A method for in vivo production of a library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element independently of the host chromosomal replication machinery. The genetic element comprises

The present invention relates to methods for in vivo production of libraries of polypeptide variants, the screening of these variants and selection of those exhibiting desired properties. The invention furthermore relates to methods for producing the desired polypeptide variants.
An increasing number of polypeptides, including enzymes and non-enzymatic proteins, are being produced industrially, for use in various industries, household, food/feed, cosmetics, medicine etc. One of the major sources for these proteins is and have been microorganism found in nature.
The classical approach for finding polypeptides with new and special properties, have been to screen wild type organisms present in nature. This has been a very successful way of procuring polypeptides to be used in such diverse areas as the above mentioned applications.
However, often it has not been possible to produce such polypeptides in sufficient amounts because the quantities produced in the natural host systems were too minute to allow a production, and even if the cost was no problem, difficulties could be encountered in providing sufficient amounts in relation to the demand (e.g. human growth hormone).
Such problems have to a large degree been overcome by the advent of recombinant techniques for the production of polypeptides. In this art polypeptides are produced by the use of biological systems. Genes encoding certain polypeptides are cloned and transferred into cells that will produce the polypeptides in quantities much larger than those, wherein they are produced in the original organism. Over the latest twenty years a large number of methods for the production of polypeptides according to such techniques have been developed.
Often, proteins from natural sources do not meet the requirements for certain applications, and it will be necessary to modify existing proteins towards certain activities or biophysical properties.
It is possible to generate new variants of a protein by classical mutagenesis of the microorganism using radiation (X-ray and UV) or chemical mutagens. However, since this approach is a very labour and time consuming process, in the same last two decades researchers have been developing improvements on existing polypeptides by using more specific and selective recombinant techniques, such as protein and genetic engineering for creating artificial diversity.
Based upon considerations using knowledge of the structure-function relationships and general protein chemistry, researchers have come a long way in designing polypeptide variants exhibiting improvements in various properties.
However, it has also been realised that the various interactions into which polypeptides take part, are so complex that rational design according to such knowledge has serious limitations, and in recent years methods employing random mutagenesis followed by screening of or selection from very large numbers of variants produced therefrom has gained interest.
For this purpose a microbial library of mutants is generated for subsequent expression and screening to determine variants possessing the desired properties.
Over the years many both in vitro and in vivo DNA mutagenesis techniques for creating high numbers of different variants of polypeptides have been developed.
Considering the fact that a typical naturally occurring polypeptide consists of between 100 and 1000 amino acids, and each may be varied in 20 ways (only to stay within the naturally occurring amino acids), the number of possible variants for a specific polypeptide is enormous. Since the main parameter that defines or measures the usefulness of a microbial collection or library used to identify improved variants of polypeptide is the number of different variants, N, which is comprised in the collection, a need for large libraries has emerged.
Especially in cases when a powerful selection system is available, the limiting factor for the identification of the desired polypeptide is the size of the library.
In in vitro systems the practical, state of art, limit for N is about 108. This is mainly due to inefficiency of transformation (introduction of DNA into the cell) of the manipulated DNA into the host organism. This number varies a lot from organism to organism: in the presently best case, E. coli, the usual efficiency of transformation of in vitro manipulated DNA, e.g. a ligation of DNA fragments or chemical treatment of DNA, leads at the most to library sizes up to 108 bacteria (Greg Winter, Current methods in Immunology 5: 253-255, 1993). Very few examples of libraries of this size have been reported.
In vitro library constructions in other prokaryotes, such as Bacillus sp., Streptococcus sp. or Staphylococcus sp. will for practical reasons be orders of magnitude below this number.
Considering eukaryotic hosts such as Saccharomyces cerevisiae or various Aspergillus sp., an even lower number of transformants can be expected from in vitro manipulated DNA.
A special case of a large library has been reported based on in vivo recombination between libraries of antibody light and heavy chains based on a specially designed system useful for that particular case (Griffiths, A. D. et al., 1994, EMBO J. 14: 3245-3260).
A number of methods are available to generate variants of a polypeptide in microorganisms in vivo, ranging from very simple, such as treating cells with chemical or physical mutagens, to rather complex, relying on cells that contain an error-prone DNA polymerase but lack the mismatch repair system which corrects the errors (Stratagene, XL1-red (mutS, mutD, mutT) Catalog #200129). But these techniques have a major drawback as the mutagenesis is not targeted to a specific part of the genome (coding for the polypeptide of interest) and high frequencies of mutations are generated also in essential genes for the cell as well as in the target gene, resulting in massive cell death, together with a high number of cells, where the mutations do not influence the polypeptide of interest. Such xe2x80x9cnoisexe2x80x9d will limit the accumulation of mutations in the target region.
It is therefore the object of the invention to provide an in vivo target region-specific mutagenesis procedure in order to produce very large numbers, N, of polypeptide variants.
A second object of the invention relates to the screening or selection of variants with the desired properties, both by existing and future technologies.
The present invention therefore relates to a method for in vivo production of a library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element comprising
i) an origin of replication from which replication is initiated,
ii) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic,
iii) a gene encoding the polypeptide of interest, independently of the host chromosomal replication machinery.
The invention furthermore relates to a method for the generation of a DNA sequence encoding a desired variant of a polypeptide of interest, wherein
i) a mutant library is produced by the above method,
ii) said library is cultivated under conditions conducive for the expression of said gene of interest to produce polypeptide variants,
iii) said variant polypeptides are screened or selected for a desired property, and hosts producing such desired variants identified and isolated,
iv) said genetic element in said hosts is sequenced to elucidate the DNA sequence of the mutant gene encoding a desired variant.
And a method for the determination of the DNA sequence encoding a desired variant of a polypeptide of interest, wherein
(i) a mutant library is produced by the above method,
(ii) said library is cultivated under conditions conducive for the expression of said gene of interest to produce variant polypeptides,
(iii) said variant polypeptides are screened or selected for a desired property, and hosts producing desired variants identified and isolated,
(iv) said genetic element in said hosts is sequenced to elucidate the DNA sequence of the mutant gene encoding a desired variant.
The screening of the library or the selection of the variants depends on the specific polypeptide and which properties thereof it is desired to improve and/or retain. It is therefore necessary to set up a screening protocol for each case. Such protocols involving a number of assays are described in the literature (Clackson et al., Nature 352:624-628, 1991, Bryan, P et al., Proteins 1:326-334, 1986).
An elegant approach to the combination of the generation of diversity and the selection of variants with the desired properties would be a combination of the in vivo method of the invention for generating the diversity with a phage display system (Greg Winter, Supra).
A specific example of a polypeptide of interest is the alkaline proteases used in the detergent industry for the removal of proteinaceous stains from fabric. In that case the screening may be performed in actual detergent compositions to investigate properties such as thermal stability, oxidation stability, storage stability, substrate specificity and affinity, stability to non-aqueous solvents, pH profile, ionic strength dependence, catalytic efficiency, and wash performance.
Furthermore the invention relates to a process for the production of a desired polypeptide variant, wherein
(i) a DNA sequence encoding a polypeptide of interest that has been determined according to the method above is introduced into a suitable host in a manner whereby it can be expressed in said host,
(ii) said host is cultivated under conditions conducive to the expression of said DNA sequence, and
(iii) said polypeptide variant is recovered.
Methods for the introduction of the DNA sequence selected into suitable host systems are described in, (Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.).
It is also within the abilities of the skilled person to select suitable growth media and other conditions for the host system selected that are conducive for the expression of the polypeptide variant od interest. Guidance hereto may f. ex. be found in (Sambrook et al., supra).
Also for the recovery of the polypeptide a large number of methods are available for the separation and purification of proteins, e.g. in (Scopes, R. K., Protein Purification (1987), Springer-Verlag)
Lastly, the invention relates to the polypeptides produced by the above method.
The invention comprises a method to construct in vivo libraries of variants in a gene of interest. The method involves the use of a genetic element, such as a bacteriophage or a plasmid that is able to replicate independently of the host chromosomal replication system. By the use of the possibility to separate the replication of the host chromosome and the replication of the genetic element (phage/plasmid), it is possible by modifications of one replication system to selectively introduce mutations in the genetic element (phage/plasmid) keeping the chromosome of the host intact. This means that the generation of variation in the gene of interest does not compromise the viability of the host.
