in a first clock cycle, then the column access command may be sent in a second clock cycle, and then some clock cycles later a burst of data may be supplied via the data channel 204. The burst of data may be supplied over several clock cycles. Typically, single DIMM data channel 205 is typically a 64-bit (8-byte) wide channel, wherein each access comprises a "burst" length of 8, thus resulting in the data channel carrying 64 bytes for each access. The length of the "burst" may refer to a number of clock cycles or phases of a clock cycle when dual-data rate (DDR) is employed. For instance, a burst length of 8 may refer to 8 clock cycles, wherein 8 bytes of data is communicated on the data channel for a given access in each of the 8 clock cycles (resulting in the data channel carrying 64 bytes of data for the access). As another example, a burst length of 8 may refer to 8 phases of a clock (e.g., when DDR is employed), wherein 8 bytes of data is communicated on the data channel for a given access in each of the 8 phases (over 4 clock cycles), thus resulting in the data channel carrying 64 bytes of data for the access.
To improve data channel bandwidth, tiling is commonly employed in memory architectures. For instance, rather than waiting for completion of a burst of data for one access operation before supplying address/control signals for a next access operation, the instructions supplied via the address/control channel 204 may be used to attempt to maintain full bandwidth utilization of the data channel 205. FIG. 3 shows an example of one traditional tiling technique. FIG. 3 shows a clock cycle 301 of a reference clock signal, wherein the illustrated example shows 20 clock cycles numbered 1-20. A clock phase 302 is also shown, wherein for each clock cycle the clock has a low phase ("L") and a high phase ("H"), as is well known. An address/control channel 303 is also shown, which corresponds to address/control channel 204 of FIG. 2. Also, in this example, a data channel 304 is shown, which corresponds to data channel 205 of FIG. 2.
The exemplary tiling technique of FIG. 3 allows for the address/control channel 303 to be used to maintain high bandwidth utilization of the data channel 304. In the illustrated example, a first memory access operation is requested, whereupon a row select command 306 is communicated from memory controller 201 to memory module 202 over address/control channel 303 during clock cycle 1. Then, during clock cycle 2, a column access command (e.g., column read or column write command) 307 for the first memory access operation is communicated from memory controller 201 to memory module 202 over address/control channel 303. After some delay, data channel 304 carries the data "burst" for the first memory access operation. For instance, beginning in the high phase of clock cycle 9 and ending in the low phase of clock cycle 13, data burst 308 carries the data for the first memory access operation. Traditionally, a single DIMM data channel, such as data channel 304, is typically a 64-bit (8-byte) wide channel where each memory access comprises a "burst" length of 8, thus resulting in the data channel carrying 64 bytes for each access. For instance, each of the 8 blocks of burst 308 (labeled 0/0/0-0/0/7) is typically an 8-byte block of data, thus resulting in burst 308 containing 64 bytes of data for the first memory access operation (read or write to/from the specified address).
A second memory access operation is requested in this example, whereupon a row select command 309 is communicated from memory controller 201 to memory module 202 over address/control channel 303 during clock cycle 5. Then, during clock cycle 6, a column access command 310 for the second memory access operation is communicated from memory controller 201 to memory module 202 over address/control channel 303. After some delay, data channel 304 carries the data "burst" for the second memory access operation. For instance, beginning in the high phase of clock cycle 13 and ending in the low phase of clock cycle 17, data burst 311 carries the data for the second memory access operation. As with the data burst 308 discussed above for the first memory access operation, data burst 311 typically has a length of 8 blocks (labeled 0/1/0-0/1/7) that are each an 8-byte block of data, thus resulting in burst 311 containing 64 bytes of data for the third memory access operation (read or write to/from the specified address).
As the example of FIG. 3 illustrates, rather than waiting for the data burst 308 for a first memory access operation to complete before providing the address/control information for the next memory access operation to be performed, the tiling technique uses the address/control channel 303 to effectively schedule the data bursts for different memory access operations back-to-back, thereby maintaining high bandwidth utilization on the data channel 304.
As also illustrated in FIG. 3, traditionally the data channel 205 of a DIMM carries a 64-byte burst of data for each memory access operation requested. Some DIMMs can support 64-byte or 32-byte accesses. That is, some DIMMs may be configured into either a 64-byte access or a 32-byte access mode. Thus, memory bandwidth may be conserved to some extent for certain memory access operations by performing a 32-byte access of the DIMM, rather than a 64-byte access (if the operation only requires access of 32 or fewer bytes). However, the full burst of either 32-bytes or 64-bytes is utilized for a single memory access operation.
In certain implementations, a plurality of DIMMs may share an address/control channel, and each DIMM may provide a separate data channel, wherein tiling may be employed on the address/control channel to maintain high bandwidth utilization on both data channels of the DIMMs. However, in these implementations, each DIMM provides only a single data channel.
As is well-known in the art, memory is often arranged into independently controllable arrays, often referred to as "memory banks." Under the control of a memory controller, a bank can generally operate on one transaction at a time. As mentioned above, the memory may be implemented by dynamic storage technology (such as "DRAMS"), or of static RAM technology. In a typical DRAM chip, some number (e.g., 4, 8, and possibly 16) of banks of memory may be present. A memory interleaving scheme may be desired to minimize one of the banks of memory from being a "hot spot" of the memory.
In most systems, memory 101 may hold both programs and data. Each has unique characteristics pertinent to memory performance. For example, when a program is being executed, memory traffic is typically characterized as a series of sequential reads. On the other hand, when a data structure is being accessed, memory traffic is usually characterized by a stride, i.e., the difference in address from a previous access. A stride may be random or fixed. For example, repeatedly accessing a data element in an array may result in a fixed stride of two. As is well-known in the art, a lot of algorithms have a power of 2 stride. This power of 2 stride gives rise to an increase in occurrences of bank conflicts because the power of 2 stride ends up accessing the same bank repeatedly. Accordingly, without some memory interleave management scheme being employed, hot spots may be encountered within the memory in which a common portion of memory (e.g., a given bank of memory) is accessed much more often than other portions of memory.
As discussed above, many compute devices, such as the Intel x86 or AMD x86 microprocessors, are cache-block oriented. Today, a cache block of 64 bytes in size is typical, but compute devices may be implemented with other cache block sizes. A cache block is typically contained all on a single hardware memory storage element, such as a single dual in-line memory module (DIMM). As discussed above, when the cache-block oriented compute device accesses that DIMM, it presents one address and is returned the entire cache-block (e.g., 64 bytes), as in the exemplary data bursts 308 and 311 discussed above with FIG. 3.
Some compute devices, such as certain accelerator compute devices, may not be cache-block oriented. That is, those non-cache-block oriented compute devices may access portions of memory (e.g., words) on a much smaller, finer granularity than is accessed by the cache-block oriented compute devices. For instance, while a typical cache-block oriented compute device may access a cache block of 64 bytes for a single memory access request, a non-cache-block oriented compute device may desire to access a Word that is 8 bytes in size in a single memory access request. That is, the non-cache-block oriented compute device in this example may desire to access a particular memory DIMM and only obtain 8 bytes from a particular address present in the DIMM.
As discussed above, traditional multi-processor systems have employed homogeneous compute devices (e.g., processor cores 104A and 104B of FIG. 1) that each access memory 101 in a common manner, such as via cache-block oriented accesses. While some systems may further include certain heterogeneous compute elements, such as accelerators (e.g., a GPU), the heterogeneous compute element does not share the same physical or virtual address space of the homogeneous compute elements. Accordingly, traditional memory interleave schemes have not attempted to address an interleave of memory accesses across heterogeneous compute elements, which may access memory in different ways, such as via cache-block and non-cache-block accesses.
U.S. Patent Application Publication No. 2007/0266206 to Kim et al. (hereinafter "Kim") proposes a scatter-gather intelligent memory architecture. Kim mentions that to avoid wasting memory bandwidth, the scatter/gather engine supports both cache line size data accesses and smaller, sub-cache line accesses. However, Kim does not appear to describe its memory architecture in detail. One of ordinary skill in the art would thus suppose that Kim may be employing the above-mentioned traditional DIMMs, which enable either a full cache line (e.g., 64 bytes) or a sub-cache line (e.g., 32 bytes) access. However, as with the traditional DIMMs, only a single data channel per DIMM appears to be supported. Kim does not appear to provide any disclosure of a DIMM architecture that provides more than a single data channel per DIMM.

