The invention relates to methods and reagents for influencing alternative RNA splicing in living cells. More particularly, the invention relates to novel means for influencing RNA splice choice in living cells using polynucleotide-based reagents that compete for binding sites in nucleotide binding proteins, and novel methods for using these reagents as therapeutics.

The spatial and temporal coordination of gene expression during embryogenesis involves a variety of regulatory mechanisms, of which those acting at the transcriptional level have been most intensively studied (Davidson et al., 1998; Gellon and McGinnis, 1998; Gray and Levine, 1996; Mannervik et al., 1999). Less is known about mechanisms that control differential production and accumulation of specific proteins at various sites in the developing embryo at the post-transcriptional level, causing the RNA transcript to be spliced appropriately, or regulating transport of the spliced mRNA to the cytoplasm. Recent interest in the role of differential splicing in development and the factors and mechanisms by which it is accomplished (Chabot, 1996; Lopez, 1998), and a growing understanding of the determinants of nucleo-cytoplasmic transport of particular mRNAs (Piñol-Roma and Dreyfuss, 1992; Siomi and Dreyfuss, 1997; Weis, 1998), has set the stage for a systematic analysis of how these RNA processing factors contribute to regional and cell type specificity in the embryo.
The ribonucleoprotein hnRNP A1 is of particular interest in this regard, as it functions in both RNA splice site selection and nucleus-to-cytoplasm transport of mRNA. In its capacity as a splicing factor, this protein modulates 5′ splice site selection in a group of gene products, some of which contain a well-characterized RNA sequence determinant (Burd and Dreyfuss, 1994). Among these are the pre-mRNAs of the HIV type 1 tat protein (Del Gatto-Konczak et al., 1999), FGF receptor 2 (FGFR2) (Del Gatto-Konczak et al., 1999), and hnRNP A1 itself (Chabot et al., 1997). In its role in nucleus-to-cytoplasm transport, hnRNP A1 acts as a "shuttle" protein (Piñol-Roma and Dreyfuss, 1992), and is characterized by a novel amino acid motif termed M9, which contains both nuclear localization and nuclear export activities (Michael et al., 1995; Siomi and Dreyfuss, 1995).
The known functions of hnRNP A1 as an RNA shuttle protein and in splice choice selection are exerted in a gene product-specific fashion (Dreyfuss et al., 1993). The tissue-restricted spatiotemporal patterns in the protein's expression reported are therefore likely to be a causal component of the process by which cell types become distinctive from one another during organogenesis. A subclass of primary transcripts (including hnRNP A1 itself, Chabot et al., 1997; Mayeda et al., 1994) is differentially spliced by a process that depends on hnRNP A1. Control of splice choice appears to involve the antagonism of constitutive splicing factors such as SF2/ASF by members of the hnRNP A/B family of proteins (Mayeda and Krainer, 1992, Mayeda et al., 1993, 1994; Del Gatto-Konczak et al., 1999). Once spliced, these RNAs are transported into the cytoplasm by a process that involves hnRNP A1 and transportin 1 (Nakielny and Dreyfuss, 1998, Nakielny et al., 1999). This implies that the regulation of hnRNP A1 levels within living cells during development plays a key role in cell type diversification.
Earlier studies have surveyed the distribution of hnRNP A1 in a limited set of adult cell types (Kamma et al., 1995; Faura et al., 1995) including the developing germ cells of postnatal mice. A study by the inventors and their colleagues documenting the characterization of the sequence of chicken hnRNP A1 and its spatiotemporal and organ-specific expression during embryogenesis is hereby incorporated by reference in its entirety. (Bronstein et al., 2001). hnRNP A1 protein is abundantly expressed in early stage epithelia such as skin, extraembryonic membranes, and neuroectoderm; epithelioid tissues, such as liver; as well as "secondary" epithelia and epithelioid tissues derived from mesenchymes, e.g., heart muscle, skeletal muscle, kidney tubules, sinusoidal vascular endothelium, and precartilage condensations. It is not clear, however whether this pattern represents an authentic expression theme, or simply the prevalence both of epithelioid tissues in the early embryo and of hnRNP A1 expression. (Bronstein et al., 2001).
The expression of hnRNP A1 in differentiating neuroectoderm and dorsal root ganglia broadly coincides with patterns of expression of members of the Hu class of RNA binding protein genes in the chicken (Wakamatsu and Weston, 1997) and it is significant in this regard that the Hu family of proteins results from extensive alternative splicing of Hu gene products during neurogenesis (Okano and Darnell, 1997). However, while the expression of the two RNA binding proteins may be partly overlapping, they are not entirely so: the typical DRG cell nuclei expressing hnRNP A1 are larger than those expressing Hu and vertebral body cartilage expresses hnRNP A1 but not Hu.
The transcription of natural antisense RNA cognate to exonic sequences of the hnRNP A1 gene in many of the same tissues that are producing sense transcript is an unusual phenomenon, but one that is not as rare as previously thought (Dolnick, 1997; Vanhee-Brossollet and Vaquero, 1998). Antisense RNA probably functions as a post-transcriptional inhibitor of gene expression (Knee and Murphy, 1997). This regulatory mechanism may be particularly relevant during development—natural antisense transcripts of several developmentally active growth factors—fibroblast growth factor-2 (Savage and Fallon, 1995), bone morphogenetic protein-2 (Feng et al., 1997), and transforming growth factor-2 (Coker et al., 1998)—have been detected at significant levels, the first two in embryonic tissues. There is one previous report of differential expression of natural antisense RNA expression of a splicing factor gene (Sureau et al., 1997). Moreover, since hnRNP A1 can promote RNA-RNA strand annealing (Cobianchi et al., 1993; Idriss et al., 1994) it is itself a potential component of natural antisense regulatory mechanisms (Oberosler and Nellen, 1997).
The organ- and tissue-specific sense and antisense hnRNP A1 RNA expression patterns seen at different stages are consistent with the idea that antisense expression may be playing a regulatory role during development. For example, in kidney and liver virtually all cells at the early stages express the sense transcript. But whereas the protein product is also broadly distributed in liver the more limited distribution of the protein in kidney may be related to the more localized distribution of antisense RNA during development of this organ.
Because hnRNP A1 helps regulate nuclear-cytoplasmic transport and alternative splicing for well-defined classes of transcripts, its own regulation can provide the basis for post-transcriptional control of the partitioning of organ primordia into distinct gene expression domains. It is therefore significant that the hnRNP A1 gene is widely transcribed throughout the early embryo and its encoded protein is subject to numerous demonstrated and potential autoregulatory effects at the post-transcriptional level: it helps splice its own pre-mRNA, it may transport its own mRNA from the nucleus to the cytoplasm, and it may participate in the regulation of its own synthesis by its gene's antisense transcript. Small changes in the balance of any of these processes, or of other possible but speculative ones, such as unmasking of maternally inherited hnRNP A1, or even transfer of the protein from one cell to another, could thus activate a post-transcriptional cascade leading to the local expression of hnRNP A1, and with it the expression of its target gene products.
The recent recognition that a large proportion of the genes constituting the human genome are alternatively spliced (Ewing and Green, 2000) (a recent estimate indicates that 38% of human mRNAs contain possible alternative splice forms; Bretta et al., 2000) highlights the centrality of the developmental regulation of hnRNP A1 and other nonconstitutive splicing factors in the generation of complexity in vertebrate organisms.
Classes of hnRNP Proteins