DNA replication is a highly accurate process, and the misincorporation rate of the chromosomal replication in E. coli has been estimated to be in the order of 10xe2x88x9210 pr. base pr. round of replication. The base pairing carried out during DNA replication leads to a preference for the polymerase to incorporate the correct base at a certain position, which accounts for approximately 105 of the overall replication fidelity. If an incorrect base has been incorporated, the polymerase will stall and a 3xe2x80x2-5xe2x80x2 exonuclease will often remove the 3xe2x80x2 misincorporated base. This part denoted proof-reading accounts for approximately 102 of the overall replication fidelity. The repair system of the cell accounts for the last 103 of the fidelity rate.
Replication of the chromosome in E. coli is for the most part carried out by DNA polymerase III holoenzyme, which is a multi-protein complex containing 10 different polypeptides including a polymerase (alpha sub-unit, polC gene) and a 3xe2x80x2-5xe2x80x2 exonuclease (dnaQ gene).
A further polymerase, DNA polymerase I (DNA pol I, polA gene), contains three different activities, viz. a DNA polymerase activity, a 3xe2x80x2-5xe2x80x2 exonuclease activity, and a 5xe2x80x2-3xe2x80x2 exonuclease activity despite the fact that it is one single polypeptide. This polymerase has several functions in the cell. Besides DNA repair, DNA pol I is also needed for the chromosomal DNA replication, as it is involved in the assembly of DNA fragments during synthesis of the lagging DNA strand. However, it replicates only a very minor portion of the genome.
This polymerase is furthermore involved in initiation of DNA replication of certain classes of plasmids, e.g. ColEI origin of replication-based plasmids such as pBR322 in Escherichia coli and Gram-negative bacteria or pAMxcex21 like plasmids in Gram-positive bacteria. Such plasmids may be able to replicate completely through the activity of DNA polymerase I without DNA polymerase III being present in active form, e.g. if this enzyme is dysfunctional due to genetic causes, e.g. temperature sensitive variants at a non-permissive temperature), or under conditions where only limited amounts of DNA Polymerase III are present in the cell.
It is well known that certain mutations lead to a decrease (or an increase) in the fidelity of DNA replication. These mutations have been mapped to reside mainly in polymerases, exonucleases or in elements of the repair system. Unfortunately, most of these mutations alter/impair the fidelity rate for the complete genome present, and such non-targeted mutations are not desirable.
One example of such a mutation could be an inactivation of the 3xe2x80x2-5xe2x80x2 exonuclease activity of DNA pol I.
However, according to the invention use is being made of the fact that some elements in the replication system may be temporarily xe2x80x9cswitchedxe2x80x9d off, fully or partially, thereby stopping or greatly slowing down the replication of the genome, while replication of certain genetic elements as defined herein is continued.
An E. coli strain containing a temperature sensitive DNA pol III (i.e. the polymerase xcex1-sub-unit or another temperature sensitive sub-unit that render the holoenzyme conditionally non-functional), or a function required for initiation of chromosomal replication, such as DnaA, an error prone DNA pol I and a colEI based plasmid containing a gene of interest, is an example of a genetic system according to the invention designed to specifically introduce mutations in the plasmid (and the gene of interest).
In such a system raising the temperature to a non-permissive value will have the effect that DNA pol III ceases to function fully, while the error prone DNA pol I will retain its function and replicates the plasmid with reduced fidelity resulting in mutated copies of the plasmid.
Since the generation of mutations is random, each cell will generate unique mutations and upon lowering the temperature, the temperature sensitive function will become active again, and normal replication of the cells continue.
A variation would be an E. coli strain with temperature sensitive alleles of polIII and polI and an inducible expression (by a ts repressor (temperature-sensitive) or by chemical induction) of the error-prone polymerase. At a restrictive temperature and the presence of the inducer mutations will accumulate in the genetic element. At permissive temperature and the absence of the inducer, the complete systems functions as the wild type cell.
Accordingly the invention in its first aspect relates to a method for in vivo production of a mutant library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element comprising
i) an origin of replication from which replication is initiated,
ii) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic,
iii) a gene encoding the polypeptide of interest, independently of the host chromosomal replication machinery.
The invention consequently comprises a method for in vivo production of a library in cells comprising a multitude of mutated genetic elements comprising
A) providing a cell having
i) an error-prone polymerase that independently of the chromosomal replication machinery of said cell will replicate all or a part of a genetic element comprising
a) an origin of replication from which replication is initiated,
b) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic,
c) a gene encoding the polypeptide of interest, and
ii) a chromosomal replication machinery that can be reversibly induced to be substantially non-functional,
B) growing such a cell under conditions conducive to its replication to obtain a multitude of ancestral cells,
C) reversibly inducing said chromosomal replication machinery in said ancestral cells to be substantially non-functional for a period of time sufficient to allow for the replication of said genetic element by said error-prone polymerase to generate mutations in said genetic element,
D) reversibly inducing said chromosomal replication machinery in such mutated cells to be substantially functional, and
E) growing such mutated cells under conditions conducive to their replication.
In this context the expression xe2x80x9cmutant libraryxe2x80x9d means a set of cells, bacteria or phages (typically 105 to 1013 cells or phages) that differs with respect to one particular gene encoding a polypeptide of interest. Typically one would like to introduce one or more different amino acid alterations in this particular polypeptide in each member of the library.
In this context the expression xe2x80x9cerror-prone polymerasexe2x80x9d means a polymerase that during DNA replication will incorporate mistakes (one of the wrong nucleotides in a given position or cause a deletion or an insertion of one or several nucleotides) with higher frequency than the polymerase normally used for this purpose (e.g. E. coli DNA pol I, Bacillus subtilis DNA pol I, T4 DNA polymerase, T7 DNA polymerase).
The expression xe2x80x9cancestral cellxe2x80x9d here means such cells wherein no mutations have been introduced. In some embodiments of the invention the mutation cycle may be reiterated, and in that case, such cells that were initially mutated become ancestral cells for the second mutation cycle, etc.
In this context the expression xe2x80x9chost chromosomal replication machineryxe2x80x9d means the DNA polymerase or DNA polymerase holoenzyme that is mainly responsible for the replication of the host chromosome, e.g. DNA polymerase III in E. coli.
In this context the expression xe2x80x9cgenetic elementxe2x80x9d means a small (from 1 or 2 kilo bases to 100 kilo bases) entity consisting of RNA or DNA, that is able to replicate independently, i.e. it contains an origin of replication. The genetic element would typically be a bacteriophage, a phagemid, or a plasmid. The genetic element must also according to the invention comprise a gene encoding the polypeptide of interest, and it may further comprise a genetic marker, e.g. a gene conferring resistance towards an antibiotic.
A virus, a retrovirus, or a transposon that is able to replicate independently of the host replication machinery, e.g. retrotransposons could also be used as the xe2x80x9cgenetic elementxe2x80x9d.
Kim and Loeb (1995, PNAS 92: 684-688) have demonstrated that HIV reverse transcriptase (HIV-RT) is able to complement E. coli DNA pol I with respect to chromosomal DNA replication and initiation of plasmid DNA replication. The misincorporation rate of HIV-RT (and related retroviral reverse transcriptases) is several orders of magnitude higher than the rate of DNA pol I, i.e. 10xe2x88x923 to 10xe2x88x924 misincorporations pr. base pr. round of replication. The use of such a polymerase in stead of a mutated error prone E. coli DNA pol I in an embodiment of the invention would significantly increase the frequency of replication errors in the system described above.
In a further embodiment of the invention the mutation cycle described above can be reiterated, i.e. the mutagenic polymerase switched on and off several times, thereby generating even more mutants. Such a step could furthermore help the segregation of plasmids if a multicopy plasmid is used as the genetic element.
In certain genetic elements one can envision that only the part of the genetic element located in the vicinity of the origin of replication is replicated by the error-prone polymerase. In such cases, the gene of interest should be situated within this region.
In a specific embodiment of the invention the genetic element is a phage, wherein the gene encoding the polypeptide of interest is positioned at a locus where the polypeptide upon expression is displayed from the surface of the phage, whereby a screening can be performed directly (see Greg Winther, supra). To ensure the correspondence between DNA sequence of the phage and the protein displayed the primary phage stock should be passed through wild type E. coli, at low multiplicity of infection, prior to selection or screening.
To further increase the frequency of mutations, the method of the invention comprises embodiments where the method is used in conjunction with a repair deficient host, e.g. mutL, mutS, mutH, or a combination of mutator genome types.
In this context the expression xe2x80x9crepair deficient hostxe2x80x9d means a cell containing one or more alterations in genes encoding proteins known to be directly or indirectly involved in the DNA repair. The result of such mutations is that a higher frequency of introduced mutations (by the polymerases, chemicals, X-ray, UV light, etc.) will not be repaired and will be xe2x80x9cpermanentlyxe2x80x9d incorporated in the genome, the so called mutator phenotype. Examples of such genes are mutL, mutS, mutH, mutT.
As the genetic element one could as indicated above use a phagemid in stead of a plasmid in order to couple the variant generation to a display system, e.g. M13, fI, fd.
In this context the expression xe2x80x9cphagemidxe2x80x9d means a plasmid that besides its plasmid origin of replication contains a phage origin of replication. phagemids are dependent of the conditions able to replicate as a plasmid or as a phage (upon infection with a helper-phage).