Multiple data channel memory module architecture


A method, apparatus, and computer program product are disclosed for selectively prohibiting speculative conditional branch execution. A particular type of conditional branch instruction is selected. An indication is stored within each instruction that is the particular type of conditional branch instruction. A processor then fetches a first instruction from code that is to be executed. A determination is made regarding whether the first instruction includes the indication. In response to determining that the instruction includes the indication: speculative execution of the first instruction is prohibited, an actual location to which the first instruction will branch is resolved, and execution of the code is branched to the actual location. In response to determining that the instruction does not include the indication, the first instruction is speculatively executed.

1. Technical Field
The present invention is directed to data processing systems. More specifically, the present invention is directed to a method, apparatus, and computer program product for selectively prohibiting speculative conditional branch execution.
2. Description of Related Art
Superscalar processors enable concurrent execution of instructions. Superscalar processors can be implemented within symmetric multiprocessing (SMP) systems, simultaneous multi-threading (SMT) systems, or other types of computer systems. A symmetric multiprocessing (SMP) data processing system has multiple processors that are symmetric such that each processor has the same processing speed and latency. An SMP system has one operating system that divides the work into tasks that are distributed evenly among the various processors by dispatching one software thread of work to each processor at a time. Thus, a processor in an SMP system executes only one thread at a time.
A simultaneous multi-threading (SMT) data processing system includes multiple processors that can each concurrently execute more than one thread at a time per processor. An SMT system has the ability to favor one thread over another when both threads are running on the same processor.
Known computer systems, including SMP systems, SMT systems, and other systems, typically speculatively execute conditional branch instructions in order to improve processing efficiency within the systems. A fetch engine in the processor speculates past a branch instruction in order to supply a continuous instruction stream to the decode, dispatch, and execution pipelines in order to maintain a large window of potentially executable instructions.
Instruction fetch performance depends on a number of factors. Branch prediction accuracy has been long recognized as an important factor in determining fetch performance.
Modern microprocessors routinely use a plurality of mechanisms to improve their ability to efficiently fetch past branch instructions. The prediction mechanisms allow a processor to fetch beyond a branch instruction before the outcome of the branch is known. For example, some mechanisms allow a processor to speculatively fetch beyond a branch before the branch's actual target address has been computed. These techniques use run-time history to speculatively predict what the actual target address will be. Thus, these techniques speculatively predict which instructions should be fetched by predicting what the actual target address will be.
A significant pipelined delay penalty may result from the execution of "conditional branch" instructions. Conditional branch instructions are instructions which dictate the taking of a specified conditional branch in response to a particular outcome of the processing of one or more other instructions. The conditional branch will be either taken or not taken. If the conditional branch is taken, processing will pass to a particular target address that is not the next sequential address in the code that is being processed. The instructions that are stored starting at this non-sequential target address will then be processed. If the conditional branch is not taken, processing will fall through to the next sequential address in the code. The instructions that are stored starting at this next sequential address are then processed.
Conditional branch instructions can be speculatively processed by predicting in advance whether the conditional branch instruction will be taken or not taken. If it is predicted that the conditional branch will be taken, the instructions that are stored starting at the particular non-sequential target address are speculatively executed. If it is predicted that the conditional branch will not be taken, the instructions that are stored starting at the next sequential address are speculatively executed. When the conditional branch instruction is actually resolved, it becomes known whether the conditional branch instruction will be taken. If the prediction was correct, processing continues with the speculatively executed instructions being completed. If the prediction was incorrect, the speculatively executed instructions must be flushed from the processor and the correct instructions retrieved and executed. Flushing speculatively executed instructions consumes power and processing resources unnecessarily.
With power consumption becoming an ever more critical aspect of microprocessor-based system design, mechanisms to prevent unnecessary work are of significant value to the design. Current microprocessors perform a significant amount of unnecessary work by incorrectly predicting branch instructions in which the wrong instructions past the branch are fetched, decoded, and executed only to be flushed when it is discovered that the predicted target address did not turn out to be the actual target address. Thus, the speculatively executed instructions are on the wrong path.
In modern microprocessors, branch instructions are typically predicted to be taken or not taken early in the decoding phase of instruction processing. If the actual condition on which the branch is dependent turns out to be opposite of the predicted value, the instructions which followed the branch need to be flushed and the execution of the program resumed with the correct path for the branch.
In a power sensitive environment, speculating on branches when the prediction accuracy is low is a poor trade off of performance verses power, thus lowering the performance per watt of power used. When the processor is in a simultaneous multithreaded (SMT) mode, inaccurate speculation consumes power as well as cycles that another thread in the processor could have exploited.
Branch prediction accuracy can vary widely depending upon many factors. If the consumption of power is an important factor, turning off branch prediction can reduce power substantially but may also substantially reduce performance.
Therefore, a need exists for a method, system, and computer program product for preventing speculative execution for particular types of branch instructions that tend to be unreliably predicted in order to save power and improve processor performance.

Selectively prohibiting speculative execution of conditional branch type based on instruction bit

International Business Machines Corporation


An adjustable channel installation bracket for supporting pre-cast drain channels on rebar studs with a support member securable thereto by set screw receptacles formed in the support member and having biased engagement plates engaged to the support member which capture the drain channel therebetween.

1. Field of the Invention
The present invention relates to brackets utilized in the installation of pre-cast drain channels for surface water drainage. When graded ground is provided with engineered surface water drainage, a wide variety of pre-formed trench drains are available to choose from to simplify the drainage surface construction. Pre-formed channels offer design flexibility, ease of installation, and cost savings over hand forming methods. The channels are positioned sequentially, creating a continually shaped channel run. Installation of the channels requires them to be suspended or positioned in a drain trench usually secured to an adjacent concrete pour form or a structure to be embedded in the concrete. The channel must be secured in a rigidly fixed position so that it will maintain proper alignment and height during the concrete pour.
In a preferred method of pre-formed channel installation, concrete is poured into the drain trench to surround the bottom and sides of the channel so that the top of the channel is level with the top of the finished concrete drain surface. Reinforcing bar stock, rebar, is secured in position to be embedded in the concrete below and/or surrounding the channel. A bracket is secured to the rebar to hold the channel in position with respect to the rebar and thereby with respect to the finished drainage surface. Securing the channels to the rebar permits accurate alignment of the channels and prevents them from floating during the concrete pour. The bracket which secures the channel to the rebar, to hold the channel in position for the concrete pour, is the subject of the present invention.
2. Description of the Prior Art
A primary type of installation bracket utilized for positioning a pre-formed channel in a drain trench preparatory to a concrete pour has a specialized configuration. A pair of uniquely formed securing clamps is positioned in opposing relation to engage lateral projections disposed on the sides of a pre-formed channel. The clamps are mounted on a U-shaped stand which is secured to the underlying rebar. The stand forms two upward projecting legs which serve to maintain the clamps at the proper height and keep the channels from floating during the concrete pour. A securing bolt, extending between the clamps and the two legs of the U-shaped stand, biases the securing clamps toward the sides of the channel and keeps the entire bracket attached to the channel. The lower crossbar of the U-shaped stand has a pair of holes to permit the stand to be mounted on a pair of upward projecting ends of rebar or studs mounted in the drain trench which is to be filled with concrete.
The present invention is an adjustable channel installation bracket. It comprises a support member having a pair of receptacles formed at the ends thereof for engaging a pair of free-standing positioning members. A means is provided for adjustably securing the support member to the positioning members. A pair of channel engagement plates are adjustably engaged to the support member for infinitely adjustable positioning therealong. The plates project perpendicularly from the support member and are formed for engaging a pre-formed channel The bracket includes a tensioning means for biasing the plates to capture a channel therebetween.
It is therefore an important object of the present invention to provide a new and novel bracket for securing pre-formed drain channels to structural rebar.
It is another object of the present invention to provide a design for a bracket for installing pre-formed channels which permits adjustable height positioning on the underlying and upward projecting supporting structure.
It is a further object of the present invention to provide a design for a bracket for installing pre-formed channels which permits unlimited lateral movement of the side clamps for installing different width channels.
It is still another object of the present invention to provide a design for a bracket which permits infinite lateral adjustment of the side clamps for permitting perfect lateral alignment of the successive channels.
It is yet a further object of the present invention to provide a simplified design for a bracket for installing pre-formed channels which can be made from sheet and bar stock metal.
And it is yet another object of the present invention to provide a new and novel bracket for installing pre-formed channels which is relatively inexpensive to manufacture and does not require complex or costly machining tools.
Other objects and advantages of the present invention will become apparent when the apparatus of the present invention is considered in conjunction with the accompanying drawings.