In eukaryotes, heterogeneous nuclear RNAs (hnRNAs), which are the products of RNA polymerase II, are extensively processed to produce messenger RNAs (mRNAs). mRNA processing includes capping, splicing, and polyadenylation (Dreyfuss et al., 1993) and involves the association of the hnRNAs with nuclear proteins collectively known as ribonucleoprotein (RNP) complexes (Dreyfuss et al., 1993; Michael et al., 1995). RNPs that directly bind to hnRNAs are classified as the hnRNPs and are involved in the splicing and shuttling of pre-mRNAs. Others are categorized into special classes such as small nuclear ribonuclear proteins (snRNPs) and include the U snRNPs (Bandziulis et al., 1989; Dreyfuss et al., 1993; Dreyfuss et al., 1988; Luhrmann, 1990; Steitz, 1988; Zieve and Sauterer, 1990). The mature transcript produced from the hnRNA-hnRNP-snRNP complex is transported to the cytoplasm by specific hnRNPs where it may associate with yet another set of RNPs involved in translational regulation and mRNA stability (Bandziulis et al., 1989; Dreyfuss et al., 1993; Luhrmann, 1990; Steitz, 1988; Zieve and Sauterer, 1990).
hnRNP proteins are highly conserved throughout the vertebrates, as well as having sequence homologies in the invertebrate Drosophila (Amrein et al., 1988; Robinow and White, 1988) (Bell et at., 1991; Dreyfuss et at., 1993; Inoue et at., 1990; Kay et at., 1990; Roth et al., 1991; Voelker et al., 1990; Von Besser, 1990), and are the most abundant proteins found in the nucleus (Dreyfuss, 1986, Dreyfuss, 1993). In HeLa cells two-dimendional gel electrophoresis has resolved 20 major groups of proteins. These proteins are designated as the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 (˜34 kDa) to hnRNP U (˜120 kDa), and categorized by structural motifs (Cobianchi, 1990; Dreyfuss et al., 1993; Matunis et al., 1992; Pinol-Roma et at., 1988). Furthermore, sequence analysis has determined that hnRNPs have one or more RNA-binding modules referred to as the RNP motif or RNA Recognition Motif (RRM) in addition to at least one other auxiliary domain (Dreyfuss et al., 1993). The RNP motif contains two consensus sequences, RNP1 and RNP2, within a domain of approximately 90 amino acid residues that are located about 30 amino acids from each other (Dreyfuss et al., 1993; Dreyfuss et al., 1988). The RNP 1 module is an octapeptide, Lys/Arg-Gly-Phe/Tyr-Gly/Ala-Phe-Val-X-Phen/Tyr, SEQ ID NO: 7 in the Sequence Listing (Adam et al., 1986; Dreyfuss et al., 1993), while the RNP2 module is a hexapeptide rich in aromatic and aliphatic amino acids and is less well conserved (Dreyfuss et al., 1993; Dreyfuss et al., 1988). Both of these consensus sequences are directly related to RNA binding (Dreyfuss et al., 1993; Merrill et al., 1988).
Functional and structural categories of human hnRNPs include:                (i) hn RNP A2/B1 complexes with the snRNPs and plays a role in splicing pre-mRNAs. Though localized in the nucleus of most tissues, A2 is also found in the cytoplasm of the squamous epithelium of the skin and the esophagus, and abundant amounts of A2 are found in the medulla, but not the cortex of the adrenal gland. Both A2 and B1 are found throughout spermatogenesis while A1 expression is repressed in spermatocytes (Kamma et al., 1999).        (ii) hnRNP C1 is involved in the post-translation base change of cytosine to uracil in the apolipoprotein (apo) B mRNA which codes for the catalytic subunit APOBEC-1, a protein involved in splicesome assembly. C1 may regulate apoB mRNA editing thus restricting the activity of the catalytic subunit (Greeve et al., 1998).        (iii) hnRNP D is involved in the immunoglobulin heavy chain recombination process by binding to the switching regions in conjunction with a B cell-specific duplex DNA binding factor (Dempsey et al., 1999), while transcriptional regulation of the complement receptor 2 (CR2) is achieved by hnRNP DOB through its binding of both single and double stranded DNA (Tolnay et al., 1997; Tolnay et al., 1999).        (iv) hnRNP K may play a role in cytosine-rich pre-mRNA metabolism and cell cycle progression. Highly upregulated levels of K have been found in transformed keratinocytes (Dejgaard et al., 1994).        (v) hnRNP H, H′ are posttranslationally cleaved to produce the C-terminal proteins H(C) and H(C′) both having a molecular weight of 35 kDa with localization primarily in the nucleus. In contrast, hnRNP F varies with its localization depending on the cell type and is predominantly cytoplasmic in some cells which may be important its function (Honore et al., 1999).        (vi) Autoantibodies of hnRNP A1, A2, B have been found in individuals with connective tissue diseases. In addition to the A/B proteins, hnRNP I has been found in patients with systemic sclerosis (SSc) and in particular, in individuals with pre-SSc or limited SSc. The A/B and I protein complexes may elicit autoimmune responses (Montecucco et al., 1996).        (vii) The hnRNP L protein, having an unknown function, is found both as a component of the hnRNP complex as well as in discrete nonnucleolar structures of the nucleoplasm in HeLa cells (Pinol-Roma et al., 1989).        (viii) Finally hnRNP R, an hnRNP P-like protein, was isolated from yet another individual with autoimmune symptoms and may be a component of subcellular particles that are found in autoimmune diseases (Hassfeld et al., 1998). This protein may have some relationship to the gene product of the TLS/FUS gene, an RNA binding protein identical to hnRNP P2, and first identified as a fusion protein in human myxoid liposarcomas (Calvio et al., 1995; Crozat et al., 1993; Hassfeld et al., 1998; Rabbitts et al., 1993).
In addition, the hnRNP classes of RNA-binding proteins have been shown to be developmentally important in many embryonic tissues including the formation and maintenance of the nervous system (Dreyfuss et al., 1993), sex determination in Drosophila melanogaster (Bandziulis et al., 1989; Del Gatto-Konczak et al., 1999; Lynch and Maniatis, 1995; Lynch and Maniatis, 1996), neuronal splice activation (Del Gatto-Konczak et al., 1999; Min, 1997) and maintenance (Dreyfuss, 1993), and epithelial/mesenchymal differentiation (Johnson and Williams, 1993). In Drosophila, the embryonic lethal abnormal visual (ELAV) system proteins are required for correct differentiation and maintenance of neurons. In mammals the ELAV-like neuronal RNA-binding proteins HuB, HuC, and HuD are implicated in neuronal development and differentiation in both the central and peripheral nervous systems (Akamatsu et al., 1999; Kasashima et al., 1999). In other systems such as the human immunodeficiency virus (HIV-1) hnRNPs are involved in regulating exon 2 of the tat splicing gene (Del Gatto-Konczak et al., 1999; Si, 1997).
hnRNP A1
The hnRNP A1 protein contains two RNP consensus motifs, a glycine-rich auxiliary domain at its carboxy-terminus (Burd and Dreyfuss, 1994; Burd et al., 1989; Buvoli et al., 1990; Merrill et al., 1988), as well as an RGG box, also at its carboxy-terminus (Kiledjian and Dreyfuss, 1992). In addition to these motifs, the hnRNP A1 class of proteins contain a nuclear localization signal, within a domain of approximately 38 amino acids at the carboxy-terminal region of the protein (Michael et al., 1995). This motif, referred to as M9, is a novel nuclear localization signal (NLS)/nuclear export signal (NES) and is not homologous to the classical nuclear localization signal (NLS) found, for example in either the large T antigen of the SV40 virus or the bipartite basic NLS of nucleoplasmin (Izaurralde et al., 1997b; Kalderon et al., 1984; Michael et al., 1995; Robbins et al., 1991; Weighardt et al., 1995). The presence of the M9 motif allows hnRNPs to shuttle continuously between the nucleus and the cytoplasm (Dreyfuss et al., 1993). hnRNPs of the A1, A2/B1, D, E, I and K classes have this capability, while those of the C1, C2, and U class are found restricted to the nucleus (Izaurralde et al., 1997b; Michael et al., 1995; Pinol-Roma and Dreyfuss, 1992). Furthermore, hnRNP A1 is found bound to the poly (A)+ tail of RNA polymerase II transcripts in both the nucleus and the cytoplasm and data suggest that the hnRNP A1 protein is transported out of the nucleus with the mature message during the export process (Pinol-Roma and Dreyfuss, 1992). FIG. 1a shows the cDNA sequence designated SEQ ID NO:1 and FIG. 1b shows the amino acid sequence of chicken hnRNP A1 (indicated by CHKA1) designated SEQ ID NO:2 compared to the human hnRNP A1 amino acid sequence (indicated by HUMA1) designated SEQ ID NO:3.