A phagemid based system would involve the construction of a phagemid containing:
1) a plasmid origin of replication, e.g. ColEI
2) a M13 phage origin of replication
3) a chimeric gene consisting of the gene of interest fused to the gene encoding GIII protein. (Ørum, P. et al., Nucl. Acid. Res. 21: 4491-4498, 1993).
The first step would be the generation of diversity by growing/maintaining an E. coli strain transformed with this phagemid as described above. The second step would be the infection with the helper phage in order to create single stranded phagemid that will be packed into phage particles. The phages displaying the variant proteins can then be subjected to a selection procedure.
Also, certain bacteriophages such as T4 or T7 in Eschericia or SPOII and Phi29 in Bacillus contain their own DNA polymerases, and according to the invention one could envision embodiments where the genetic element described above is a bacteriophage containing an error-prone DNA polymerase.
According to the invention the error-prone polymerase is typically selected from the group comprising DNA polymerase I or reverse transcriptases. A preferred error-prone polymerase is a variant of E. coli DNA polymerase I or HIV reverse transcriptase.
As a polypeptide of interest a large number is possible, and especially such polypeptides exhibiting biological activities could be mentioned. Among these are enzymes, hormones, receptors, blood-clotting factors, anti-microbial agents, and other such polypeptides important for the prophylaxis and treatment of various disorders and diseases in humans and animals.
Also, enzymes used for industrial purposes could be mentioned. Among such industrial enzymes, enzymes belonging to the groups carbonyl hydrolases, carbohydrases, oxidoreductases, transferases, phytases, anti-microbial polypeptides, oxidoreductases, isomerases, lyases, and ligases.
In this context the expression xe2x80x9ccarbonyl hydrolasexe2x80x9d means enzymes that hydrolyze compounds containing a xe2x80x94C((xe2x95x90O)xe2x80x94X group, where X is oxygen or nitrogen.
Specific classes of enzymes belonging to the group of carbonyl hydrolases are such as hydrolases (lipases) and peptide hydrolases (proteases).
Proteases are here meant as enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).
Examples include proteases selected from those classified under the Enzyme Classification (E.C.) numbers:
3.4.11 (i.e. so-called aminopeptidases), including 18.104.22.168 (Prolyl aminopeptidase), 22.214.171.124 (X-pro aminopeptidase),
220.127.116.11 (Bacterial leucyl aminopeptidase), 18.104.22.168 (Thermophilic aminopeptidase), 126.96.36.199 (Lysyl aminopeptidase),
22.214.171.124 (Tryptophanyl aminopeptidase), 188.8.131.52 (Methionyl aminopeptidase).
3.4.21 (i.e. so-called serine endopeptidases), including 22.214.171.124 (Chymotrypsin), 220.127.116.11 (Trypsin), 22.214.171.124 (Cucumisin),
126.96.36.199 (Brachyurin), 126.96.36.199 (Cerevisin) and 188.8.131.52 (Subtilisin);
3.4.22 (i.e. so-called cysteine endopeptidases), including
22.214.171.124 (Papain), 126.96.36.199 (Ficain), 22.214.171.124 (Chymopapain),
126.96.36.199 (Asclepain), 184.108.40.206 (Actinidain), 22.214.171.124 (Caricain) and 188.8.131.52 (Ananain);
3.4.23 (i.e. so-called aspartic endopeptidases), including
22.214.171.124 (Pepsin A), 22.214.171.124 (Aspergillopepsin I), 126.96.36.199 (Penicillopepsin) and 184.108.40.206 (Saccharopepsin); and
3.4.24 (i.e. so-called metallo endopeptidases), including 22.214.171.124 (Bacillolysin).
Examples of relevant subtilisins comprise subtilisin BPNxe2x80x2, subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, thermitase, aqualysin, Bacillus PB92 protease, proteinase K, Protease TW7, and Protease TW3.
Specific examples of such readily available commercial proteases include Esperase(copyright), Alcalase(copyright), Neutrase(copyright), Dyrazym(copyright), Savinase(copyright), Pyrase(copyright), Pancreatic Trypsin NOVO (PTN), Bio-Feed(trademark) Pro, Clear-Lens Pro (all enzymes available from Novo Nordisk A/S).
Examples of other commercial proteases include Maxatase(copyright), Maxacal(copyright), Maxapem(copyright) marketed by Gist-Brocades N.V., Opticlean(copyright) marketed by Solvay et Cie. and Purafect(copyright) marketed by Genencor International.
It is to be understood that also protease variants are contemplated as the polypeptide of interest. Examples of such protease variants are disclosed in EP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomas et al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol. Biol., 193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500, WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo Nordisk A/S), WO 91/00345 (Novo Nordisk A/S), EP 525 610 (Solvay) and WO 94/02618 (Gist-Brocades N.V.).
The activity of proteases can be determined as described in xe2x80x9cMethods of Enzymatic Analysisxe2x80x9d, third edition, 1984, Verlag Chemie, Weinheim, vol. 5.
Lipases are here meant as enzymes classified under the Enzyme Classification number E.C. 3.1.1 (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).
Examples include lipases selected from those classified under the Enzyme Classification (E.C.) numbers:
3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including (184.108.40.206) Triacylglycerol lipases, (18.104.22.168.) Phospholipase A2.
Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are incorporated herein by reference:
Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis var. thermoidea and H. insolens (U.S. Pat. No. 4,810,414)
Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps. fluorescens (WO 89/04361), or Ps. plantarii or Ps. gladioli (U.S. Pat. No. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps. pseudoalcaligenes (EP 218 272) or Ps. mendocina (WO 88/09367; U.S. Pat. No. 5,389,536).
Fusarium, e.g. F. oxysporum (EP 130,064) or F. solani pisi (WO 90/09446).
Mucor (also called Rhizomucor), e.g. M. miehei (EP 238 023).
Chromobacterium (especially C. viscosum)
Aspergillus (especially A. niger).
Candida, e.g. C. cylindracea (also called C. rugosa) or C. antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO 89/02916).
Geotricum, e.g. G. candidum (Schimada et al., (1989), J. Biochem., 106, 383-388)
Penicillium, e.g. P. camembertii (Yamaguchi et al., (1991), Gene 103, 61-67).
Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R. niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem 56, 716-719) or R. oryzae.
Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992) or B. pumilus (WO 91/16422).
Specific examples of readily available commercial lipases include Lipolase(copyright), Lipolase(trademark) Ultra, Lipozyme(copyright), Palatase(copyright), Novozym(copyright) 435, Lecitase(copyright) (all available from Novo Nordisk A/S).
Examples of other lipases are Lumafast(trademark), Ps. mendocina lipase from Genencor Int. Inc.; Lipomax(trademark), Ps. pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) from Unilever; Bacillus sp. lipase from Solvay enzymes. Other lipases are available from other companies.
It is to be understood that also lipase variants are contemplated as the polypeptide of interest. Examples of such are described in e.g. WO 93/01285 and WO 95/22615.
The activity of the lipase can be determined as described in xe2x80x9cMethods of Enzymatic Analysisxe2x80x9d, Third Edition, 1984, Verlag Chemie, Weinhein, vol. 4, or as described in AF 95/5 GB (available on request from Novo Nordisk A/S).
In this context the expression xe2x80x9ccarbohydrasexe2x80x9d means all enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five and six member ring structures (i.e. enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)). Also included in the group of carbohydrases according to the invention are enzymes capable of isomerizing carbohydrates e.g. six member ring structures, such as D-glucose to e.g. five member ring structures like D-fructose.
Examples include carbohydrases selected from those classified under the Enzyme Classification (E.C.) numbers:
xcex1-amylase (220.127.116.11) xcex2-amylase (188.8.131.52), glucan 1,4-xcex1-glucosidase (220.127.116.11), cellulase (126.96.36.199), endo-1,3(4)-xcex2-glucanase (18.104.22.168), endo-1,4-xcex2-xylanase (18.104.22.168), dextranase (220.127.116.11), chitinase (22.214.171.124), polygalacturonase (220.127.116.11), lysozyme (22.214.171.124), xcex2-glucosidase (126.96.36.199), xcex1-galactosidase (184.108.40.206), xcex2-galactosidase (22.214.171.124), amylo-1,6-glucosidase (126.96.36.199), xylan 1,4-xylosidase (126.96.36.199), glucan endo-1,3-xcex2-D-glucosidase (220.127.116.11), xcex1-dextrin endo-1,6-glucosidase (126.96.36.199), sucrose xcex1-glucosidase (18.104.22.168), glucan endo-1,3-xcex1-glucosidase (18.104.22.168), glucan 1,4-xcex2-glucosidase (184.108.40.206), glucan endo-1,6-xcex2-glucosidase (188.8.131.52), arabinan endo-1,5-xcex1-arabinosidase (22.214.171.124), lactase (126.96.36.199), chitonanase (126.96.36.199) and xylose isomerase (188.8.131.52).
Examples of relevant carbohydrases include xcex1-1,3-glucanases derived from Trichoderma harzianum; xcex1-1,6-glucanases derived from a strain of Paecilomyces; xcex2-glucanases derived from Bacillus subtilis; xcex2-glucanases derived from Humicola insolens; xcex2-glucanases derived from Aspergillus niger; xcex2-glucanases derived from a strain of Trichoderma; xcex2-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1,4-xcex1-D-glucosidases (glucoamylases) derived from Aspergillus niger; xcex1-amylases derived from Bacillus subtilis; xcex1-amylases derived from Bacillus amyloliquefaciens; xcex1-amylases derived from Bacillus stearothermophilus; xcex1-amylases derived from Aspergillus oryzae; xcex1-amylases derived from non-pathogenic microorganisms; xcex1-galactosidases derived from Aspergillus niger; Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulases derived from Humicola insolens; cellulases derived from Trichoderma reesei; cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger; dextranases derived from Penicillium lilacinum; endoglucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus; xcex2-galactosidases derived from Kluyveromyces fragilis; xylanases derived from Trichoderma reesei;
Specific examples of readily available commercial carbohydrases include Alpha-Gal(trademark), Bio-Feed(trademark) Alpha, Bio-Feed(trademark) Beta, Bio-Feed(trademark) Plus, Bio-Feed(trademark) Plus, Novozyme(copyright) 188, Carezyme(copyright), Celluclast(copyright), Cellusoft(copyright), Ceremyl(copyright), Citrozym(trademark), Denimax(trademark), Dezyme(trademark), Dextrozyme(trademark), Finizym(copyright), Fungamyl(trademark), Gamanase(trademark), Glucanex(copyright), Lactozym(copyright), Maltogenase(trademark), Pentopan(trademark), Pectinex(trademark), Promozyme(copyright), Pulpzyme(trademark), Novamyl(trademark), Termamyl(copyright), AMG (Amyloglucosidase Novo), Maltogenase(copyright), Sweetzyme(copyright), Aquazym(copyright) (all enzymes available from Novo Nordisk A/S). Other carbohydrases are available from other companies.
It is to be understood that also variants of such carbohydrases are contemplated as the polypeptide of interest.