In a child seat and a method for child-seat detection, a radio-identification chip, which is configured solely as a transmitter module, is introduced in the child seat, preferably in the fabric. Due to the automatically emitted radio signal, the child seat is detected by corresponding receiver devices in the vehicle.

A transponder system for child-seat detection is known from German Patent Application No. DE 198 21 501. This system utilizes radio signals to detect a child seat, the child-seat detection in the vehicle seat transmitting a radio signal to the transponder in the child seat, which then retransmits this signal in changed form. The child-seat identification is carried out on the basis of the retransmitted radio signal.

Child seat and method for recognition of a child seat


A method in which a crude chlorinated product of a phenylpropanolamine, preferably prepared by reacting thionyl chloride with the phenylpropanolamine, is purified by contacting an aqueous solution of the crude product with carbon. The carbon-treated solution of the crude chlorinated product of a phenylpropanolamine is catalytically hydrogenated to the corresponding amphetamine derivative.

1. Field of the Invention
The invention relates to the preparation of d- or d,l-amphetamines.
2. Description of Related Art
U.S. Pat. No. 6,399,828 identifies two prior art techniques for synthesizing amphetamine from norephedrine.
In one approach, norephedrine first is refluxed with hydrogen iodide and red phosphorus. In the other approach, norephedrine is chlorinated using thionyl chloride and then the chlorinated product, following recrystallization from a methanol-isopropyl ether solution, is subjected to hydrogenolysis (catalytic hydrogenation) over a palladium-on-carbon catalyst.
Tomie et al., Chem. Pharm. Bull., Vol. 24, No. 5, pp. 1033-1039 (1976) describes a synthesis of threo-2-amino-1-chloro-1-phenylpropane from norephedrine by reaction with thionyl chloride in chloroform (trichloromethane —CHCl3). Following in vacuo concentration and an ether wash, the product was recrystallized from an ethanol solution.
Knie{hacek over (z)}o et al., Collection Czechoslovak Chem. Commun., Vol. 46, No. 3, pp. 717-724 (1981) describes a synthesis of threo-1-chloro-1-phenyl-2-propylammonium chloride from norephedrine by reaction with thionyl chloride in benzene (C6H6). Following vacuum concentration ("the mixture was taken down"), the product was recrystallized from an ethanol solution.
Workers continue to look for improved ways of making amphetamines, such as from phenylpropanolamines, such as norephedrine.


This invention relates to an aluminum nitride sintered body comprising aluminum nitride crystal grains, in which the grains have a given content of a rare earth element (a conversion content as an oxide thereof), a given average grain size, and a given spin amount per unit mg of aluminum obtained from a spectrum as measured by an electron spin resonance method. The aluminum nitride sintered body is used in an electronic functional material and an electrostatic chuck.

1. Field of the Invention
This invention relates to an aluminum nitride sintered body having a relatively small temperature dependence of volume resistivity as well as an electronic functional material or an electrostatic chuck employing the same, respectively.
2. Description of Related Art
At present, an electrostatic chuck is used for attracting and holding a semiconductor wafer in film forming processes for the semiconductor wafer such as transportation, light exposure, chemical vapor deposition (CVD), sputtering and the like, or steps of micromachining, cleaning, etching, dicing and the like. As a substrate for the electrostatic chuck, dense ceramics have been used. Particularly, a halogenous corrosive gas such as CIF.sub.3 or the like is frequently used as an etching gas or a cleaning gas in an apparatus for the manufacture of the semiconductor. A dense aluminum nitride has a high corrosion resistance against the above halogenous corrosive gas. And the aluminum nitride is also known to be a high thermal conductive material and to be high in the thermal shock resistance. Therefore, it is considered that it is preferable to make the substrate of the electrostatic chuck for the semiconductor manufacturing apparatus from an aluminum nitride sintered body.
In general, when the aluminum nitride is used as a substrate material for the electrostatic chuck, it is preferable that a volume resistivity is within a range of 10.sup.8 -10.sup.13 .OMEGA..multidot.cm at operating temperatures. However, the volume resistivity of aluminum nitride extremely decreases, for example, from 10.sup.16 .OMEGA..multidot.cm to not more than 10.sup.7 .OMEGA..multidot.cm over a range of room temperature to 600.degree. C., so that it is impossible to develop the stable operation as the electrostatic chuck in such a wide temperature range. Therefore, a service temperature of the electrostatic chuck is limited to a range of, for example, 200-400.degree. C.

Aluminum nitride sintered body, electronic functional material, and electrostatic chuck


The present invention relates to an in-vessel or down-hole optical imaging sensor or system for operating in structures which may contain media with different spectral transmission characteristics. The imaging sensor of the present invention selectively emits and/or detects two or more independently controllable wavelengths or wavebands. The imaging sensor comprises an illuminator for emitting radiation of a specified wavelength or waveband through a medium to a target, at least one detector for detecting the radiation deflected by said target and at least one amplifier for providing non-linear amplification of the detector output.

1). Field of the Invention
The present invention relates to an optical system image sensor operating in structures which may contain media with different spectral transmission characteristics; for example, in vessels containing both crude oil and water, either by rendering all media transparent simultaneously, or, on command, by rendering one or more of the media opaque to allow its detection.
2). Discussion of Related Art
In the oil industry, amongst others, it is necessary to inspect surfaces for cracks, corrosion, scale or other defects or characteristics, to examine welds to establish the integrity of a structure and ascertain the need for repair or replacement. It is desirable to use a single sensor to inspect internal surfaces of structures such as tanks, wells and pipelines containing crude oil and water, and also distinguish between oil and water, without emptying, flushing or cleaning the structure. It is also desirable to inspect surfaces coated with oil or wax in air.
Image sensors operating in structures containing fluids transparent in the visible region of the electromagnetic spectrum such as water are well-known, and disclosed, for example, in EP0846840, EP0264511, and WO0206631.
Operation may be extended to opaque fluids by flushing the vessel with a transparent fluid in the vicinity of the image sensor, and a method for doing this is disclosed in U.S. Pat. No. 4,238,158.
An image sensor operating directly in fluids which are opaque in the visible region of the spectrum but transmit energy at other wavelengths, for example, crude oil, is disclosed in GB2332331B. Transmission in these fluids may be limited, restricting operation of a practical sensor to close range.
The absorption at a given wavelength varies widely for different crude oils, but the general shape of each plot of absorption against wavelength is very similar and transmission "windows" occur at the same wavelengths in the spectrum, as shown in U.S. Pat. No. 5,266,800 which discloses a method for using infrared absorption measurements to discriminate between different crude oils.
As well as discriminating between crude oils, is also possible to distinguish between other fluids by measuring their spectral absorption characteristics, as disclosed in U.S. Pat. No. 4,994,671.
It is an object of the present invention to enable an image sensor to operate within, and also by remote command or autonomous internal control to discriminate between, media such as crude oil and water, which have transmission bands in different regions of the spectrum.