FIG. 2 illustrates the structure of the human core hnRNP proteins A1, A1.sup.B, A2 and B1. The RNP-2 and RNP-1 conserved submotifs of RRM1 and RRM2, and the G domains of each protein are shown. hnRNP A1 (SEQ ID NO. 8) and A1.sup.B or hnRNP A2 (SEQ ID NO. 9) and B1 (SEQ ID NO. 10) are identical except for extra amino acid regions indicated by boxes. The sequences of the RNP-1 and RNP-2 submotifs are aligned. The dots in the alignment indicate amino acid identities. All recombinant proteins are in authentic form except for post-translational modifications. The numbers indicate the position of amino acid residues from the initiation codon Met1. Based on published cDNA sequences (Burd, 1989; Buvoli, 1990). After Mayeda et al. (1994).
hnRNP A1 and Splice Choices
In a multi-step process, uracil rich small nuclear ribonuclear proteins (U snRNPs) in association with the core hnRNPs A1, A2, B1, B2, C1, C2, and C3 (classified by increasing molecular weight), bind to the pre-mRNAs in an ordered manner at specific sequences forming the spliceosome (Beyer et al., 1977; Chung and Wooley, 1986; Del Gatto, 1996; Dreyfuss, 1986; Kumar et al., 1986; Mayeda and Krainer, 1992). Alternative splicing allows for the functional and structural diversity of gene products by the addition or deletion of elements as small as a single amino acid (as seen in the Pax-3 and Pax-7 gene products) (Lopez, 1998). Additional means of obtaining protein variants from a single transcript in a cell-specific manner include splice activation and splice repression (Del Gatto-Konczak et al., 1999).
Alternative splicing may involve the use of alternative 5′ or 3′ splice sites, optional exons, exclusive exons, or retained introns (Lopez, 1998). Except for intron retention, splicing patterns are under competitive control of splicing proteins (Lopez, 1998). Splice activation may involve multi-protein complexes on pre-mRNAs. An example of this is seen in the activation of the female specific dsx exon of Drosophila melanogaster by the female specific proteins, tra (transformer), tra-2 and SR (splice regulator proteins rich in arginine/glycine repeats) (Del Gatto-Konczak et al., 1999; Lynch and Maniatis, 1995; Lynch and Maniatis, 1996; Wang et al., 1998). In the mouse, the c-scr exon N1 is activated by the KSRP splicing factor (KH-type splicing regulator) (Min, 1997; Wang and Manley, 1997) which induces the assembly of five other proteins including hnRNP F (a pre-mRNA splicing factor which is associated with the TATA-binding protein, essential for transcription initiation (Del Gatto-Konczak et al., 1999; Min, 1997; Yoshida et al., 1999). This multiprotein complex activates the intronic splicing enhancer that splices the neuronal specific c-scr N1 exon in vitro (Del Gatto-Konczak et al., 1999; Min, 1997).
Splice repression involves protein binding to an intronic 3′ splice site and is seen in the female-specific Sxl protein of Drosophila. This interaction effectively blocks U2 snRNP and U2AF (U2 snRNP auxiliary factor) (Del Gatto, 1996; Del Gatto-Konczak et al., 1999; Lopez, 1998; Valcarcel et al., 1993). Other protein complexes may use exon sequences for splice repression.
Vertebrate genes including the human fibroblast growth factor receptor 2 gene (fgfr2), and the human immunodeficiency virus type 1 (HIV-1) tat gene contain exons that have sequences acting as exonic splice silencers (ESS) (Amendt et al., 1994; Amendt et al., 1995; Baba-Aissa et al., 1998; Caputi et al., 1994; Caputi et al., 1999; Del Gatto, 1995; Del Gatto, 1996; Del Gatto-Konczak et al., 1999; Gallego et al., 1996; Graham et al., 1992; Si, 1997).
The ESS of the human FGFR2 pre-mRNA contains a UAGG sequence in the kgfr exon (keratinocyte growth factor receptor-exon 8)(Del Gatto, 1996; Del Gatto and Breathnach, 1995; Del Gatto-Konczak et al., 1999). This sequence has homology to the high affinity consensus sequence 5′-UAGGGA/U-3′ recognized by hnRNP A1 (Del Gatto-Konczak et al., 1999). In in vitro studies, Del Gatto-Konczak et al. (1999) have demonstrated that hnRNP A1 can modulate splice choices by binding to a 10 mer ESS designated S10 (5′-UAGGGCAGGC-3′, SEQ ID NO: 5 in the Sequence Listing) or to a 6 mer ESS designated S6 (5′-UAGGGC-3′).
In in vitro studies, RNA molecules containing the splicing silencer sequence from the human fibroblast receptor 2 kgfr exon (IIIb) were capable of directing splice choice selection by the recruitment hnRNP A1 (Del Gatto-Konczak et al., 1999). When the following point mutations were introduced into the S6 ESS UCGGGC or UACGGC a two-fold decrease in hnRNP A1 binding was detected (Del Gatto-Konczak et al., 1999). Furthermore, it was determined that the targeting of hnRNP A1 to the ESS domain was through the glycine-rich motif at the C-terminus of the protein. In the human hnRNP A1 protein, the glycine-rich domains are found between residues 189-320: the RGG motif is specifically located at residues 189-247, followed by another glycine-rich motif from residues 239-320 (Del Gatto-Konczak et al., 1999). Silencing of the k-sam (kgfr) exon in these in vitro studies required the entire glycine-rich motif By examining the corresponding sequence in the chicken kgfr exon (IIIb exon 8) of fgfr2 it has been determined that the sequence corresponding to the human ESS is 5′-UAGGGAGGGC-3′, SEQ ID NO: 6 in the Sequence Listing).
Studies involving hnRNP A1 proteins demonstrated that it is capable of promoting RNA molecules to base pair into double stranded structures, therefore influencing pre-mRNA splicing by snRNPs (Burd and Dreyfuss, 1994; Buvoli et al., 1992; Eperon et al., 1993; Kumar and Wilson, 1990; Munroe and Dong, 1992; Pontius and Berg, 1990; Portman and Dreyfuss, 1994). In in vitro assays hnRNP A1, as well as the RNA binding protein splicing factor 2 (ASF/SF2) (a member of the SR nuclear phosphoprotein family) were capable of making splice choices at the 5′ splice site of pre-mRNAs that contain multiple 5′ splice sites and are essential for constitutive splicing (Caceres et al., 1997; Caceres et al., 1998; Del Gatto, 1996; Fu, 1995; Ge and Manley, 1990; Krainer et al., 1990; Manley and Tacke, 1996; Mayeda et al., 1993; Mayeda and Krainer, 1992; Mayeda et al., 1994; Munroe and Dong, 1992; Zahler et al., 1993).
In vitro studies suggest that hnRNP A1 and ASF/SF2 may act antagonistically and that the hnRNP A/B family of splicing proteins regulates the SR family both in vitro and in vivo (Caceres et al., 1998; Caceres et al., 1994; Mayeda and Krainer, 1992; Yang et al., 1994). In in vitro experiments, excess hnRNP A1 favored the distal 5′ splice site, in contrast to excess ASF/SF2 favoring proximal 5′ splice sites in a concentration-dependent manner resulting in alternate splicing patterns of many genes in specific cell types (Del Gatto, 1996; Mayeda et al., 1993; Mayeda and Krainer, 1992; Mayeda et al., 1994; Munroe and Dong, 1992). Burd and Dreyfuss (1994) have shown that the consensus sequence 5′-UAGGGA/U-3′ is a high affinity binding site of hnRNP A1 and that this sequence is similar to the 5′ and 3′ splice sites in vertebrate pre-mRNAs. In addition, the ability of hnRNP A1 to bind to this consensus sequence increased if it was duplicated and separated by two nucleotides, resulting in a dissociation constant of 1×10−9 M. While hnRNP A1 proteins are capable of binding to other pre-mRNA sites, binding affinity varies greatly over a >100 fold range, therefore classifying these proteins as sequence specific RNA binding proteins (Burd and Dreyfuss, 1994).
hnRNP A1 is also involved in self-splicing. The 4.6 kb human hnRNP A1 mRNA containing 10 exons encodes for the 34 kDa hnRNP A1 protein. The pre-mRNA for hnRNP A1 can be differentially spliced to produce the A1 form and A1B form (Buvoli et al., 1990). It has been shown that the human hnRNP A1B protein (FIG. 2) with an apparent molecular weight of 38 kDa, corresponds to the protein previous designated as hnRNP B2 (Buvoli et al., 1990). The A1B splice variant which contains an extra exon in the C-terminal region glycine-rich region (156 bp; 52 amino acids) has a higher affinity for ssDNA than the 34 kDa form though its abundance in the cell is only ˜5% that of hnRNP A1 (Buvoli et al., 1990).