The activity of carbohydrases can be determined as described in xe2x80x9cMethods of Enzymatic Analysisxe2x80x9d, third edition, 1984, Verlag Chemie, Weinheim, vol. 4.
Oxidoreductases are here meant to be enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).
Examples include oxidoreductases selected from those classified under the Enzyme Classification (E.C.) numbers: Glycerol-3-phosphate dehydrogenase (NAD+) (220.127.116.11), Glycerol-3-phosphate dehydrogenase NAD(P)+ (22.214.171.124), Glycerol-3-phosphate 1-dehydrogenase (NADP) (184.108.40.206), Glucose oxidase (18.104.22.168), Hexose oxidase (220.127.116.11), Catechol oxidase (22.214.171.124), Bilirubin oxidase (126.96.36.199), Alanine dehydrogenase (188.8.131.52), Glutamate dehydrogenase (126.96.36.199), Glutamate dehydrogenase (NAD(P)+) (220.127.116.11), Glutamate dehydrogenase (NADP+) (22.214.171.124), L-Amino acid dehydrogenase (18.104.22.168), Serine dehydrogenase (18.104.22.168), Valine dehydrogenase (NADP+) (18.104.22.168), Leucine dehydrogenase (184.108.40.206), Glycine dehydrogenase (126.96.36.199), L-Amino-acid oxidase (184.108.40.206.), D-Amino-acid oxidase(184.108.40.206), L-Glutamate oxidase (188.8.131.52), Protein-lysine 6-oxidase (184.108.40.206), L-lysine oxidase (188.8.131.52), L-Aspartate oxidase (18.104.22.168), D-amino-acid dehydrogenase (184.108.40.206), Protein disulfide reductase (22.214.171.124), Thioredoxin reductase (188.8.131.52), Protein disulfide reductase (glutathione) (220.127.116.11), Laccase (22.214.171.124), Catalase (188.8.131.52), Peroxidase (184.108.40.206), Lipoxygenase (126.96.36.199), Superoxide dismutase (126.96.36.199)
Said Glucose oxidases may be derived from Aspergillus niger.
Said Laccases may be derived from Polyporus pinsitus, Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera.
Bilirubin oxidases may be derived from Myrothechecium verrucaria.
The Peroxidase may be derived from e.g. Soy bean, Horseradish or Coprinus cinereus.
The Protein Disulfide reductase may be any mentioned in any of the DK patent applications no. 768/93, 265/94 and 264/94 (Novo Nordisk A/S), which are hereby incorporated as reference, including Protein Disulfide reductases of bovine origin, Protein Disulfide reductases derived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived from Escherichia coli.
Specific examples of readily available commercial oxidoreductases include Gluzyme(trademark) (enzyme available from Novo Nordisk A/S). However, other oxidoreductases are available from others.
It is to be understood that also variants of oxidoreductases are contemplated as the polypeptide of interest.
The activity of oxidoreductases can be determined as described in xe2x80x9cMethods of Enzymatic Analysisxe2x80x9d, third edition, 1984, Verlag Chemie, Weinheim, vol. 3.
In this context transferases are enzymes classified under the Enzyme Classification number E.C. 2 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).
The transferases may be any transferase in the subgroups of transferases: transferases transferring one-carbon groups (E.C. 2.1); transferases transferring aldehyde or residues (E.C. 2.2); acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4); transferases transferring alkyl or aryl groups, other that methyl groups (E.C. 2.5); transferases transferring nitrogenous groups (2.6).
In a preferred embodiment the transferease is a transglutaminase E.C 184.108.40.206 (Protein-glutamine xcex3-glutamyltransferase).
Transglutaminases are enzymes capable of catalysing an acyl transfer reaction in which a xcex3-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of mono-substituted xcex3-amides of peptide-bound glutamic acid. When the epsilon-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transferases form intramolecular or intermolecular xcex3-glutamyl-xcex5-lysyl crosslinks.
Examples of transglutaminases are described in the pending DK patent application no. 990/94 (Novo Nordisk A/S).
The transglutaminase may the of human, aminal (e.g. bovine) or microbially origin.
Examples of such transglutaminases are animal derived transglutaminases, FXIIIa; microbial transglutaminases derived from Physarum polycephalum (Klein et al., Journal of Bacteriology, 174, p. 2599-2605); transglutaminases derived from Streptomyces sp., including Streptomyces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Streptoverticillium sp., including Streptoverticillium mobaraense, Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (Motoki et al., U.S. Pat. No. 5,156,956; Andou et al., U.S. Pat. No. 5,252,469; Kaempfer et al., Journal of General Microbiology, 137, 1831-1892; Ochi et al., International Journal of Sytematic Bacteriology, 44, 285-292; Andou et al., U.S. Pat. No. 5,252,469; Williams et al., Journal of General Microbiology, 129, 1743-1813).
It is to be understood that also transferase variants are contemplated as the polypeptide of interest.
The activity of transglutaminases can be determined as described in xe2x80x9cMethods of Enzymatic Analysisxe2x80x9d, third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10.
In this context phytases are enzymes classified under the Enzyme Classification number E.C. 3.1.3 (Phosphoric Monoester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).
Phytases are enzymes produced by microorganisms which catalyse the conversion of phytate to inositol and inorganic phosphorus
Phytase producing microorganisms comprise bacteria such as Bacillus subtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomyces cerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species).
Examples of phytases include phytases selected from those classified under the Enzyme Classification (E.C.) numbers: 3-phytase (18.104.22.168) and 6-phytase (220.127.116.11).
The activity of phytases can be determined as described in xe2x80x9cMethods of Enzymatic Analysisxe2x80x9d, third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10, or may be measured according to the method described in EP-A1-0 420 358, Example 2 A.
In the present context an anti-microbial polypeptides may be any polypeptide exhibiting anti-microbial activities, such as anti-fungal, anti-bacterial, and/or anti-insecticidal activity.
Such polypeptides may also exhibit other activities such as enzymatic activity.
Examples of anti-microbial polypeptides according to the invention include: fungicidally active polypeptides derived from the mold genus Curvularia described in WO 94/01459 (Novo Nordisk A/S); anti-bacterial polypeptides described in EP 403.458 (Kabigen AB); anti-microbial proteins isolated from the Mirabilis seed, described in WO 92/15691 (Imperial Chem Ind. PLC); anti-bacterial polypeptides isolated from an extract of pig small intestine, described in WO 92/22578 (Boman et al.); polypeptide with yeast lethal action accumulated by yeast of Hansenula spp. as described in JP-60130599; Phytolacca insularis antiviral protein, which can be used as an anti-microbial described in U.S. Pat. No. 5,348,865 (Jin Ro LTD.); bacteriolytic enzymes preparations derived from Nocardiopsis dassonvillei described in U.S. Pat. No. 5,354,681 (Novo Industri A/S).
Examples of other anti-microbial polypeptides are maganinin, protegrin, defensin, pseudomycin, mutanolysin and N-acetylmuramidase.
The present invention in a further aspect relates to a method for generating a DNA sequence encoding a desired variant of a polypeptide of interest, wherein
(i) a mutant library is produced by the above method,
(ii) said library is cultivated under conditions conducive for the expression of said gene of interest to produce variant polypeptides,
(iii) said variant polypeptides are screened or selected for a desired property, and hosts producing desired variants identified and isolated,
(iv) the DNA encoding said variants is isolated.
The present invention in a still further aspect relates to a method for the determination of a DNA sequence encoding a desired variant of a polypeptide of interest, wherein
(i) a mutant library is produced as described above,
(ii) said library is cultivated under conditions conducive for the expression of said gene of interest to produce variant polypeptides,
(iii) said variant polypeptides are screened or selected for a desired property, and hosts producing desired variants identified and isolated,
(iv) said genetic element in said hosts is sequenced to elucidate the DNA sequence of the mutant gene encoding a desired variant.
This aspect of the invention can be performed by making dilutions of the library (e.g. in microtiter plates) and culturing these, whereby populations are made each originating from one member of the library, and the variant polypeptide produced from each of the populations screened for the desired properties. Alternatively the library might be plated on agar-plates containing a desired growth medium that allows for the screening of or selection for desired properties of the variant polypeptide.
If the phage display method is used, the screening or selection is performed directly with the phages.
The criteria used for the selection will vary according to the end use of the polypeptide variant of interest, but properties typically being tested may include solubility and half-life in various media, antigenicity and allergenicity, thermal stability, oxidation stability, storage stability, substrate specificity and affinity, stability to non-aqueous solvents, pH profile, ionic strength dependence, catalytic efficiency, and compatibility with other components of envisaged end products wherein the polypeptide variant will form a part.
For enzymes to be used in detergents further properties to be investigated are, wash performance and compatibility with various surfaces, especially fabrics.
Numerous other criteria could be mentioned.
Upon identification of populations that produce variant polypeptides fulfilling the criteria selected, the DNA encoding the polypeptide variant of interest is isolated and sequenced by use of methods well known in the art.
The invention furthermore comprises a process for the production of a desired polypeptide variant, wherein
(i) a DNA sequence determined as indicated above is introduced into a suitable host in a manner whereby it can be expressed in said host,
(ii) said host is cultivated under conditions conducive to the expression of said DNA sequence, and
(iii) said polypeptide variant is recovered.
The present invention can be used with any cell, especially any microbial cell, but it is often suitable to use a prokaryote, especially a bacterium, preferably of the genus Bacillus, etc.
Among the Bacilli it is preferred to use a strain chosen from the group comprising B. lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, etc.
For some uses it is preferable to use a microbial cell which is a fungus, especially a filamentous fungus, preferably of the genus Aspergillus, Trichoderma, etc.
Among the Aspergilli it is preferable to use a strain chosen from the group comprising A. oryzae, A. niger, A. awamori, etc.
Among the Trichoderma it is preferable to use a strain chosen from the group comprising T. reseei, etc.
In yet other situations it is more expedient to use a mammalian cell chosen from the group comprising BHK, etc. cells.