A system (10) is disclosed for facilitating the healing of traumatized tissue and broken or fractured bone. The system (10) establishes an electric field between a pair of electrodes (14) positioned on opposite sides of the patient site (12), resulting in the production of an alternating current having the desired frequency and amplitude characteristics in the tissue or bone. Specifically, the system (10) includes a resonator (32) formed by an inductor (36) coupled in series with the resistor (R1) and capacitor (C1) of an equivalent circuit (34) representing the patient site (12), the electrodes (14), and any gaps (30) therebetween. This resonator (32) also includes a capacitor (C2) designed to prevent spurious, high-frequency oscillations. The resonator (32) is operated by a free-running oscillator (16), which maintains the operation of the resonator (32) at its resonant frequency. The oscillator (16) includes a forward network (38), having a CMOS inverter (42), and a feedback network (40), including an open-loop operational amplifier (52), and provides a simple, stable, and efficient form of operation free from spurious, high-frequency oscillations.

The use of electric current to facilitate the healing of traumatized tissue and broken or fractured bone has been recognized for some time. The stimulative effect of such current appears to occur whether the flow of current is induced naturally, by internal body mechanisms, or artificially, by external sources. While the natural flow of current produced by electrochemical, myoelectric, and piezoelectric-like body mechanisms advantageously facilitates healing without external circuitry, is some instances it is desirable to expedite the recuperative process by artificially supplementing the natural current flow.
A variety of different techniques have been devised for establishing supplemental electric currents in tissue and bone. Briefly, such techniques can be grouped according to both the type of current developed and the manner in which the current is established. Considering first the type of current product, the current may be characterized as either a direct (DC) current, having an amplitude that is substantially constant as a function of time, or an alternating (AC) current, which exhibits a time-dependent amplitude variation. The use of AC current is preferred because it can be established in a variety of ways, discussed below. DC current, on the other hand, can be induced only by providing a direct electrical connection between the tissue or bone and the energy source.
The manner in which the auxiliary stimulating current is induced offers several alternative forms of classification. First, such techniques can be categorized as being either invasive or noninvasive, depending on the connections provided to the patient. Invasive techniques involve the application of electric current directly to the site of the trauma or fracture through electrodes implanted at the site. While this approach minimizes the electric potential required to generate a particular desired current in the tissue or bone, it also involves the expense and risk of infection attendant surgical implantation procedures. As a result, noninvasive procedures, in which the flow of electric current in the tissue or bone is induced by apparatus external to the patient, are preferred.
The noninvasive establishment of an AC current in tissue or bone can be further grouped according to the electric principle involved in its production. For example, a resistive approach involves the conduction of current directly to the patient through special electrodes coupled to the patient's skin via conductive gel. This technique has the disadvantage of requiring good electrical contact between the electrodes and the skin, necessitating the periodic replacement of the conductive gel on the electrodes.
A second, or inductive, approach employs magnetic fields to establish the desired AC current in the tissue or bone. Specifically, this approach involves the application of an electric current to magnetic coils positioned proximate the patient. The flow of current through the coils produces a magnetic field in the patient's bone or tissue, resulting in the establishment of the desired alternating therapeutic current. This approach has a number of disadvantages including the required use of bulky magnetic coils and an energy source having an output whose frequency and waveform are sufficient to induce the desired stimulating currents at the patient site. The inductive approach also involves relatively large energy losses attributable to the heating of the coil windings produced by the flow of current therethrough.
The third technique for noninvasively establishing an alternating current in the patient's tissue or bone can be referred to as the "capacitive" or electric field approach. This technique typically employs a pair of electrodes that are placed on opposite sides of the treatment site and are insulated from the patient's skin. Energy applied to the electrodes establishes an electric field between the electrodes, normal to the skin. It is this electric field that induces the alternating therapeutic current at the treatment site.
While this approach overcomes the difficulties outlined above with respect to the resistive and inductive techniques, the capacitive production of therapeutically effective levels of electric current at the patient treatment site traditionally presents several additional problems. For example, the patient's skin normally contributes a series capacitive reactance to the equivalent electric circuit representative of the elements between the electrodes. In addition, a much larger variable capacitive reactance is exhibited by the insulative, dielectric "gaps" between the electrodes and the patient.
The combined series capacitive reactance of the elements between the electrodes has been a problem for several reasons. First, this reactive component seriously attenuates the current flow produced by a given potential applied to the electrodes. To understand how this energy loss arises, it may be helpful to briefly review the dynamics of interaction between the stimulating current and the patient. To be therapeutically effective, the electric current must have a frequency that is low enough to ensure its penetration to the site of the fracture or trauma. This requirement is imposed because a high-frequency current will concentrate near the dermal region of the patient in response to a mechanism known as the "electromagnetic skin effect." Other, biological, mechanisms that limit the efficacy of relatively high-frequency current also likely exist.
At therapeutically effective frequencies, the peak energy stored electrostatically in both the electrode-to-skin interface and the epidermis during one cycle of the alternating voltage potential applied to the electrodes is substantially larger than the energy absorbed by the tissue and bone being treated. This stored energy is typically either dissipated within the source or radiated as electromagnetic energy, resulting in a system inefficiency or energy loss. As a result, high levels of reactive power are required to establish the desired therapeutic current.
As an alternative to the use of higher voltages to compensate for energy losses, an inductive reactance can be employed to produce a resonant circuit that reduces the energy losses. For example, a series-connected inductor can be used to recapture the stored energy and apply it to the treatment site during the next cycle of the alternating voltage applied to the electrodes. As a result, only a relatively small amount of energy is dissipated and radiated.
Even with energy losses reduced, the capacitive technique of establishing therapeutic current in tissue and bone still presents several problems. As noted previously, the capacitive reactance exhibited by the dielectric gaps between the electrodes and the patient represents a rather large and variable electrical impedance to the drive circuit. The addition of the inductor to form a resonant circuit having a high quality factor Q, provides a significant reduction in impedance when the circuit is operated at its resonant frequency. Because the capacitive portion of the circuit may vary substantially in response to both the condition of the patient and movement between the electrodes and patient, when a fixed inductance is employed the circuit can be maintained at resonance only by adjusting the frequency of the driver to correspond to the resonant frequency of the circuit as it varies with the changing capacitance. Alternatively, a variable inductive reactance can be used to negate the effect of the changes in capacitance, leaving the resonant frequency of the circuit unchanged.
In U.S. Pat. No. 4,459,988 (Dugot) a circuit is disclosed that employs the former technique. Specifically, a portion of the patient positioned between stimulating electrodes is included in a series resonant circuit incorporating positive feedback to maintain the frequency of the stimulating signal at the resonant frequency of the circuit. The Dugot approach, however, suffers from several disadvantages. First, the disclosed implementation is relatively complex and involves a large number of components. Positive feedback is required to stabilize the circuit with respect to frequency and automatic gain control is preferably employed to regulate the amplitude of the signal generator's output. In addition, the output produced by the circuit may be subject to spurious and multiple high-frequency oscillations decreasing the efficiency of the system. The use of a variable inductive reactance to maintain a constant resonant frequency in the presence of capacitive changes disadvantageously requires the use and expense of some form of adjustable inductor and feedback to control it.
In light of the preceding remarks, it would be desirable to provide a method and apparatus for noninvasively establishing a regenerative electric current in traumatized tissue and broken or fractured bone. It would further be desirable to employ a capacitive technique of establishing such a current that is simple, does not require a high-voltage potential to overcome large variable gap capacitances, is inherently stable with respect to both frequency and amplitude, and rejects high-frequency spurious oscillations.