More recently, Blanchette and Chabot (1997) have shown that alternative splicing of the hnRNP A1 pre-mRNA yields the A1 and A1B forms via 5′ splice selection and exon skipping, and that this process requires conserved elements. Studies have shown that the addition of the alternate exon 7B in the mature mRNA produces the hnRNP A1B protein (Buvoli et al., 1990). Furthermore, Blanchette and Chabot have demonstrated that the conserved intron element (CE1) upstream from exon 7B favors distal 5′ splice site selection. SR proteins, including SF2, which favor the proximal 5′ splice selection site, require U1 snRNP and U2AF when involved in the 5′ splice site stimulation of a 3′ splice site, as seen in the male specific 3′ splice site of tra in Drosophila (Blanchette and Chabot, 1997; Valcarcel et al., 1993). Interestingly, the CE1 element does not interfere with U1 snRNP binding and led to the discovery of an additional element CE610, which is located downstream from exon 7B. CE610 is also involved in distal 5′ splice site selection by secondary structure formation and exon skipping (Blanchette and Chabot, 1997). Since the SR family of splice selection proteins and hnRNP A1 act antagonistically for 5′ splice choices, where the SRs choose the 5′ proximal site and the hnRNPs the 5′ distal site (Weighardt et al., 1996), hnRNP A1 may be involved in modulating its own splicing (Blanchette and Chabot, 1997; Chabot et al., 1997; Del Gatto-Konczak et al., 1999; Mayeda et al, 1994).
Fibroblast Growth Factors (FGFs), Fibroblast Growth Factor Receptors (FGFRs), and FGFR-2 Splice Variants
Fibroblast growth factors (FGFs) are important mitogens in both cell proliferation and differentiation, but in some cases may act as antagonists and inhibit differentiation. Examples of FGF induced differentiation are seen in the stimulation of pre-adipocyte fibroblasts (Broad and Ham, 1983; Johnson and Williams, 1993; Serrero and Khoo, 1982), and hippocampal neurite outgrowth (Johnson and Williams, 1993; Walicke et al., 1986). Developmental roles have been demonstrated in embryonic mesodermal induction in Xenopus (Kimelman and Kirschner, 1987; Slack et al., 1987), and the inhibition of differentiation of myotubes has been shown in skeletal muscle (Linkhart et al., 1981). In addition to acidic FGF (aFGF or FGF1) and basic FGF (bFGF or FGF2), the family of FGFs, including keratinocyte growth factor (KGF) have been shown to stimulate the proliferation of mesenchymal and neuroectodermal cell types (Burgess and Maciag, 1989; Johnson and Williams, 1993). Using immunohistochemical analysis on chick embryo sections, FGF2 has been localized to the heart, myotome, limbs and muscles (Han, 1997; Joseph-Silverstein, 1989) as well as to the notochord, neural tissue, gut cells, and tubules in the mesonephric and metanephric kidneys (Dono and Zeller, 1994; Han, 1997). In addition to the previously mentioned tissues, Han (1997) localized this mitogen to the developing pharyngeal arches, specifically the maxilla and mandible. FGF2 plays an important role in morphogenesis and pattern formation in the vertebrate limb (Han, 1997; Noji et al., 1993; Riley et al., 1993; Savage et al., 1993), as well as in kidney development (Dono and Zeller, 1994; Han, 1997).
Receptors for the 19 known fibroblast growth factors (FGFs) (Hu et al., 1998; Ohbayashi et al., 1998) include the tyrosine kinase fibroblast growth factor receptors (FGFRs) (Johnson and Williams, 1993), the CFR receptor or cytosine rich FGFR, (Burrus and Olwin, 1989) and the heparan sulfate proteoglycans (HSPGs). In chicken, the genes for fgfrs1, 2, and 3 and 4 (fgfr-related kinase or frek) as well as the kgfr (exon IIIb-keratinocyte growth factor receptor) and bek (exon IIIc-bacterial expressed kinase) splice variants for receptors 1 and 2 have been cloned (Szebenyi et al., 1995). The vertebrate FGFRs contain the domains as described by Johnson and Williams, (1993). Modifications of FGFR isoforms are due to alternative splicing of the pre-mRNAs for each gene. In a schematic representation of human FGFR1, the extracellular region of the molecule has the following domains including a signal peptide region at its N-terminus, followed by three immunoglubulin-like (Ig-like) domains with an acid box between domain I and II. A membrane-proximal region precedes the transmembrane (TM) region. On the intracellular side, two tyrosine kinase domains that are separated by a kinase insert follow a juxtamembrane (JM) domain, and at the C-terminus is a C-tail domain. The third Ig-loop of FGF receptor 2 is involved in the chondrogenic process and can contain either the IIIa and IIIb (kgfr) or IIIa and IIIc (bek) exonic sequences (Johnson and Williams, 1993).
Using chick limb micromass culture, Szebenyi et al. (1995) have looked at changes in the expression of the FGFRs in differentiated cartilage and have found transcripts for fgfr1 in undifferentiated proliferating mesenchyme, fgfr2 in precartilage condensations, and fgfr3 in differentiating cartilage nodules suggesting spatiotemporal regulation in limb development. Binding of the FGFs to their receptors plays an important role in limb development through the regulation of cell survival, proliferation, and precartilage cell differentiation (Fallon et al., 1994; MacCabe et al., 1991; Niswander et al., 1993; Schofield and Wolpert, 1990; Szebenyi et al., 1995; Watanabe and Ide, 1993).
The messenger RNA splice variants IIIb (kgfr) and IIIc (bek) from fgfr1 and fgfr2, as well as fgfr3 were detected in nuclease protection assays on chicken limbs (Szebenyi et al., 1995). In addition, micromass cultures of stage 23-24 wing buds and in situ hybridization of stage 18, 23, 26, and 36 wings showed a spatial distribution of messenger RNA for fgfr1, 2, and 3. Furthermore, the probes for fgfr1 and 2 contained sequences for both the kgfr and bek splice variants and did not allow for the in vivo or in vitro detection of either of these isoforms. This is critical since there is a cell type specific role for the fibroblast growth factor receptor 2 isoforms, (FGFR2) kgfr (exon 8-IIIb) and bek (exon 9-IIIc), in precartilage differentiation. In addition, fibroblast growth factors (FGFs) influence cell function in a tissue-specific or developmental manner that can lead to defects such as craniosynostosis and syndactyly (Del Gatto, 1996; Mayeda and Krainer, 1992; Oldridge et al., 1999). This differential splicing of the pre-mRNAs produced by a single gene allows for the production of splice variants and results in forms that respond to the different growth factor isoforms in a highly specific manner. Cells that will differentiate into epithelia splice only the kgfr exon, while mesenchymal cells, including fibroblasts, as well as other cell types including endothelial cells, splice the 5′ distal bek exon (Del Gatto, 1996; Fallon et al., 1994; Johnson and Williams, 1993; Rubin et al., 1989; Szebenyi et al., 1995).
Mutations in the various receptors cause various skeletal defects (Oldridge et al., 1999). Pfeiffer syndrome, a mutation in the fgfr1 gene, presents with craniosynostosis as well as limb defects; Crouzon syndrome, a result of a mutation in the fgfr2 gene, presents with limb abnormalities; and type II achondroplasia or dwarfism, is caused by a mutation in the fgfr3 gene (Szebenyi et al., 1995). Apert syndrome or acrocephalosyndactyly type 1, presents with head, hand, and foot abnormalities (Anderson et al., 1999). Oldridge et al. (1999) looked at mutations in the fgfr2 gene of 260 unrelated Apert syndrome patients and found that 258 individuals have a missense mutation in exon 7 which lies between the 2nd and 3rd Ig-like loop domains. The remaining two individuals had an ˜360 bp insertion of an Alu-element either 5′ to exon 9 or within exon 9, which arose as de novo mutations in the paternal chromosome (Oldridge et al., 1999). Exon 9 corresponds to the 3rd Ig-like loop domain and contains the bek (IIIc) sequence (Oldridge et al., 1999). In early studies involving the role of the fgfr2 splice variants IIIb (exon 8) and IIIc (exon 9), Rubin et al. (1989) found that keratinocytes expressed the FGFR2 IIIb isoform and were stimulated by KGF. In addition, fibroblasts and endothelial cells expressed the FGFR2 IIIc isoform, and responded to FGF2 (bFGF) (Johnson and Williams, 1993; Rubin et al., 1989). In an RNA analysis of fibroblasts obtained from two Apert and two Pfeiffer syndrome patients having mutations in exon 9, severity of limb abnormalities directly corresponded to ectopic expression of the of the IIIc-kgfr form of the FGFR2. These data provided evidence of the role of signal transduction pathways through the KGFR form of the receptor in relation to syndactyly in Apert syndrome (Oldridge et al., 1999; Park et al., 1995; Wilkie et al., 1995).