Method for in vivo production of a mutant library in cells

Abstract: iii) a gene encoding the polypeptide of interest. Also methods for the generation of a DNA sequence encoding a desired variant of a polypeptide of interest, and for the determination of such a DNA sequence are described.


A portable analgesic system comprises a liquid medicine supply unit which stores a given amount of liquid medicine temporarily and then discharges the liquid medicine automatically, a first three-way connector connected to an outlet of the liquid medicine supply unit, an intermittent injection line for liquid medicine connected to one outlet of the first three-way connector, a continuous injection line for liquid medicine connected to the other outlet of the first three-way connector, and a second three-way connector for intercommunicating with outlets of the both lines.

An analgesic plays an important role in pain control and especially in cases involving chronic pain. It has heretofore been necessary for a patient to be hospitalized for a long time or to visit a hospital as an out-patient
The need for frequent injection of liquid medicine such as an analgesic or the like is onerous both to a patient and in terms of time and efficiency to a hospital. Consequently, an automatic injection system has been developed which injects liquid medicine to an in-patient periodically and automatically. However, conventional systems are large, expensive, and physically limit a patient.
Therefore, a convenient automatic injection system which is not physically limiting and can be applied to in patients, out-patients, and home-patients has been desired for a long time.
Thus, the applicant has proposed a portable analgesic system (PCT/JP94/00608) which satisfies the above requirements This portable analgesic system comprises a continuous injection for liquid medicine which discharges liquid medicine continuously for a given period of time; a switch valve connected to an outlet of said injector for shutting off discharge of liquid medicine from said injector; a three-way connector to an outlet of said switch valve and having three ways; a flexible reservoir connected to one way of said three-way connector for storing liquid medicine; and a pressure-check valve connected to another way of said three-way connector for opening a flow passage only when a pressure of liquid medicine in said reservoirs rises above a given value.
In the portable analgesic system, a continuous injector for liquid medicine which has a given capacity and a given self-maintaining discharge time is prepared beforehand and a given kind and amount of liquid medicine is injected into the injector in accordance with the condition of a patient. Then, the switch valve is opened to transfer liquid medicine to the reservoir for a given period of time. During such a transference of medicine, a patient can freely move with the system being attached to the body.
A period of time for transferring liquid medicine from the injector to the reservoir coincides with a self-maintaining remedy effect time of liquid medicine. When pain is felt, a patient compresses the reservoir to inject the liquid medicine contained in the reservoir into the body through the pressure-check valve.
In preparation for any subsequent pain, the injector can then be supplied with liquid medicine by a licensee either at the patient's home or at a hospital.
This system has a distinctive effect on the specific condition of patient's disease. However, there are intermittent occasions when the injection of a given amount of liquid medicine into a patient's body is required and therefore administered, e.g., when the patient experiences an irregular or sudden pain. This occurs in parallel with the periodical continuous injection for liquid medicine and in accordance with the ingravescence of the state of the disease, the state of the affected body part and the like.
In this case, a continuous injector for liquid medicine is used in parallel with the portable analgesic system described above. Such a continuous injector for liquid medicine has been developed (Japanese Patent No. 1384289) by the present applicant. This injector includes an inlet portion for liquid medicine, an outlet portion for liquid medicine, and a balloon which connects the inlet portion to the outlet portion. Liquid medicine injected in the balloon flows out through the outlet portion for a long period of time.
However, when using the portable analgesic system in parallel with the continuous injector for liquid medicine, it is necessary to attach to a patient's body two continuous supply units which have substantially the same structures. This is onerous to a doctor or a nurse and is an encumbrance to a patient.


A reel assembly (10) having a spool (20) comprising a drum core (22) and side disc flanges (24) attached to the drum core (22) for retaining a wound hose (26) on the spool (20), and a release mechanism (120) operatively engaging the spool (20) via a drive shaft (110). The release mechanism (120) has a pair of rotatable hubs (132, 134) arranged in axial alignment and having a clamping spring (136) fitted around the rotatable hubs (132, 134) and arranged to clamp the hubs (132, 134) together in fixed rotational relation, wherein the clamping spring (136) opens to releases the rotatable hubs (132, 134) when the spool (20) is rotated in a payout direction so that the rotatable hubs (132, 134) disengage and the spool (20) can freewheel.

Industrial hose reels have various applications, such as in firefighting, and general purpose use in industrial plants. For such industrial reel devices, it is desirable for the reel to wind on a hose under control, yet pay out freely as required. Power-operated devices have been used to supply this functionality by relying upon an electromagnetic clutch mechanism which is activated or deactivated as the case may be by an operator when the hose is to be wound or payed out.
As will be appreciated, such reels are often required to be used to respond to emergencies when time is of the essence, and when there are distractions competing for attention. Under such conditions, operators can inadvertently overlook the need to deactivate a drive mechanism to pay out a wound hose for use. Besides delay and inconvenience, this can also result in operator injury as a consequence of vigorously pulling on a wound hose in an attempt to get the reel assembly paying out hose, when the hose reel is not yet released. Moreover, electromechanical drive devices tend to be oversized and underpowered as a consequence of striking an unsatisfactory compromise between the power required to wind a hose at full pay out, and the limited space available in an industrial hose reel assembly. There is in light of the foregoing a need for reel assemblies that at least attempt to address these and other limitations of existing reel assemblies. More particularly, there is a need for a reel assembly that allows for controlled spooling as hose is wound, but which permits freewheeling payout.

Automatically releasable hose reel assembly


Methods for the reversal of hemorrhagic shock or hemorrhagic trauma. Methods for restoring mean arterial pressure to a normal range and reducing trauma adverse risks in a patient through the administration of oxygen reduced blood compositions.