Method and apparatus for capacitively regenerating tissue and bone


A method for introducing a foreign matter into a cell, includes the steps of placing a small particle carrying a foreign matter at a part of a cell surface of a living cell, boring a hole in a cell wall and/or a cell membrane by irradiating and treating said part of the cell surface with a laser beam, and introducing the foreign matter into the living cell.

1. Field of the Invention
The present invention relates to a method for introducing a foreign matter into a living cell. Particularly, the invention relates to such a foreign matter into a living cell with use of a laser.
2. Related Art Statement
As the methods for introducing foreign matters such as genetic substances into living cells, there are known a method for directly introducing a foreign matter into a living cell with use of a fine glass tube (microinjection method), a method for applying a cell-fusing technique (a liposome fusion method and a protoplast fusion method), an introducing method using an infecting process (Agrobacterium method), a membrane permeability-promoting method (electropolation method), etc. when broadly classified.
According to the microinjection method, a fine micropipette is prepared with use of a glass tube, connecting it with a micromanipulator, and introducing a liquid of a foreign matter into a cell one by one under observation with a microscope. This method is a relatively excellent foreign matter-introducing method for animal cells having no cell walls.
As the cell fusion technique-applying methods, there are the liposome method, the protoplast fusion method, etc. According to the protoplast fusion method, a colon bacterium (Escherichia coli) or the like having a coloned plasmide is converted to a protoplast, which is fused with a target cell with ethylene glycol or the like, thereby introducing a gene into the cell. According to the liposome fusion method, a foreign matter is introduced into a target cell by using a liposome as a carrier.
As the infection process-utilizing introduction method, there is known a method for introducing a gene by using an Agrobacterium carrying a Ti plasmid.
According to the electropolation method based on the promotion of the membrane permeability, a cell is suspended in a solution of a foreign matter, and DC pulses at a high voltage upon the resultant solution or suspension, and thereby the foreign matter is introduced into the cell.
There is also known a gene gun method in which a fine metal particle coated with DNA is thrown into a cell of an organism, and thereby a gene is introduced into the cell through a cell wall and/or cell membrane. Furthermore, there is recently known a method for introducing a foreign matter into a cell by using a laser. These conventional methods for introducing the foreign matters into the cells are used mainly for transforming the target cells through the introduction of the foreign genes.
However, with recent progresses in the biotechnology, there have been the possibilities in diagnosis and treatment of diseases, molecular breeding of agricultural products imparted with various tolerances, production of useful products including livestock, etc. through introducing, into living cells, foreign matters other than genes, e.g., organella such as chloroplasts, nuclei, chromosomes and mitochondoria, physiologically active materials and indicators, functional proteins. Further, production of directly transformed bodies having undergone de-differentiation nor re-differentiation is conceivable in the case of plant, for example, by each introducing a foreign matter in a specified one cell of a living tissue. Thus, molecular breeding of many useful plants of which dedifferentiation or re-differentiation system has not been established are expected. Production of transformed products in which a plurality of characters are simultaneously transformed by simultaneously introducing plural kinds of foreign matters into a cell or cells is expected as well.
From this point of view, there are problems of difficulties in introducing foreign matters other than genes in the case of a gene-introducing method utilizing agrobacteria carrying Ti plasmides, a protoplast fusion method and a gene gun method among the conventional foreign matter-introducing methods. Further, a microinjection method, a liposome method and an electropolation method have a possibility of introducing foreign matters other than genes into cells, but the liposome method and the electropolation method require protoplast conversion when applied to plant cells, so that these methods have a problem in that there is difficulty in introducing a foreign matter into a specified one cell of a specific tissue. The microinjection method has a problem in that there is extremely difficult because skill is needed to handle a plant cell having a cell wall tougher than that of an animal cell. On the other hand, a laser method can introduce a foreign matter into a given target cell, but the conventional laser method has a problem in that there is difficulty in stably introducing foreign matters having extremely small physical strength, such as artificial chromosomes, or macrostructural bodies such as organella into cells.
Therefore, there have been strongly desires for development of a method which is easy to manipulate and has general-purpose applicability, which transforms a specified one cell as a target cell, and which can introduce a foreign matter such as not only genes but also foreign matters having extremely small physical strength, such as artificial chromosomes, or foreign matters such as macrostructural bodies such as organella into living cells irrespective of species of organisms. However, such a method for introducing the foreign matter into the living cell has not been known yet.

Method for introducing foreign matters into living cells


In a first aspect, the invention involves a reactive support useful for automated synthesis of oligonucleotides. The reactive support comprises a label moiety (e.g. hapten) covalently bonded via a stable bond to a trifunctional spacer. The labeled trifunctional spacer complex is covalently bonded to a solid support via a cleavable bond. One arm of the trifunctional spacer attaches the solid phase; another arm attaches the label; while the third arm provides a hydroxyl group useful for synthesizing a labeled oligonucleotide. Upon synthesis, the cleavable bond is broken, yielding the labeled oligonucleotide. Methods for labeling oligonucleotides and useful kits are also described.

Labeled oligonucleotides find utility in a number of applications, including DNA sequencing, diagnostic detection or quantitation and forensic science. Typically, the labeled oligonucleotide is allowed to hybridize or anneal with nucleic acid present in the sample and the presence or absence of label is detected following separation steps.
Many mechanisms and schemes have been used to introduce labels into oligonucleotides. For a comprehensive review of these methods see Goodchild, Bioconjugate Chemistry, 1(3):165-187 (1990). According to Goodchild, past researchers have labeled oligonucleotides at both internal and terminal locations; by enzymatic and chemical synthetic means; and utilizing a single or many marker molecules per oligonucleotide. Methods involving incorporation of marker moieties at internal locations in the oligonucleotides are generally less preferable due to their less predictable hybridization behavior. For this reason, end labeled oligonucleotides are often preferred, particularly for automated detection systems. Similarly, methods for incorporating multiple label moieties into an oligonucleotide are less preferred for stoichiometric reasons. For some applications, it may not be critical that the labeled oligonucleotides are poorly characterized in terms of exact positioning and number of label molecules. However, for automated diagnostic detection, it is desireable that each oligonucleotide have a single marker moiety, preferably a haptenic "hook", at an end location.
Methods for placing a single marker, or hapten capable of reacting with an antibody or other specific binding member, at a terminal position on an oligonucleotide have been described in the literature. Most commonly, a linker member containing a primary amine or other nucleophilic group is attached at the 3' or 5' terminus to enable conjugation to one of numerous electrophilic detectable markers. Alternatively, terminal deoxynucleotidyl transferase, ligase and phosphoramidite chemistry have been used to attach direct labels or reactive linkers to oligonucleotides.
For example, Kempe, et al., Nucleic Acids Research 13(1):45-57 (1985) describe methods for post-synthesis biotinylation of 3' termini. A first method involves oxidation of the 3' hydroxyl to a 3' aldehyde, followed by condensation with alkyl diamines to provide a reactive amine for condensation with biotin. In a second method, biotin is attached to the 3' end by RNA ligase.
Zuckermann, et al., Nucleic Acids Research 15(13): 5305-5321 (1987) describe nucleosides modified to incorporate a linker arm having a disulfide link. The disulfide link is used to attach the modified nucleoside to a solid support. The nucleoside is then able to undergo oligonucleotide synthesis according to the phosphoramidite or phosphotriester methods. Following synthesis, the disulfide is cleaved and the free sulfhydryl group may be used to attach a fluorescent reporter to the probe.
Nelson, et al., Nucleic Acids Research 17(18): 7187-7194 (1989) describe a control pore glass solid support having incorporated therein a multifunctional agent having both a masked amino group and a protected hydroxyl group. The hydroxyl group can be used for standard synthesis of oligonucleotides as is known in the art. The protected amino group can be used after synthesis and cleavage of the oligonucleotide for coupling to a reporter molecule. This approach is very much like the commercially available 3' Amine-On CPG.TM. (Clontech, Palo Alto, Calif.)
Each of the above methods have been used successfully to incorporate detectable marker compounds into 3' end positions of oligonucleotides. However, none is readily adaptable to automated synthesis such that the label moiety can be added to the oligonucleotide prior to cleavage from the support. In each case, the oligonucleotide must undergo one or more additional steps to couple the label to the cleaved oligonucleotide. This is undesirable if commercial quantities of labeled oligonucleotides are to be produced by automated synthesis.
Accordingly, the present invention seeks to overcome the problems associated with prior methods, and to provide a solid support and a method for 3' end labeling of oligonucleotides which is amenable to automated chemical synthesis. A method for preparing such a novel support is also described.