FIG. 4 illustrates FGFR2 with positional mutations, polymorphic nucleotides, and primers using in the Oldridge study. Top shows leader sequence (L), acid box (A), three Ig-like domains (IgI, IgIII, and IgIII), a transmembrane region (TM), and a split tyrosine-kinase domain (TK1 and TK2). Exons 8 and 9 encode for the alternative splice variants of the second a half of the IgIII domain, which is depicted by the IgIIIb (kgfr isoform) and IgIIIc (bek isoform) respectively. Positional mutations of two Apert syndrome patients (1 and 2) with Alu insertions, as well as two patients with Pfeiffer syndrome (3 and 4) with nucleotide substitutions are also shown. After Oldridge et al., (1999)
Alternative Splicing
An embodiment of the present invention describes a method and reagents that influence alternative splicing in living cells. Alternative splicing is a mechanism by which a single gene may eventually give rise to several different proteins. Alternative splicing is accomplished by the concerted action of a variety of different proteins, termed "alternative splicing regulatory proteins," that associate with the pre-mRNA in the cell nucleus, and cause distinct alternative exons to be included in the mature mRNA. These alternative forms of the gene's transcript give rise to distinct isoforms of the specified protein. The virulence of the HIV virus associated with AIDS depends on particular alternative splice choices, and several cancers, rheumatoid and osteoarthritis, and other inflammatory diseases, exhibit aberrant splice choices when compared to corresponding non-diseased tissues.
An embodiment of the present invention describes a novel means for influencing splice choice in living cells using polynucleotide-based reagents that compete for binding sites in alternative splicing regulatory proteins, and novel methods for using these reagents as therapeutics.
An embodiment of the present invention contains the following novel aspects, which will be taken up in order:                1. A novel method for influencing splice choice in living cells using polynucleotide-based reagents that compete for binding sites in alternative splicing regulatory proteins.
Sequences in pre-mRNA molecules that bind to alternative splicing regulatory proteins can be found in introns or exons, and are known by the terms intronic splicing silencers or enhancers, and exonic splicing silencers or enhancers (ISS, ISE, ESS, ESE). No published paper in the Medline database reports the introduction into living cells of polynucleotide-based competitors for ISS, ISE, ESS, or ESE binding sites in alternative splicing factors. Burd, C. G., and Dreyfuss, G. (1994) identified a 20-mer RNA sequence that binds the alternative splicing factor hnRNP A1, but this was work done with isolated protein and nucleic acid components, not within living cells. Blanchette, M., and Chabot, B. (1999) and Breathnach and co-workers (Del Gatto, F., and Breathnach, R. , 1995; Del Gatto, F., Gesnel, M. C., and Breathnach, R., 1996; Del Gatto, F., Plet, A I, Gesnel, M. C., Fort, C., and Breathnach, R., 1997; Del Gatto-Konczak, F., Olive, M., Gesne, M. C., and Breathnach, R., 1999; have investigated the effects of various ISS, ISE, ESS, ESE-related sequences in splice choice, but all these experiments have been done in cell-free extracts, not within living cells.                2. Novel methods for using the reagents described above as therapeutics.
Although it has been recognized for some time that the life cycle of the AIDS virus HIV involves alternative splicing (Amendt, B. A., Si, Z. H., and Stoltzfus, C. M., 1995; Si, Z., Amendt, B. A., and Stoltzfus, C. M., 1997; Si, Z. H., Rauch, D., and Stoltzfus, C. M., 1998; Del Gatto-Konczak, F., Olive, M., Gesnel, M. C., and Breathnach, R., 1999; none of these, nor any other, studies propose treating the disease with competitors of ISS, ISE, ESS, or ESEs.
However, it is likely that if agents that competed with alternative splicing regulatory proteins such as hnRNP A1 for the HIV tat protein ESS were introduced into HIV infected cells, as shown in the Dissertation of one of the inventors "Avian hnRNP A1, an mRNA Shuttle Protein-Exon Splicing Silencer: Developmental Regulation and Role in Chondrogenesis" (Department of Cell Biology and Anatomy, New York Nedical College), which is herein incorporated by reference in its entirety, indicates that the method is feasible and effective, and that the the viral infection would be attenuated (Purcell, D. F., and Martin, M. A., 1993). Indeed, splicing of HIV-1 pre-mRNA must be inefficient to provide a pool of unspliced messages which encode viral proteins and serve as genomes for new virions (Caputi, M., Mayeda, A., Krainer, A. R., and Zahler, A. M., 1999), and virus production is arrested in a natural HIV variant that has an aberrant ESS (Wentz, M. P., Moore, B. E., Cloyd, M. W., Berget, S. M., and Donehower, L. A., 1997).
With regard to cancer, it has been found that certain tumors, such as mammary carcinomas (Stickeler, E., Kittrell, F., Medina, D., and Berget, S. M., 1999) and colon adenocarcinomas (Ghigna, C., Moroni, M., Porta, C., Riva, S., and Biamonti, G., 1998) contain levels of hnRNP A1 and other alternative splicing regulatory proteins that are altered relative to related normal tissues. Moreover, this abnormality is reflected in aberrant splicing patterns of certain alternatively spliced gene products, such as the cell adhesive protein CD44, although the specific role of splice variants of CD44 in tumorigenicity and metastasis is unresolved (Sneath, R. J., and Mangham, D. C., 1998).
The neoplastic state is characterized by numerous other gene products that show aberrant alternative splicing patterns. These include the extracellular matrix protein fibronectin (Midulla, M. Verma, R., Pitnatelli, M., Ritter, M. A., Courtenay-Luck, N. S., and George, A. J., 2000), the proteolytic enzyme cathepsin B (Keppler, D., and Sloane, B. F., 1996), the breast cancer susceptibility gene BRCA2 (Bieche, I., and Lidereau, R., 1999), and the apoptosis-associated gene products Bcl-x (Xerri, L., Hassoun, J., Devilard, E., Birnbaum, D., and Birg, F., 1998), Bax (Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J., 1993), and caspase 2 (Ich-1) (Jiang, Z. H., and Wu, J. Y., 1999). The apparent causal relationship of some of these aberrant splicing patterns to the neoplastic state, coupled with the emerging evidence that tumors express abnormal levels of alternative splicing regulatory proteins, suggest that treatment with agents that specifically inhibit these regulatory proteins, such as those methods and reagents disclosed and claimed herein, represent a promising approach to cancer therapy.
Inflammatory diseases such as rheumatoid and osteoarthritis also involve protein (e.g., CD44) that exhibit abnormal alternative splicing patterns (Croft, D. R., Dall, P., Davies, D., Jackson, D. G., McIntyre, P., and Kamer, L. M., 1997; Boyle D. L., Shi, Y., Gat, S., and Firestein, G. S., 2000), and it is reasonable to hypothesize that the resulting aberrant proteins, among which are secreted and cell surface molecules, contribute to the immune-mediated manifestations of these diseases. Again, these data suggest that treatment with agents that specifically target alternative splicing factors represent a promising therapeutic approach.
Several publications have suggested using an antisense strategy to alter splicing patterns as therapeutics for cancer and ceratin other diseases (but not AIDS) (Sierakowska, H., Gorman, L., Kang, S. H., and Kole, R. (2000); Mercatante, D., and Kole, R., 2000). The invention described herein is not an antisense strategy, and has many advantages over such a strategy.
Current treatment of AIDS uses multiple reagents (AXT, protease inhibitors) directed against different biological functions of HIV. The method and reagents according to an embodiment of the present invention are directed against a distinct cell-virus interactive function, alternative splicing, and should add productively to the spectrum of agents available for treatment of this disease. Current treatment for cancer involves the use of agents that are frequently highly toxic and nonspecific. The method and reagents according to an embodiment of the present invention will constitute therapeutics with high specificity for a biological function, alternative splicing, that is aberrant in many cancers.