In 2010, there were 5.1 million deaths from injuries, surpassing the number of deaths due to HIV, tuberculosis, and malaria combined (3.8 million). See Norton, et al., "Global Health Injuries" in The NEAT 368:1723-30 (2013) ("Norton 2013") (hereby incorporated by reference in its entirety). Injuries include unintentional injuries (e.g., road- traffic incidents, falls, and bums) and intentional injuries (e.g., self-harm, interpersonal violence, war and conflict). See Norton 2013. The number of deaths from injuries increased by 24% between 1990 and 2010, worldwide, and increased 23% between 2000 and 2010, in the United States. See Norton 2013. Additionally, at least 20% of all trauma deaths are the result of survivable injuries and are therefore preventable with optimal care. Fox et al., "Earlier Endpoints are Required for Hemorrhagic Shock Trials Among Severely Injured Patients." Shock, 47:567-73 (2017) (hereby incorporated by reference in its entirety). The percentage of preventable deaths make it imperative to develop therapy for avoidable complications which lead to mortality.
Penetrating wounds (e.g., gunshot or stabbing) and blunt trauma (e.g., fall or automobile accident) are major causes of hemorrhagic trauma. The resulting shock is a condition of inadequate oxygen supply to tissues from massive hemorrhage causing oxygen debt, anaerobic metabolism and raise of plasma lactate level. Failure to reverse shock by restoring circulation and oxygen delivery can result in permanent tissue damage, multiple organ failure and mortality.
Clinical sequelae of hemorrhagic trauma and shock include mortality from exsanguination within several hours of trauma, as well as after 24 hours from morbidity from trauma and massive transfusion. Such morbidity includes multiple organ failure including lung, kidney, liver from acute traumatic coagulopathy or inflammation, and infection/sepsis from transfusion related immune modulation; both morbidities are heightened by lower quality of transfused blood products as well as higher volume of transfused pRBC.
One approach for treating hemorrhage shock is the use of crystalloids for resuscitation. However, the use of crystalloids result in increased morbidity and mortality by causing trauma induced coagulopathy. For at least this reason, early administration of blood components is advocated for reversing shock caused by hemorrhagic trauma. Packed red blood cells (pRBCs) are transfused into a hemorrhagic trauma patient to restore lost blood volume, restore oxygen carrying capacity in patients and restore oxidative metabolism in tissue from anaerobic metabolism. However, the use of pRBCs is not without risk of complications, including antigen mismatch, pathogen transmission, circulatory overload, and degradation of pRBCs during ex vivo storage.
When stored conventionally, stored blood undergoes a steady deterioration which is associated with various storage lesions including, among others, hemolysis, hemoglobin degradation, and reduced ATP and 2,3-DPG concentrations. When transfused into a patient, the effects of the steady deterioration during storage manifest, for example, as a reduction in the 24-hour in vivo recovery. The rapid decrease in the hematocrit that results from reduced 24-hour recovery, when severe, can result in delayed hemolytic transfusion reaction (DHTR). Other complications, for example systemic inflammatory response syndrome (SIRS), transfusion related acute lung injury (TRALI), and transfusion related immunomodulation (TRIM) are associated with transfusion of stored blood, though identification of the underlying causes has remained unclear.
Even when transfused within the current 6-week limit, stored RBCs tend to exhibit lower quality (e.g. increased fraction of RBCs removed; compromised oxygen exchange capacity; reduced deformability) and increased toxicity, often manifested as the clinical sequelae of transfusion therapy. A large and growing number of articles in the literature supports this view. See Zimring, "Established and theoretical factors to consider in assessing the red cell storage lesion," Blood, 125:2185-90 (2015); Zhu et al., "Impaired adenosine-5′-triphosphate release from red blood cells promotes their adhesion to endothelial cells: a mechanism of hypoxemia after transfusion," Critical care medicine, 39:2478-86 (2011); Weinberg et al., "Red blood cell age and potentiation of transfusion-related pathology in trauma patients," Transfusion, 51:867-73 (2011); Spinella et al., "Does the storage duration of blood products affect outcomes in critically ill patients?" Transfusion 51:1644-50 (2011); Roback et al., "Insufficient nitric oxide bioavailability: a hypothesis to explain adverse effects of red blood cell transfusion," Transfusion, 51:859-66 (2011); Reynolds et al., "The transfusion problem: role of aberrant S-nitrosylation," Transfusion, 51:852-8 (2011); Kim-Shapiro et al., "Storage lesion: role of red blood cell breakdown," Transfusion, 51:844-51 (2011); Jy et al., "Microparticles in stored red blood cells as potential mediators of transfusion complications," Transfusion, 51:886-93 (2011); Hod et al., "Transfusion of human volunteers with older, stored red blood cells produces extravascular hemolysis and circulating non-transferrin-bound iron," Blood, 118:6675-82 (2011); Flegel et al., "Does prolonged storage of red blood cells cause harm?" British journal of haematology 165:3-16 (2014); Redlin et al., "Red blood cell storage duration is associated with various clinical outcomes in pediatric cardiac surgery," Transfusion medicine and hemotherapy: offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin and Immunhamatologie 41:146-51 (2014); Rogers et al., "Storage duration of red blood cell transfusion and Clostridium difficile infection: a within person comparison," PLoS One 9:e89332 (2014); Spinella et al., "Properties of stored red blood cells: understanding immune and vascular reactivity," Transfusion 51:894-900 (2011); Brown et al., "Length of red cell unit storage and risk for delirium after cardiac surgery," AnesthAnalg, 119:242-50 (2014); Wang et al., "Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia," Transfusion, 54:1712-24 (2014); Liu et al., "Mechanism of faster NO scavenging by older stored red blood cells," Redox biology, 2:211-9 (2014); Prestia et al., "Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice," Transfusion 54:2842-51 (2014); D'Alessandro et al., "An update on red blood cell storage lesions, as gleaned through biochemistry and omics technologies," Transfusion, 55:205-19 (2015) (hereby incorporated by reference in their entireties). An extensive body of in vitro studies unequivocally shows the degradation of RBCs (storage lesions) during conventional storage. A body of emerging metabolomic studies show the development of storage lesions at the molecular level. See Roback et al., "Metabolomics of AS-1 RBCs Storage," Transfusion medicine reviews (2014); D'Alessandro et al., "Metabolomics of AS-5 RBCs supernatants following routine storage," Vox sanguinis (2014); D'Alessandro et al., "Routine storage of red blood cell (RBC) units in additive solution-3: a comprehensive investigation of the RBC metabolome," Transfusion 55:1155-68 (2015); D'Alessandro et al., "Red blood cell storage in additive solution-7 preserves energy and redox metabolism: a metabolomics approach," Transfusion (2015); Wither et al., "Hemoglobin oxidation at functional amino acid residues during routine storage of red blood cells," Transfusion (2015); D'Alessandro et al., "Citrate metabolism in red blood cells stored in additive solution-3," Transfusion (2016); D'Alessandro et al., "Omics markers of the red cell storage lesion and metabolic linkage," Blood Transfus, 15:137-44 (2017) (hereby incorporated by reference in their entireties). There is a need for reducing or preventing this degradation to increase the efficacy of transfusions (more O2 delivery to peripheral tissues immediately after transfusion) and to reduce mortality due to hemorrhagic trauma.
Oxidative damage initiates many RBC storage lesions in conventionally stored blood and their downstream consequences; thus, methods to reduce the extent of oxidative stress are required to reduce the RBC storage lesions. A number of approaches have been developed aimed at minimizing storage lesions and improving transfusion outcomes. Approaches include additive solutions (for example, U.S. Pat. No. 4,769,318 to Hamasaki et al. and U.S. Pat. No. 4,880,786 to Sasakawa et al. U.S. Pat. No. 6,447,987 to Hess et al.), frozen storage (see U.S. Pat. No. 6,413,713 to Serebrennikov Chaplin et al., "Blood Cells for Transfusion," Blood, 59: 1118-20 (1982), and Valeri et al., "The survival, function, and hemolysis of human RBCs stored at 4 degrees C. in additive solution (AS-1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4 degrees C. in sodium chloride and glucose solution for 24 hours," Transfusion, 40: 1341-5 (2000)) (hereby incorporated by reference in their entireties).
One approach that has proven successful in improving blood quality and extending its utility is through the depletion of oxygen and storage under anaerobic conditions. Among the benefits of storing blood under oxygen depleted conditions are improved levels of ATP and 2,3-DPG, and reduced hemolysis. U.S. Pat. No. 5,624,794 to Bitensky et al., U.S. Pat. No. 6,162,396 to Bitensky et al., and U.S. Pat. No. 5,476,764 to Bitensky (hereby incorporated by reference in their entireties) are directed to the storage of red blood cells under oxygen-depleted conditions. U.S. Pat. No. 5,789,151 to Bitensky et al. is directed to blood storage additive solutions (hereby incorporated by reference in its entirety). U.S. Pat. No. 6,162,396 to Bitensky et al. (the '396 patent) (hereby incorporated by reference in its entirety) discloses anaerobic storage bags for blood storage that comprise an oxygen impermeable outer layer, a red blood cell (RBCs) compatible inner layer that is permeable to oxygen, and having an oxygen scrubber placed between the inner and outer layers.
Storing blood under oxygen depleted conditions can also result in reduced microparticle levels, reductions in the loss of deformability, reduced lipid and protein oxidation and higher post transfusion survival when compared to blood stored under conventional conditions. See Yoshida et al., "The effects of additive solution pH and metabolic rejuvenation on anaerobic storage of red cells," Transfusion 48:2096-2105 (2008) and Yoshida, T., et al. "Reduction of microparticle generation during anaerobic storage of red blood cells. Transfusion", 52, 83A (2012) (hereby incorporated by reference in their entireties). Anaerobically stored RBCs further provide higher 24-hour in vivo recovery after autologous transfusion, higher 2,3-DPG and ATP levels, lower hemolysis, and beneficial remodeling of metabolic pathway. See Reisz et al. "Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells," Blood, 128:e32-42 (2016); and Yoshida et al., "Extended storage of red blood cells under anaerobic conditions," Vox sanguinis 92:22-31 (2007) (hereby incorporated by reference in their entireties).
In the present disclosure, we demonstrate that oxygen reduced (OR) or oxygen and carbon dioxide reduced (OCR) blood from rats provides improved ATP and 2,3-DPG during storage compared to conventionally stored blood, as has been previously demonstrated using human blood. Thus, OR or OCR rat RBCs are expected to have similar reductions in microparticles, improved deformability, reduced lipid and protein oxidation and higher post transfusion survival.
Here we demonstrate for the first time that OR and OCR blood in rats provides for surprising improvements in clinical outcomes when transfused to treat hemorrhagic trauma. Using a rat hemorrhagic shock resuscitation model, we show that OR or OCR RBCs provide for reduced organ damage relative to conventionally stored blood. In addition, OR or OCR RBCs provide for reversal of the shock state using smaller pRBC volumes. Finally, OR or OCR RBCs, when transfused to treat hemorrhagic shock more rapidly stabilized hemodynamics compared to conventionally stored pRBC of same storage duration.
OR and OCR RBCs provide for improved methods for treatment of trauma resulting in exsanguination to reduce mortality and morbidity over conventionally stored blood. OR and OCR RBCs provide for reduced organ failure, including reductions in levels of markers of lung and liver damage. OR and OCR RBCs further provide reductions in the amounts of blood necessary to restore and stabilize hemodynamic function. Thus, OR and OCR RBCs can provide for reducing the volume of RBCs required for transfusion therapy when treating hemorrhagic trauma. The improved quality of OR and OCR, in addition to the previously demonstrated improvements to the ability of stored RBCs to deliver oxygen, also provides for unexpected reductions in organ damage, morbidity, and mortality associated with trauma.

Methods and treatment of trauma adverse events with oxygen reduced blood


The fluid expansion engine uses a liquid working fluid contained by primary pressurized cylinders. A heat exchange system alternately cycles hot and cold through the primary pressurized cylinders. As a result, the liquid working fluid in the cylinders reciprocally expands and contracts. The work done by the fluid expansion engine is extracted via a hydraulic pump and gearbox connected to secondary pressurized cylinders attached to the primary pressurized cylinders.

1. Field of the Invention
The present invention relates to heat engines, and particularly to a fluid expansion engine that converts heat-driven expansion of a liquid without phase change into mechanical or electrical power.
2. Description of the Related Art
Energy in general and electrical energy in particular are essential for human activities. This energy can be expensive due to the cost of needed fuel and the complexity of required conversion systems. In addition most power-generating systems can have adverse effect on the environment. Providing simple and cost effective methods for energy production can improve the quality of life for less fortunate people. Alternative energy production methods may also improve environmental quality, which currently is under great constraint due to high energy demand.
Thus, a fluid expansion engine solving the aforementioned problems is desired.

KUWAIT INSTITUTE FOR SCIENTIFIC RESEARCH


A matching circuit and an antenna apparatus which, though small-sized, can be used in a radio apparatus which transmits and receives radio waves having a wide range of frequencies. A matching circuit includes an inductance element formed of a chip inductor connected in series with an antenna body, a first capacitance element formed of a trimmer capacitor, and a second capacitance element formed of a chip capacitor, which capacitance elements are connected between respective ends of the inductance element and a ground terminal, and thus in parallel to the antenna body. One end of the inductance element is connected to the power supply terminal of the antenna body, and the other end is connected to a power source for applying a voltage to the antenna body. The first capacitance element is connected between a ground and the connection point of the power supply terminal of the antenna body and the inductance element. The second capacitance element is connected between a ground and the connection point of the inductance element and the power source.

1. Field of the Invention
The present invention relates to a matching circuit and an antenna apparatus employing the same and, more particularly, to a matching circuit and an antenna apparatus for use in a radio apparatus which transmits and receives radio waves having a wide range of frequencies, for example, a television, a radio or a pager.
2. Description of the Related Art
Generally, as antenna bodies used in radio apparatus which transmit and receive radio waves having a wide range of frequencies, for example, televisions (90 to 800 MHz), there are Yagi and Uda antennas, whip antennas and the like. However, in the case of Yagi, Uda and whip antennas, since the bandwidth is widened so as to receive radio waves having a wide range of frequencies, the antenna become greatly enlarged.
As compared with the above antennas, an antenna apparatus 50 shown in FIG. 8 has been proposed to achieve a small size. Shown in FIG. 8 are an antenna body 51, a mounting board 52 for mounting the antenna body 51, grounding patterns 53 formed on the mounting board 52, and a transmission line 54 formed similarly on the mounting board 52. The grounding patterns 53 are connected to a ground. Further, the transmission line 54 is connected to a power source V, and the power source V is connected to a ground.
For example, this antenna body 51, as shown in FIG. 9, comprises a rectangular-parallelopiped insulator 55 in which insulator layers (not shown) made of insulator powder of alumina, steatite or the like are laminated, a conductor 56, made of silver, silver-palladium or the like, formed in a coil shape inside the insulator 55, a magnetic substance 57, made of magnetic powder of ferrite powder or the like, formed inside the insulator 55 and the coil-shaped conductor 56, and external connection terminals 58a and 58b which are coated and printed to the extension end (not shown) of the conductor 56 after the insulator 55 is baked.
However, in the above-described conventional small-sized antenna apparatus, the bandwidth is narrow, and therefore, the antenna apparatus cannot be used in a radio apparatus which transmits and receives radio waves having a wide range of frequencies.
The present invention has been achieved to solve the above-described problem. It is an object of the present invention to provide a matching circuit and an antenna apparatus employing the same, which, though small-sized, can be used in a radio apparatus which transmits and receives radio waves having a wide range of frequencies.