Methods, kits, and reactive supports for 3' labeling of oligonucleotides


Method of laying a tire belt ply on a form, using one or two transporters each having gripping assemblies, for gripping and moving the gripping sectors situated at the two longitudinal ends of a belt ply. The front and rear edges are aligned, after having determined the angular geometry of the ply edges by means of a suitable device, by independently moving at least one gripping sector of each of the front and rear areas. This method makes it possible to align edges having high curvatures and to lay a belt ply on a curved form.

The invention relates to a method, and an apparatus for implementing the method, for shaping and depositing a tire belt ply on a laying drum.
The manufacture of radial carcass tires makes it necessary to deposit, generally during the step of producing and assembling the uncured tire, a set of belts whose function is, amongst other things, to provide the clamping of the crown of the tire. These belt plies are formed from rectilinear cords, often metallic, coated in a rubber mixture, disposed parallel to each other and forming a given angle with the longitudinal direction of the ply.
The requirements of productivity and quality have for many years been leading tire manufacturers to seek a way for automating these laying operations. Thus the practice which consists of first depositing the belt plies on generally cylindrical laying forms and manually adjusting edge to edge the front and rear edges of the ply, and then gripping the crown belt thus produced by means for example of a transfer ferrule and bringing it onto an outer casing previously shaped in the form of a torus, has become widespread.
However, there remains a great difficulty in producing, without the intervention of a skilled operator, an edge to edge joining of the two ply ends with the precision compatible with the quality requirements of modern tires, avoiding superimpositions, offsets or openings.
Several prior art methods for automatically depositing the belt ply on a cylindrical form have been disclosed. These methods describe apparatus and methods whose aim is to control both the ply length, the angular geometry of the ply edge and the positioning of the ply at the time of laying.
Thus, Okuyama et al. U.S. Pat. No. 4,769,104 proposes a method comprising a set of movable arms for aligning and shaping the edge of a ply previously deposited on a magnetic belt. The necessarily rectilinear shape of the arms does not make it possible to correct curved ply edges. In addition, the successive action of the arms tends to modify the previously achieved alignment of the boundary at the time of the alignment of the edge, causing an offset at the bringing together of the two ends of the ply once deposited on the form.
Published EP 0 649 730 reports on a method in which the shape of the ply is analysed by cameras. By comparison with a predetermined theoretical shape, an automatic control system determines the transverse movements to be effected at the time of the deposition of the ply from an unwinding belt to a magnetic belt. This system nevertheless proves ineffective in precisely correcting the angular deviations of the ply edge with respect to a pre-established value.
U.S. Pat. No. 4,857,123 discloses a method in which the geometry of the ply edge is assessed by measuring the difference between the successive positions of a floating blade able to move in the direction perpendicular to the cutting edge, and a predetermined theoretical cutting line. The angular correction is made by a manipulator gripping the ply edge and effecting a controlled rotation about an axis perpendicular to the plane of the ply and passing substantially through the center of the edge, making the toe and heel of the ply undergo identical angular corrections.
U.S. Pat. No. 5,092,946 improves this last method by proposing depositing the ply on a succession of magnetic belts for holding the ply edge in position at each step of the laying cycle. These methods prove relatively effective for correcting rectilinear edges but do not make it possible to juxtapose two ply ends exhibiting a marked curvature of the edges edge-to-edge without causing superimposition, separation or opening.
It will be noted that the difficulties encountered during attempts at automation of the operation of juxtaposition of the edges of two ply ends relate to the very nature of the product, which has a high tendency to vary its length and/or the angle of the cords under the action of a transverse or longitudinal stress. In addition, the release of the stresses after the cutting has a tendency to deform the edge of the ply by curving the cords in a random manner particularly in the area of the toe. The ply is then said to stick-up.


A method of screening for binding partners of a specific molecule. The method employs a chimeric protein having at least two different binding regions; one containing at least a portion of the specific molecule or an analog thereof, and the other containing a binding region of an immunoglobulin chain. In a preferred embodiment, the method is used for rapidly screening member compounds of a combinatorial library for potential biological activity.