Splice choice antagonists as therapeutic agents


Apparatus operable for dispensing toothpaste from an outlet opening at one end of a collapsible toothpaste containing tube is disclosed. The present apparatus includes a base member having a nozzle mounted adjacent a first end. The nozzle is adapted for cooperatively receiving and holding the one end of the collapsible toothpaste tube and has a passage therethrough for dispensing toothpaste from the tube. A pinch roller assembly is mounted to the base member for movement therealong between a location spaced from the nozzle and a location adjacent the nozzle, the pinch roller assembly including a pair of pinch rollers defining a space therebetween adapted for receiving the toothpaste tube in a generally collapsed condition. The outer surface of at least a first of the pinch rollers is adapted for engaging the tube so as to be capable of moving along the tube when rotated. The apparatus further includes a drive cable having one end attached to the first pinch roller for drivingly rotating it, the drive cable having an opposite end connected in rotatably driven communication with a gear motor or other rotatable power source. In operation, the rotatable power source is energizable for rotatably driving the drive cable and the connected pinch roller to move the pinch roller assembly along the toothpaste tube for collapsing a portion of the tube to pressurize the toothpaste therein and dispense the toothpaste through the nozzle.

A wide variety of both manually operable and powered devices for dispensing toothpaste from a tube are well known in the art.
However, all of the known toothpaste dispensing devices utilize mechanisms for pinching or squeezing the toothpaste tube to collapse it, which mechanisms are observed to be too complex and/or too expensive to manufacture for widespread marketability. See, for example, Wilson U.S. Pat. No. 4,213,542 and Kane U.S. Pat. No. 4,403,714.
Accordingly, the present invention is directed to overcoming one or more of the problems as set forth above.


A phase difference detecting type autofocusing device capable of selectively focusing a desired one of a plurality of subjects within a field of view, by using a simple structure. The phase difference detecting type autofocusing device includes an optical system having first and second lenses, the first and second lenses having the same focal length and disposed on a first plane with the optical axes of the lenses being set in parallel. A plurality of first photosensors are disposed on a second plane in parallel with the first plane, for converting information of images focused by the first lens into electric signals; and a plurality of second photosensors are disposed on the second plane at positions covered by the second lens, for converting information of images focused by the second lent into electric signals. Each second photosensor is spaced apart in the same direction by the same distance from a corresponding one of the plurality of first photosensors.