A hydraulic bearing, having a working chamber filled with a damping fluid and a compensating chamber. The chambers are separated from one another by a dividing wall and are in fluid communication with one another via a damping channel, to damp low-frequency vibrations. The damping channel is formed by two subchannels which, in terms of technical function, are arranged in a series connection. The subchannels are arranged axially on both sides of a diaphragm that is capable of vibrating. The diaphragm forms a component of the dividing wall, and the diaphragm is provided with a channel separation and has at least one opening within the channel separation for fluid communication of the subchannels with one another.

1. Field of the Invention
The present invention relates to a hydraulic bearing having a working chamber filled with a damping fluid and a compensating chamber, wherein these chambers are separated from one another by a dividing wall and which chambers are in fluid communication with one another via a damping channel to damp low-frequency vibrations.
2. Description of the Prior Art
A bearing is shown in German Publication DE 32 25 700 C1. The dividing wall includes a diaphragm of an elastomeric material having partial areas that are moveable independently of one another, and which diaphragm is arranged between gratings.
The diaphragm in the above German publication is held sealingly at its periphery in a nozzle cage, and a damping channel surrounds the diaphragm with radial clearance on the outer peripheral side and thereby has a maximum diameter.
Therefore, the surface of the diaphragm for insulating higher-frequency vibrations is comparatively small.


An intergrated optical device comprises a vertically lasing semiconductor optical amplifier (VLSOA) and an optical element integrated onto a common substrate. Example optical elements include waveguides, unguided transparent regions, and active optical devices (including additional VLSOAs). The integrated device may be fabricated using a number of different methods, including based on selective area epitaxy, impurity induced disordering, etch and fill and silicon optical bench.

1. Field of the Invention
This invention relates generally to semiconductor optical amplifiers. More particularly, it relates to vertically lasing semiconductor optical amplifiers (VLSOAs) integrated with other optical elements on a common substrate and to methods for fabricating such integrated optical devices.
2. Description of the Related Art
Optical amplifiers, which boost the power of optical signals, are a basic building block for many types of optical systems. For example, fiber optic communications systems transmit information optically at very high speeds over optical fibers. A typical communications system includes a transmitter, an optical fiber, and a receiver. The transmitter incorporates information to be communicated into an optical signal and transmits the optical signal via the optical fiber to the receiver. The receiver recovers the original information from the received optical signal. In these systems, phenomena such as fiber losses, losses due to insertion of components in the transmission path, and splitting of the optical signal may attenuate the optical signal and degrade the corresponding signal-to-noise ratio as the optical signal propagates through the communications system. Optical amplifiers are used to compensate for these attenuations. As another example, receivers typically operate properly only within a relatively narrow range of optical signal power levels; optical amplifiers are used to boost an optical signal to the proper power range for the receiver.
Fiber amplifiers are one type of optical amplifier. They include a length of fiber which has been doped to form an active gain medium. Ions of rare-earth metals, such as erbium, are typically used as the dopant. The doped fiber is typically pumped by an optical pump at a wavelength which is preferentially absorbed by the ions but different from the wavelength of the optical signal to be amplified. The pumping results in a population inversion of electronic carriers in the active medium. Then, as the optical signal propagates through the doped fiber, it is amplified due to stimulated emission.
Semiconductor optical amplifiers (SOAs) are another type of optical amplifier. SOAs contain a semiconductor active region and an electrical current typically is used to pump the electronic population in the active region. An optical signal propagating through the active region experiences gain due to stimulated emission. Conventional SOAs are non-lasing and the gain in non-lasing SOAs typically depends on the amplitude of the optical signal. Thus, strong portions of the optical signal are amplified less than weak portions, resulting in distortion of the signal and also crosstalk between different optical signals propagating simultaneously through the amplifier. This significantly limits the use of conventional SOAs. In contrast, in lasing SOAS, the semiconductor active region is part of a laser cavity which is pumped above the lasing threshold. The gain is then clamped due to the lasing action and is fairly constant until the amplifier reaches its power limit.
Integrated optics is a term which is sometimes used to refer to the concept of an optical equivalent to electronic integrated circuits, in which many optical elements are integrated onto a common substrate. Current-day electronic circuits can implement very complex functionalities by combining a very large number of electronic elements in a single monolithic device. Electronic amplifiers are a basic building block of electronic integrated circuits. Current-day optical amplifiers, however, are not suited to play an analogous role with respect to integrated optics.
For example, fiber amplifiers are inherently too large to be useful in integrated optics. Common amplifiers used in the telecommunications industry are based on fibers which are tens of meters long. Fiber amplifiers also suffer from slow switching speeds and poor power efficiency. Furthermore, fiber amplifiers are currently manufactured using a process which is more reminiscent of electronic circuits made from hand soldered discrete components than today's highly automated semiconductor processes. As a result, not only is it difficult to produce large quantities of fiber amplifiers but the fiber amplifiers produced are fairly expensive.
SOAs also suffer from drawbacks which limit their usefulness in integrated optics. Conventional SOAs are typically packaged as discrete devices. The SOA is aligned to fiber pigtails, one for the SOA input and one for the SOA output, and the entire device is produced as a single package. This type of packaged device suffers from the same drawbacks as fiber amplifiers: large size and a labor-intensive manufacturing process.
Furthermore, in electronic integrated circuits, the various components are built up from a handful of basic building blocks: the transistor, the diode, interconnects, etc. This is not the case for optical integrated circuits. SOAs typically arc very different in design from waveguides, sources, modulators, detectors, etc. The optical elements required to build up more complex systems typically are not based on a handful of common building blocks and often even require the use of different materials systems. For example, current-day fiber communications may require the use of a multi-wavelength transmitter. Such a transmitter typically uses InP based lasers, lithium niobate modulators, a dielectric thin film wavelength division multiplexer, and a glass fiber amplifier pumped by several GaAs based lasers. Note the difference in materials and structure. Integrating these devices onto a common substrate to form an integrated transmitter is not straightforward.
Even when conventional SOAs have been successfully integrated with other optical elements, they are limited to niche applications due to their crosstalk and other performance limitations. For example, irrespective of the integration problems, conventional SOAs generally are not suited to amplifying multi-wavelength data communication signals because the crosstalk between the different wavelength signals would unacceptably degrade the signals.
Thus, there is a need for an optical amplifier which can be easily integrated with other optical elements on a common substrate. There is further a need for methods for fabricating these integrated optical devices.

Integrated optical device including a vertical lasing semiconductor optical amplifier


A MOS semiconductor memory device comprises a semiconductor substrate of a first conductive type; impurity diffused regions of a second conductive type different from the first conductive type formed into a plurality of spaced columns extending in a first direction on one surface of the semiconductor substrate and having functions of bit lines; a plurality of columns of element isolation insulating films formed on the impurity diffused regions of the second conductive type, with active regions formed therebetween; a plurality of MOS transistors formed in the active regions aligned in each of a plurality of rows extending in a second direction substantially perpendicular to the first direction, each MOS transistor including a gate formed on a part of the active region with a gate insulating film therebetween and source and drain formed in the impurity diffused regions of the second conductive type; and word lines each connected electrically to the gates of the MOS transistors aligned in each of the rows and extending in the second direction; wherein, when it is assumed that the plurality of columns are named as first and second columns alternately, the sources of the MOS transistors in adjacent two columns at both sides of the bit line of the first column are all connected to the bit line of the first column, and the drains of the MOS transistors in adjacent two columns at both sides of the bit line of the second column are all connected to the bit line of the second column.