A significant recent development in pharmaceutical drug discovery and design has been the development of combinatorial chemistry to create chemical libraries of potential new drugs. Chemical libraries are intentionally created collections of different molecules; these molecules can be made by organic synthetic methods or biochemically. In the latter case, the molecules can be made in vitro or in vivo.
Combinatorial chemistry is a synthetic strategy in which the chemical members of the library are made according to a systematic methodology by the assembly of chemical subunits. Each molecule in the library is thus made up of one or more of these subunits. The chemical subunits may include naturally-occurring or modified amino acids, naturally-occurring or modified nucleotides, naturally-occurring or modified saccharides or other molecules, whether organic or inorganic. Typically, each subunit has at least two reactive groups, permitting the stepwise construction of larger molecules by reacting first one then another reactive group of each subunit to build successively more complex and potentially diverse molecules.
By creating synthetic conditions whereby a fixed number of individual building blocks, for example, the twenty naturally-occurring amino acids, are made equally available at each step of the synthesis, a very large array or library of compounds can be assembled after even a few steps of the synthesis reaction. Using amino acids as an example, at the first synthetic step the number of resulting compounds (N) is equal to the number of available building blocks, designated as b. In the case of the naturally-occurring amino acids, b=20. In the second step of the synthesis, assuming that each amino acid has an equal opportunity to form a dipeptide with every other amino acid, the number of possible compounds N=b.sup.2 =20.sup.2 =400.
For successive steps of the synthesis, again assuming random, equally efficient assembly of the building blocks to the resulting compounds of the previous step, N=b.sup.x where x equals the number of synthetic assembly steps. Thus it can be seen that for random assembly of only a decapeptide the number of different compounds is 20.sup.10 or 1.02.times.10.sup.13. Such an extremely large number of different compounds permits the assembly and screening of a large number of diverse candidates for a desired enzymatic, immunological or biological activity.
Biologically synthesized combinatorial libraries have been constructed using techniques of molecular biology in bacteria or bacteriophage particles. For example, U.S. Pat. No. 5,270,170 and 5,338,665 to Schatz describe the construction of a recombinant plasmid encoding a fusion protein created through the use of random oligonucleotides inserted into a cloning site of the plasmid. This cloning site is placed within the coding region of a gene encoding a DNA binding protein, such as the lac repressor, so that the specific binding function of the DNA binding protein is not destroyed upon expression of the gene. The plasmid also contains a nucleotide sequence recognized as a binding site by the DNA binding protein. Thus, upon transformation of a suitable bacterial cell and expression of the fusion protein, the protein will bind the plasmid which produced it. The bacterial cells are then lysed and the fusion proteins assayed for a given biological activity. Moreover, each fusion protein remains associated with the nucleic acid which encoded it; thus through nucleic acid amplification and sequencing of the nucleic acid portion of the protein:plasmid complexes which are selected for further characterization, the precise structure of the candidate compound can be determined. The Schatz patents are incorporated herein by reference.
In other biological systems, for example as described in Goedell et al., U.S. Pat. No. 5,223,408, nucleic acid vectors are used wherein a random oligonucleotide is fused to a portion of a gene encoding the transmembrane portion of an integral protein. Upon expression of the fusion protein it is embedded in the outer cell membrane with the random polypeptide portion of the protein facing outward. Thus, in this sort of combinatorial library the compound to be tested is linked to a solid support, i.e., the cell itself. A collection of many different random polypeptides expressed in this way is termed a display library because the cell which produced the protein "displays" the drug on its surface. Since the cell also contains the recombinant vector encoding the random portion of the fusion protein, cells bearing random polypeptides which appear promising in a preliminary screen can be lysed and their vectors extracted for nucleic acid sequencing, deduction of the amino acid sequence of the random portion of the fusion protein, and further study. The Goedell patent is incorporated herein by reference.
Similarly, bacteriophage display libraries have been constructed through cloning random oligonucleotides within a portion of a gene encoding one or more of the phage coat proteins. Upon assembly of the phage particles, the random polypeptides also face outward for screening. As in the previously described system, the phage particles contain the nucleic acid encoding the fusion protein, so that nucleotide sequence information identifying the drug candidate is linked to the drug itself. Such phage expression libraries are described in, for example, Sawyer et al., 4 Protein Engineering 947-53 (1991); Akamatsu et al., 151 J. Immunol. 4651-59 (1993), and Dower et al., U.S. Pat. No. 5,427,908. These patents and publications are incorporated herein by reference.
While synthesis of combinatorial libraries in living cells has distinct advantages, including the linkage of the compound to be tested with a nucleic acid capable of amplification by the polymerase chain reaction or another nucleic acid amplification method, there are clear disadvantages to using such systems as well. The diversity of a combinatorial library is limited by the number and nature of the building blocks used to construct it; thus modified or R-amino acids or atypical nucleotides may not be able to be used by living cells (or by bacteriophage or virus particles) to synthesize novel peptides and oligonucleotides. There is also a limiting selective process at play in such systems, since compounds having lethal or deleterious activities on the host cell or on bacteriophage infectivity or assembly processes will not be present or may be negatively selected for in the library. Importantly, only peptide or oligonucleotide compounds are made in such systems; thus the diversity of the library is restricted to peptide and polynucleotide macromolecules composed of naturally-occurring monomeric units.
Other approaches to creating molecularly diverse combinatorial libraries employ chemical synthetic methods to make use of atypical or non-biological building blocks in the assembly of the compounds to be tested. Thus, Zuckermann et al., 37 J. Med. Chem. 2678-85 (1994), describe the construction of a library using a variety of N-(substituted) glycines for the synthesis of peptide-like compounds termed "peptoids". The substitutions were chosen to provide a series of aromatic substitutions, a series of hydroxylated side substitutions, and a diverse set of substitutions including branched, amino, and heterocyclic structures. This publication is incorporated by reference herein.
Other workers have used small bi- or multifunctional organic compounds instead of, or in addition to, amino acids for the assembly of libraries or collections compounds of medical or biological interest.
Using chemical synthetic methodologies to create large diverse libraries of potentially useful compounds permits the synthesis of compounds joined to a solid support of some kind. However, the use of such synthetic methods requires the ability, after synthesis, to identify the structure of the rare members of the library which are able to pass a screening process. Thus, such libraries must be rationally designed so as to permit such identification. This task becomes virtually overwhelming as the number of possible compounds grows multiplicatively.
In attempting to consider this latter point, a number of attempts have been made to devise post-screening methods of "addressing" the specific compounds that the screening process indicates as candidates for further study. One class of such addressable libraries employs a strategy of linking the individual peptides of the library with the nucleic acids encoding them. Examples of such systems, such as the use of biological entities such as bacteriophage displaying the compounds of the library or plasmid-binding proteins fused to member compounds of the library have been described above. However, this methodology is not limited to biological systems, and can be employed by the co-polymerization of the test compound and a corresponding nucleotide sequence onto a single solid support.
Another strategy involves chemically synthesizing the combinatorial libraries on solid supports in a methodical and predetermined fashion, so that the placement of each library member gives information concerning the synthetic structure of that compound. Examples of such methods are described, for example, in Geysen, U.S. Pat. No. 4,833,092, in which compounds are synthesized on functionalized polyethylene pins designed to fit a 96 well microtiter dish so that the position of the pin gives the researcher information as to the compound's structure. Similarly Hudson et al., PCT Publication No. W094/05394, describe methods for the construction of combinatorial libraries of biopolymers, such as polypeptides, oligonucleotides and oligosaccharides, on a spatially addressable solid phase plate coated with a functionalized polymer film. In this system the compounds are synthesized and screened directly on the plate. Knowledge of the position of a given compound on the plate yields information concerning the nature and order of building blocks comprising the compound. Similar methods of constructing addressable combinatorial libraries may be used for the synthesis of compounds other than biopolymers.
Another approach has been the use of large numbers of very small derivatized beads, which are divided into as many equal portions as there are different building blocks. In the first step of the synthesis, each of these portions is reacted with a different building block. The beads are then thoroughly mixed and again divided into the same number of equal portions. In the second step of the synthesis each portion, now theoretically containing equal amounts of each building block linked to a bead, is reacted with a different building block. The beads are again mixed and separated, and the process is repeated as desired to yield a large number of different compounds, with each bead containing only one type of compound.
This methodology, termed the "one-bead one-compound" method, yields a mixture of beads with each bead potentially bearing a different compound. Thus, in this method the beads themselves cannot be considered "addressable" in the same sense as in the solid phase supports and arrays described above, or as in the cellular or phage libraries. However, the compounds displayed in the surface of each bead can be tested for the ability to bind with a specific compound, and, if those (typically) few beads are able to be identified and separated from the other beads, a presumable pure population of compounds can be recovered and analyzed. Of course, this latter possibility depends upon the ability to load and extract enough information concerning the compounds on the surface of each bead to be susceptible to meaningful subsequent analysis. Such information may simply be in the form of an adequate amount of the compound of interest to be able to determine its structure. For example, in the case of a peptide, enough of the peptide must be synthesized on the bead to be able to perform peptide sequencing and obtain the amino acid sequence of the peptide.