a) Field of the Invention
The present invention relates to an autofocusing device for use with cameras or the like, and more particularly to an autofocusing device of a phase difference detecting type, capable of selectively focusing a desired one of a plurality of subjects.
b) Description of the Related Art
A phase difference detecting type autofocusing method is known as one of a number of autofocusing methods.
According to this method, two images of the single subject are focused by two lenses, and a difference in position of the two focused images changing with the distance from the lenses to the subject, is detected in the form of a phase difference.
FIGS. 7A shows an example of a conventional phase difference detecting type autofocusing device. At the back of an equivalent film plane 52 after a taking lens 51, there are disposed a condenser lens 53, a separator lens 54 and a phase difference detector in this order.
The phase difference detector is constructed of line sensors 55 and 56 such as CCDs and a signal processor 57. The line sensors 55 and 56 photoelectrically convert a pair of images of a subject focussed by the separator lens 54. The signal processor 57 discriminates an in-focus state, based upon electric signals obtained from pixels of the line sensors 55 and 56, the electric signals corresponding to a light intensity distribution on the pixels.
A pair of images focussed on the line sensors 55 and 56 comes near an optical axis 58 if the subject image is of a front focus state before the equivalent film plane 52. Conversely, the pair of images goes away from the optical axis 58 if the subject image is of a rear focus state. At an in-focus state, the pair of images takes an intermediate position between front focus and rear focus.
A phase difference detecting method is used for detecting the positions of images focused on the line sensors 55 and 56. According to this method, an in-focus state is discriminated by a relative shift amount (phase difference) between focused images, the relative shift amount corresponding to a minimum correlation value between pairs of images on the line sensors 55 and 56. A correlation value is given by the following equation (1): EQU H(m)=.SIGMA.(k=1 to n).vertline.B(k)-R(k+m-1).vertline. (1)
where .SIGMA.(k=1 to n) is a sum of functions for k=1 to k=n. k represents a k-th pixel of the line sensor 55 operating as a standard line sensor. m is an integer from 1 to 9 and represents the relative shift amount.
B(k) represents an electric signal time-sequentially outputted from respective pixels of the standard line sensor 55, and R(k+m-1) represents an electric signal time-sequentially outputted from respective pixels of the reference line sensor 56. The equation (1) is calculated for m=1 to 9 to obtain a correlation value H(1), H(2), . . . , H(9).
It is preset that an in-focus state is obtained, for example, when the correlation value H(5) takes a minimum value. If another correlation value takes a minimum value, this shift amount, i.e., a phase difference from m=5, is detected as an out-of-focus.
An example of the structure of the conventional signal processor 57 is shown in FIG. 7B. Electric signals B(k) and R(k) generated at respective pixels of the line sensors 55 and 56 are converted into digital data of 8 bits for example by a microcomputer 60, and temporarily stored in a random access memory (RAM) 61. Thereafter, a central processing unit (CPU) 60 calculates the equation (1) using the digital data stored in RAM 61.
In the autofocusing device shown in FIGS. 7A and 7B, electric charges stored in the photosensors are directly converted into, and detected as, a voltage signal which is then converted into a digital signal and stored in RAM 61, digital signals in RAM 61 being read out and calculated.

Phase difference detecting type autofocusing device including an optical system having first and second lenses


An analog-to-digital converter according to the invention is provided. The analog-to-digital converter preferably includes an analog input signal, a first reference signal, a second reference signal, and a range compression signal. The range compression signal is preferably characterized by a magnitude greater than the first reference signal and smaller than the second reference signal. In addition, when the analog input signal is sampled N times and the range compression signal is sampled N1 times, a compression factor related at least in part to the ratio N1/(N+N1) is obtained.

The input signal range of an Analog-to-Digital converter (ADC) extends between a minimum value (further referred to as the "input range lower limit" LL) and a maximum value (further referred to as the "input range upper limit" UL). As an example, in a unipolar ADC the LL usually represents the zero scale point and the UL usually describes the full-scale point. In a bipolar ADC the LL is usually the "minus full scale" point while the UL is usually the "plus full scale" point.
In the description that follows herein, the end points of the ADC external input range—i.e., the end points of the range of the analog signal transmitted to the ADC—will be referred to as LL and UL while the end points of the ADC internal input range—i.e., the end points of the range of signals that the ADC is able to process—will be referred to as LLI and ULI. When an external signal of value LL is applied at the ADC input the expected ideal conversion result is referred to as code(LL). Similarly, when an external signal UL is applied to the ADC input the expected ideal conversion result is referred to as code(UL).
Three distinct problems are present in ADCs. The first two problems are specific to oversampling converters.
First, the quantization noise of an oversampling ADC varies with the input signal and is substantially higher in the vicinity of the internal input range end points LLI and ULI. This behavior is described in detail in the paper "The Structure of Quantization Noise from Sigma-Delta Modulation" by J. C. Candy and O. J. Benjamin published in IEEE Trans. Commun., vol. COM-29, pp. 1316-1323, Sep. 1981. It is thus highly desirable to prevent the ADC operation in these regions.
Second, as the order of the modulator contained within the oversampling ADC increases, a series of stability issues arise which limit the effective range of the ADC to substantially less than the full input range. This issue is further described in the paper "Design of Stable High Order 1-bit Sigma-Delta Modulators" by T. Ritoniemi, T, Karema and H. Tenhunen published in IEEE Proc. ISCAS '90, pp. 3267-3270, May 1990. Therefore it is often necessary to operate the ADC away from the internal input range limit points LLI and ULI.
Third, the LL and UL values are in general easily available external signals like "ground" or Vref (reference voltage) or −Vref and are commonly used in evaluating and calibrating the ADC offset and gain errors. When these values are applied to the ADC input, the presence of internal ADC error sources (like gain or/and offset error) may result in a combined equivalent value outside of the ADC internal input signal range LLI-to-ULI. In this situation the conversion produces an "out of range" result. When an out of range result is obtained, the ADC output cannot be calibrated using this result. This is because the result does not contain sufficient meaningful information. Therefore it is highly desirable to use an ADC with an internal range LLI-to-ULI wider than the external range LL-to-UL.
In addition, it is important from a user point of view for the ADC to produce its minimum output code for the LL external input signal and its maximum output code for the UL external input signal. Thus any range modification and restriction that is introduced in order to solve the above mentioned issues should be transparent to the ADC user.

Range compression in oversampling analog-to-digital converters using differential input signals


A PC board ejector assembly is provided for disengaging a first PC board from a second PC board in a computer chassis. The PC boards each includes at least one connector for connecting the first PC board to the second PC board. The chassis includes a divider wall. The PC board ejector assembly includes a mounting bracket positioned on at least one of the first and the second PC boards, and a disengagement member coupled to the mounting bracket. The mounting bracket is positioned on the PC board such that movement of the disengagement member engages the member against the divider wall to disengage the PC board connectors from one another and permit removal of at least one of the PC boards. A method for disengaging a PC board including a PC board ejector assembly from a chassis is also provided. The method includes the steps of accessing a PC board including the PC board ejector assembly that is mounted in the chassis, manipulating the disengagement member of the PC board ejector assembly to disengage the PC boards from one another within the chassis, and removing that PC board from the chassis.