1. Field of the Invention
The present invention relates to a MOS semiconductor memory device, and more particularly to a structure of a MOS semiconductor memory device used as a mask MOS for writing a program during a manufacturing process.
2. Description of the Related Art
A diffused layer program system, an ion implantation program system, a contact hole program system and the like are used for writing a program in a mask ROM. These systems are described in "Design of CMOS Super Large Scale LSIs" supervised by Takuo Sugano, published on Apr. 25, 1989 by Baifukan, pp. 168-169. As to a cell area per bit, a mask ROM by the ion implantation program system has the smallest area, and a series type arrangement can be adopted therein.
FIG. 7 shows a schematic partial plan view of a conventional series type mask ROM by the ion implantation program system, and FIG. 8 shows an equivalent circuit diagram of that mask ROM. Source and drain diffused layers of MOS transistor 72 serve as a bit line 54, and each word line 52 is formed in a direction perpendicular to the bit line 54. Thus, an arrangement where the channel direction of the MOS transistor 72 forming a respective memory cell coincides with the direction of the bit line 54 is obtained. Programming is performed normally in such a manner that the threshold voltage (hereinafter abbreviated as Vth) of each of MOS transistor 72 is set in an enhancement type in advance, and only the Vth of the selected MOS transistors 72 is changed over to a depletion type by an ion implantation method for programming. In FIG. 7, 82 represents an isolation insulator, and, in FIG. 8, 62 represents a row decoder and 64 represents a column decoder.
In order to read data out of a memory cell, all the word lines are initially made at a high level and then the selected word line 52 is brought to a low level and the selected bit line 54 is brought to a high level. At this time, when the Vth of the selected memory cell is of a depletion type, an electric current flowing in the selected bit line 54 is kept flowing even if the word line 52 is brought to the low level. On the other hand, if the Vth is of an enhancement type, the electric current flowing through the selective bit line 54 is cut off. The data is read out by the method described above.
Further, FIG. 9 shows a circuit diagram of a conventional parallel type mask ROM. The circuit is structured so that a bit line 56, a MOS transistor 72 and a bit line 58 are arranged in this order repeatedly in the row direction. A drain and a source of each of the MOS transistors 72 arranged in the column direction are connected to left and right bit lines 56 and 58, and gates of the MOS transistors arranged in the row direction are connected to the same word line 52. The bit lines 56 are all grounded electrically, and selection of one of the columns is made by means of the bit lines 58.
In the case of the parallel type structure, the Vth of each MOS transistor 72 is set to an enhancement type to such an extent that the MOS transistor is turned on when the gate (word line 52) of the MOS transistor 72 is brought to a high level at time of data readout. The programming is performed by changing only the Vth of the selected memory cell by the ion implantation method to such a value that the MOS transistors 72 is not turned ON even if the word line is brought to a high level. In order to read data out of the memory cell all the word lines 52 are initially kept at a low level or opened and the bit line 58 are also kept opened. Then, only the selected word line 52 is brought to a high level, and selection of one of the columns is made by means of the bit lines 58. The memory cell is selected in such a manner for reading the data thereof through a sensor 66.
The series type mask ROM is widely used in general, since the cell size can be made smallest. Since respective MOS transistors 72 arranged in the column direction are connected in series directly to the bit line 54, however, there is the problem that the source of the MOS transistor 72 is not directly coupled with the substrate potential and hence the Vth of the transistor rises by the body effect. Moreover, as the number of the MOS transistors 72 connected in series in the column direction is increased, the total channel conductance thereof gets smaller. Therefore, there is also the problem that the reading current is restricted, and the reading speed becomes slower.
On the other hand, in the parallel type mask ROM, the sources of respective MOS transistors 72 are always grounded through the bit lines 56, and no influence by the body effect is exerted. In addition, even if the number of the MOS transistors 72 connected in the column direction is increased, the reading current flowing through to the bit line 58 is not restricted. Thus, it is possible to solve the problems of the series type mask ROM. Since two bit lines 56 and 58 are required for each memory cell, however, the memory cell size becomes larger, thereby making it difficult to integrate the memory cells at high density. A conventional parallel type mask ROM is disclosed in JP-A-63-127,496 for instance.


A system using cross polarization effects and an enhancement agent having citric or other similar alpha hydroxy acid to enhance confocal microscope reflectance images and particularly images of the nuclei of BCCs (basal cell carcinomas) and SCCs (squamous cell carcinomas) in the confocal reflectance images of excised tumor slices obtained during Mohs surgery by illuminating the tissue being imaged (a tumor slice) using polarized light. The reflected illumination is passed to a polarization analyzer, which passes the polarization component which is crossed with respect to the polarization of the illuminating light. The light from the analyzer is passed through the confocal aperture and detected. The section of the tissue either at the surface or within the tissue is scanned and the reflectance image is produced with enhanced visualization of the cellular or nuclear structure thereof thereby enabling determination of the extent of the tumor (cancerous cells) in the section. A method is also provided using the system for diagnosing cancerous cells in skin tissue.

Mohs micrographic surgery for BCCs and SCCs involves precise excision of the cancer with minimal damage to the surrounding normal skin. Conventionally, precise excision is guided by histopathologic examination for cancer margins in the excised tissue slices during Mohs surgery. Typically, 2–4 slices are excised, and there is a waiting time of 10–30 minutes for the surgeon and patient while each slice is being processed.
Confocal reflectance microscopes can noninvasively image nuclear and cellular detail in living skin to provide images of tissue sections, such a microscope is described in U.S. Pat. No. 5,880,880. The contrast in the images is believed to be due to the detected variations in the singly back-scattered light caused by variations in refractive indices of tissue microstructure. Within the epidermal (basal and squamous) cells, the cytoplasm appears bright and the nuclei as dark ovals. The underlying dermis consists of collagen bundles and that, too, appears bright with dark spaces in-between. Thus, when neoplastic epidermal cells invade the dermis as in BCCs and SCCs, confocal detection of the cancers is very difficult because the cells and nuclei lack contrast relative to the surrounding normal dermis.
Acetic acid (vinegar) has been used as an image enhancement agent which enhances the back-scatter of light from certain cells. This is described, for example, in U.S. Pat. No. 5,733,739. When a tissue is illuminated with light of one polarization and a reflected image is detected via light of cross polarization the contrast of certain cells in the detected image is enhanced, when the tissue is treated by acetic acid, especially when the image is viewed via cross-polarized light in a confocal microscope. Such a system is described in the above referenced International Application No. PCT/US00/07008.

System and method for enhancing confocal reflectance images of tissue specimens


A transceiver device comprises a transmitter to transmit signals over a plurality of conductors to a memory device. An interface receives control information from a serial communication path coupled to a controller device. The control information is provided to the memory device as the signals using the transmitter. A register stores a control parameter that specifies a drive strength adjustment to the signals to transmit over the plurality of conductors to the memory device using the transmitter.

FIG. 1 illustrates a prior art memory system that includes multiple integrated circuit memory devices 120 coupled to a memory controller 110 via a bidirectional communication channel 140. Because each memory device 120 consumes physical space along the channel, the number of memory devices that can be coupled to the channel 140, and to some extent the storage capacity of the memory system, is limited by the length of the channel 140. The length of the channel 140 is itself limited by a number of practical considerations. For example, signals attenuate as they propagate down the channel 140, constraining the channel length to one that provides a tolerable signal level at the memory IC farthest from the controller 110. Similarly, channel capacitance increases with channel length, limiting the frequency response of the channel. Accordingly, the channel length usually must be limited to support the desired operating frequency of the memory system.
One technique for increasing the number of memory devices that can be used in a memory system without unacceptable loss in signaling margin or frequency response is to use buffering circuits to segment the communication path into multiple smaller channels. Unfortunately, buffers add latency that can be problematic, particularly in synchronous memory systems which rely on deterministic timing relationships. For example, in some memory systems, memory operations are pipelined by transmitting commands in the intervening time between transmission of an earlier command (e.g., a read command) and responsive transmission of the corresponding data (e.g., the read data). When buffers are positioned along the channel's length, however, the time intervals between command and response transmissions vary arbitrarily depending on the positions of the addressed memory devices (i.e., memory devices positioned downstream from one or more buffers or repeaters exhibit greater effective response delay than memory devices coupled directly to the memory controller). This significantly complicates command pipelining.
Thus, it is desirable to provide a memory subsystem that can support a large number of memory devices without degrading the reliability and performance of the memory system.

Transceiver with latency alignment circuitry


An approach is provided for optimizing the generation of a location based search result. An optimization platform generates is also caused to initiate at least one second-location based search for at least one non-overlapping area of at least one first search area and at least one second search area to generate at least one set of new results. The optimization platform also designates at least one portion of the at least one first set of results as at least one set of old results. The optimization platform then returns at least one set of new results and the at least one set of old results as at least one second set of results for the at least one second location-based search.

Service providers, vehicle and device manufacturers (e.g., wireless, cellular, etc.) are continually challenged to deliver value and convenience to consumers by, for example, providing compelling new products and services. One area of interest has been the development of location-based services to provide users of vehicles or mobile devices (e.g., mobile phones) tools for improving their travel experience. By way of example, many vehicles and mobile devices are equipped with global positioning sensors (GPS) and navigation applications for acquiring maps and other location-based information based on user provided search criteria. Unfortunately, as the user repositions or shifts the map, another search is executed, which results in more data being acquired to update the map based on the new search results. Moreover, portions of the map presented during the initial search that overlap with the new search area are acquired again, resulting in redundancy of the data acquisition effort and more data use.

Method and apparatus for performing an optimized location based search


A connector cover for a wire harness includes a main cover composed of a hinged pair of half case bodies and a secondary cover composed of a hinged pair of semicylindrical bodies. The main cover is formed with an opening for affixing to the back of the connector and a circular opening with a circular ridge elongating inside surface for passing the wire extending from the connector when the half case bodies are closed. The secondary cover has both ends opened for receiving the wire harness and is formed with a cylindrical opening with a channel grove extending therearound for passing the wire when the semicylindrical bodies are closed, and is connected to the main cover such that the circular ridge of the main cover fits in the channel groove around the cylindrical opening. Accordingly, the secondary cover can rotate with respect to the main cover.

1. Field of the Invention
The present invention relates to a connector cover which protectively covers a wire harness and an electrical connector such as a socket plug at the connecting members therebetween, and is particularly suited to covering the electrical connector members joined with the wire bundle extending from the wire harness for use in automobiles and other similar applications.
2. Description of the Prior Art
The connecting members of electrical wire harness and the corresponding connectors, or electrical socket plugs, are commonly covered and protected by a rubber or PVC cover to protect the connecting members from grease and other problems, particularly when the connection is related to important vehicle control circuits and passenger safety circuits.
Covers are likewise used to protect the wire bundles to a wire harness from edges and burrs in the vehicle and from damage during such operations as tightening bolts.
However, in the case that the rubber cover is installed to the connecting member while it is connected with the wire harness and the electrical socket plugs, the wire connecting member with the socket plug must be disconnected from the working table in which the wire harness is connected to the socket plug. Then, a grommet spreader or other device is used to spread the cover for fitting the connecter member in. This can be a tedious task. With PVC covers, for which a spreader cannot be used, the terminals must be crimped to the wire, the wire then passed through the cover, and finally the terminals crimped to the wire inserted to the connectors, a time-consuming operation that requires the cover to the installed before the connection is made. As thus described, productivity is low during the wire harness production and connection process.