For synthetic chemical libraries, not limited to the one-bead one-compound method, in which the compounds of interest are not naturally-occurring peptides or oligonucleotides, analysis can be a tedious and difficult undertaking. In these cases, a code made from easily synthesized and analyzed "tag" molecules (for example, amino acids or other small multifunctional molecules, such as halogenated aromatics) can be co-synthesized with the compounds comprising the library. After a screening procedure, the tag can be "uncoded" to elucidate the structure of the compounds of interest. The code can be relatively arbitrary, so that the structure of any test compound made of building blocks, in which the building block members are able to be designated as corresponding, for example, to an amino acid (or dipeptide, tripeptide etc.), can be determined in this way.
As described above, the construction of combinatorial libraries provides researchers the opportunity to construct a vast number of potential chemical candidates to answer basic and applied structure-function questions, such as, without limitation: the relationship between a ligand and its receptor, a given antibody and its antigen and an enzyme and substrate. However, the ability to generate large libraries of potential drug compounds overwhelms most available screening methods. Thus, a bottleneck of this emerging and powerful technology remains adequate high-throughput screening procedures to identify the few compounds which are potential candidates for further study from among the thousands, millions or billions of other compounds in the library.
When the combinatorial library is to be screened for the presence of therapeutic or diagnostic agents, candidate compounds are generally initially screened for their ability to bind to a particular member of biological binding partners. By "binding partners" is meant that two or more compounds are able to join under appropriate biological or in vitro conditions to form a specific complex. Examples of such binding partners are, without limitation, antibody and antigen, ligand and receptor, and enzyme and substrate. At times, either ligand or receptor, or both may be comprised of a complex of more than one compound or polypeptide chain. For example, in the case of tumor necrosis factor .alpha. (TNF.alpha.), the soluble ligand TNF appears to bind to its receptor in the form of a TNF homotrimer; each TNF trimer can bind three copies of the receptor and clustering of the TNF receptor is thought to be required for it to exert its biological effects. Each and all polypeptide chains involved in the binding of the TNF trimer to the clustered receptors are considered individual binding partners.
One common screening method currently applied consists of coating a solid support, such as the wells of a microtiter dish, with the specific molecule for which a binding partner is sought. The library member compounds are then labeled, plated onto the solid support, and allowed to bind the library members. After a wash step, the binding partner complexes are then detected by detection of the label joined to the bound library members. This type of procedure is particularly well suited to combinatorial libraries wherein the member compounds are provided in a solution or medium. This method can be somewhat labor intensive and, in order to achieve the high throughput required to screen such large numbers of test compounds, may as a first step require screening pools of test compounds, followed by one or more rescreening step in order to specifically identify the compound of interest. The situation can also be reversed, so that the library members are allowed to coat individual wells and are probed with the specific molecule.
In cases wherein the combinatorial library is to contain antibody analogs or peptides targeted to a given epitope, the library members may contain a portion of an antibody recognized by a secondary antibody able to be detected, for example in an enzyme-linked immunological assay (ELISA) or by virtue of being directly or indirectly labeled, for example with a radionuclide, a chemiluminescent compound, a fluor, and enzyme or dye.
Tawfik et al ., 90 Proc. Natl . Acad. Sci. 373-77 (1993) describe a method of screening a library of antibodies (in this case, from a hybridoma library generated using a mimic of the transition state intermediate of an enzymatic reaction) for the presence of rare antibodies having a desired catalytic activity. The screening compound, in this case the enzyme substrate, was immobilized on 96 well microtiter dishes. Supernatants from each clone were placed into separate wells under conditions promoting the enzymatic reaction. The products of the enzymatic reaction, still immobilized to the microtiter dish, were assayed by the use of product-specific monoclonal antibodies. Again, this type of screening process is quite labor-intensive and may necessitate repetitive screening of pools of test compounds in order to achieve high throughput of large libraries.
In the cellular or phage display libraries and "one-bead one-compound" synthetic libraries described above the library members can be screened for the ability to bind a specific binding partner (e.g., a receptor) which is labeled with a detectable fluor, such as fluorescein or phycoerythrin. Because each particle (for example, a cell or a bead) displays only one species of test compound, the fluorescently labeled particles can be detected and sorted using a fluorescence activated cell sorter (FACS). An enriched population of positive beads or particles can then be rescreened, if necessary, and individually analyzed. This strategy can be employed using cells displaying the test compounds or beads on which the test compounds are synthesized. However, this method also suffers from a lack of ease of use, and is time intensive.
Whether screening is by the panning procedure previously described or by binding of labels to the solid phase bound test compounds, a common screening procedure is by competitive binding of the test compounds in the presence of a detectable control ligand, often the natural ligand for the specific binding partner to which the test compounds are intended to be directed. Again, this method can be quite labor-intensive and requires the generation of a standard curve and correlation of the data obtained from the competition experiments with the standard curve in order to generate meaningful data. Thus, competition assays are unable to yield easily interpreted and rapid results in an initial screen of thousands or millions of different library members.
ELISA and similar assay formats are useful when the library members are derivatives of antibodies and contain variable regions directed against known antigens. However, these methods may not be as useful in a non-competitive (i.e., direct) format where neither the specific binding partner nor the desired test compounds are antibodies or contain an available epitope against which a secondary antibody can be easily generated.
Biochemical tools have been generated consisting of chimeric peptides containing portions of a peptide ligand and specific domains of an antibody. Such agents have been devised mainly as therapeutic aids to the delivery of drugs within a patient's body. Especially in the case of peptide drugs, such as soluble agonists of cytokines and other such agents, therapeutic agents or drugs often have a short systemic half-life which reduces the stability of such drugs in vivo. This reduced stability may, in some cases, be counteracted by higher or more frequent dosages, but this may lead to such undesirable consequences as drug tolerance, toxic effects, and high cost of the drug to the patient.
One strategy for overcoming these shortcomings, particularly with regard to the use of systemic biochemical angonists, has been the use of fusion peptides, which have a longer half life in the circulatory system. These fusion peptides generally contain a binding partner, such as a cytokine receptor, fused to part of an immunoglobulin chain. The immunoglobulin chain acts as molecular camouflage, reducing the opportunity for the binding partner to be recognized as a "foreign" antigen by the organism.
Thus, Shin, et al., 92 Proc Nat'l Acad. Sci. 2820-24 (1995) employed fusion peptides made by constructing recombinant vectors having the gene encoding human transferrin fused, in frame, to the 3' end of a chimeric mouse-human IgG3 gene encoding variable and constant regions. The resulting fusion molecules were able to bind antigen (dansyl) and the purified transferrin receptor, and were able to enter the brain parenchyma of rats using the transferrin receptor for transport from the circulatory system. The remaining variable region of the antibody could contain other optional specificities, thus the site is available for secondary targeting of the molecule, such as for therapeutic purposes, once across the blood-brain barrier.
Evans and coworkers, 180 J. Exp. Med. 2173-79 (1994), using molecular cloning techniques, reported the construction of a fusion protein containing extracellular portions of the p75 high affinity receptor or, alternatively the p55 low affinity receptor, specific for tissue necrosis factor alpha (TNF.alpha.-R) fused to a constant region of human IgG. The soluble, non-fusion forms of the TNF receptors are known to be rapidly degraded in vivo. Cells were transformed with vectors expressing portions of heavy immunoglobulin chain fused to each of TNF receptors. The fusion peptide was more stable than the soluble receptor in serum. Moreover, the fusion peptides were secreted as dimers containing two heavy chains bound by disulfide linkages. The dimers were able to bind the TNF trimers (a naturally-occurring conformation of TNF.alpha.) in two separate areas and thus with higher affinity than is possible when the fusion peptide is in the soluble monomeric form.
Other fusion proteins containing a ligand or receptor and an antibody portion have been used in the search for effective therapeutic agonists to humoral agents. In Fountoulakis et al., 270 J. Biol. Chem. 3958-64 (1995) the extracellular domain of the human interferon .gamma. receptor was expressed as a fusion protein with the IgG hinge, C.sub.H 2 and C.sub.H 3 domains, and was shown to bind interferon, compete for interferon binding to the cell surface receptor of tissue culture cells, and inhibit interferon-mediated antiviral activity. Due to the immunoglobulin portion of the fusion protein, the protein was expressed in Chinese Hamster ovary cells as a disulfide-linked homodimer. The dimer was able to bind interferon more strongly than the soluble receptor monomer.
In Pitti, et al., 31 Molec. Immunol. 1345-51 (1994) the human interleukin-1 (IL-1) receptor was expressed in transfected human cells as a fusion protein containing the hinge and Fc regions of the IgG heavy chain. This fusion peptide was reported to have an extended pharmacological half-life in the circulatory system of mice and to bind IL-1.
Crowe et al., 168 J. Immunol. Meth. 79-89 (1994) expressed a gene containing coding sequences of the extracellular domain of the human lymphotoxin a receptor fused to a gene segment encoding the constant portion of human IgG heavy chain. The fusion protein was cloned into a baculovirus vector and expressed in both insect cells and African green monkey kidney cells as a dimer. The IgG portion of the fusion peptide was used as a ligand for affinity purification of the fusion peptide, and also enabled disulfude facilitated dimerization of the fusion peptides to provide a high-affinity ligand for lymphotoxin.
These latter five references are incorporated by reference herein.

Compositions and methods for screening drug libraries