The present invention relates to the field of computers and computer hardware. More particularly, the present invention relates to a PC board ejector assembly for removing a PC board from a computer, and to a method for using the PC board ejector assembly.
Computer systems typically include a central processing unit (CPU) that consists of one or more printed circuit (PC) boards, including motherboards and input/output (I/O) boards interconnected by one or more multi-pin connectors and cables. The multi-pin connectors, in turn, are configured to include of six rows of pins having forty-eight pins per row. Often times, a CPU consists of a motherboard having one or more male connectors and an I/O board having corresponding female connectors configured to be mated with the male connectors of the motherboard. The shell of the CPU typically includes a cabinet and a divider centrally disposed within the cabinet. The divider typically includes one or more cutouts and a series of bosses that extend from each side of the divider.
A CPU is typically constructed by mounting the motherboard into the center divider such that one or more male connectors extend through the cutouts in the divider onto an opposite side thereof. The board is secured in place using screws that are inserted through openings in the motherboard and threaded into the bosses on the divider. The I/O board is then mounted in the CPU on the opposite side of the divider by friction fitting the female connectors onto the male connectors of the motherboard. The I/O board is then similarly secured in place by inserting screws through openings on the board and threading the screws into the bosses on the divider. Connectors effective for connecting the motherboard and the I/O board are manufactured, for example, by Teradyne of Nashua, N.H.
The connection formed when the connectors on the motherboard and the I/O board are mated is typically very tight. A strong connection is beneficial as it ensures that the communication between the motherboard and the I/O board will not be interrupted or fail. However, the strength of this connection also makes the separation and removal of either or both of these boards very difficult. A service technician must typically exert up to approximately 80 pounds of force to extract or disengage the I/O board from the motherboard. This force typically must be exerted in a very confined area as the board connectors are typically disposed in a very confined spaced within the CPU. As a result, when a board is serviced, an attempt to remove the board by hand increases the chances of damaging the board and/or injuring the service technician. The use of a device such as a screwdriver or a pair of pliers does not facilitate the removal of the board. To the contrary, in fact, the use of such tools increases the possibility of injury to the technician and introduces further risk of damage to the I/O board due to excess flexion.
This invention addresses the disadvantages described above by providing a positive acting PC board ejector assembly for disengaging a first PC board, such as a motherboard, from a second PC board, such as an I/O board, in a chassis. The PC board ejector assembly permits removal of the board without the need for tools or equipment, and without the risk of injury to the technician or damage to the PC board. The PC board ejector assembly of the present invention also functions to reinforce the PC board to minimize the risk of damage that typically occurs during its removal from the computer.
The PC board ejector assembly includes a mounting bracket and a disengagement member coupled to the mounting bracket. The mounting bracket is positioned on at least one of the first PC board and the second PC board such that actuation of the disengagement member engages the member against the chassis to disengage the first PC board from the second PC board. The mounting bracket preferably defines an opening such that the disengagement member moves through the opening to disengage the PC boards.
The present invention further provides a method for disengaging a first PC board from a second PC board in a chassis. The method includes the steps of accessing one of the first and the second PC boards in the chassis, actuating the disengagement member of that PC board to engage the member against the chassis to disengage the PC boards from each other, and removing that PC board from the chassis.

PC board ejector assembly and method for use


The invention relates to a method of bonding together two wafers made of materials selected from semiconductor materials by providing two wafers each having a surface that is suitable for molecular bonding; and conducting plasma activation of at least one surface of one of the wafers by directing plasma species onto the surface(s) being activated while controlling activation parameters of the plasma to provide kinetic energy to the species sufficient to create a disturbed region of controlled thickness beneath the surface(s) being activated. Advantageously, the surface of each wafer is activated for optimum results while the controlling of the activation parameters also serves to control the maximum depth of the disturbed region in the surfaces. During plasma activation, the activation parameters are controlled to modify the kinetic energy of the species contained in the plasma in order to create one or a plurality of disturbed regions of controlled thickness in the thickness of a surface region of the wafer(s) whose surface is being activated.

The present invention relates to a method of bonding two wafers made out of materials selected from semiconductor materials, the method making use of plasma activation of the surface of at least one of the two layers that are to be bonded together.
Methods of this type are generally known. The term "bonding" is used herein to mean bonding by molecular bonding during which two perfectly plane surfaces are bonded together without applying any adhesive—and this being possible at ambient temperature. The quality of the bond is characterized in particular by its bonding energy, which represents the connection force between the two bonded-together wafers.
In order to consolidate bonding by molecular bonding between two wafers, the bonded-together wafers are subjected to heat treatment. Such heat treatment enables the bonding energy of the two wafers to be raised to a value of at least about 500 millijoules per square meter (mJ/m2), where this corresponds to the values that are typically desired. In conventional manner, such heat treatment is generally carried out at a temperature of at least about 900° C. (which in the context of the present specification defines the "high temperature" domain). When bonding between a wafer made of Si and a wafer made of Si or of SiO2, the bonding energy is maximized when heat treatment is carried out at temperatures of the order of 1100° C. to 1200° C.
"Plasma activation" of a surface for bonding is defined as exposing a surface to a plasma (which can be carried out in particular under a vacuum or at atmospheric pressure) prior to putting the surfaces for bonding together into contact. More precisely, in prior art activation techniques, the surface of a wafer for activation is exposed to a plasma during an exposure step in which the exposure parameters are controlled so that each is set at a given respective value, which values remain unchanged during plasma activation.
First order "exposure parameters" are as follows:
power density; this is the density of the power feeding the plasma, which represents a power density per unit area in watts per square centimeter (W/cm2), and is sometimes also referred to in the present specification by the term "power", for simplicity;
pressure (pressure in the enclosure containing the plasma); and
the nature and the flow rate of the gas fed into the enclosure. Such activation serves in particular to perform bonding by molecular bonding, obtaining high bonding energies without requiring any recourse to heat treatment that would necessarily need to be carried out at high temperatures.
Plasma activation thus makes it possible to obtain high bonding energies between two wafers, at least one of which is activated prior to bonding, and after applying heat treatment over relatively short durations (e.g. about 2 hours (h)), and at temperatures that are relatively low (e.g. about 600° C., or less). Such activation is thus advantageous for stabilizing a structure comprising two bonded-together wafers, when it is desired to avoid subjecting the structure to temperatures that are too high (as applies in particular to heterostructures, defined as being structures comprising layers made of materials having coefficients of thermal expansion that are significantly different). Such activation can also be advantageous for obtaining high bonding forces at a given temperature.
Such activation is thus advantageous for making multilayer structures involving the bonding together of two wafers. Transfer methods (in particular methods of the SMART-CUT® type as described in general terms in the work "Silicon-on-insulator technology: materials to VLSI", 2nd edition (Jean-Pierre Colinge)) constitute one example of an application that can benefit from plasma activation to enhance bonding. The present invention is therefore directed at providing improvements in such methods.

Method of bonding two wafers of semiconductor materials

S.O.I.Tec Silicon on Insulator Technologies


A thrust needle roller bearing includes a plurality of needle rollers, and an annular retainer having a plurality of pockets for retaining the needle rollers. The plurality of pockets accommodate the needle rollers arranged in multiple rows shifted from each other in a radial direction of the retainer, and the retainer is made of synthetic resin. The thrust needle roller bearing is light, can reduce friction between the retainer and the needle rollers, and has high durability.

1. Field of the Invention
The present invention relates to a thrust needle roller bearing for use in a compressor of an air conditioner of an automobile.
2. Description of the Background Art
A thrust needle roller bearing is formed of needle rollers, a retainer and a bearing washer, and the needle rollers make line-contact with the bearing washer so that it can achieve a high load capacity and a high rigidity although a bearing projection area is small. Therefore, the thrust needle roller bearings have been widely used as structures for bearing thrust loads in compressors of air conditioners of automobiles. Conditions of use are becoming more severe due to driving with lean lubrication or high-speed rotation.
For example, Japanese Patent Laying-Open No. 2002-70872 has disclosed a conventional thrust needle roller bearing, in which inflow and/or outflow properties of lubricant or lubricating oil are improved for increasing an amount of lubricant supplied per time. This thrust needle roller bearing will now be described with reference to FIGS. 21A–21C.
Referring to FIGS. 21A–21C, a thrust needle roller bearing 50 is formed of a plurality of needle rollers 80 and two annular retainers 60 and 70. Each of two retainers 60 and 70 has a plurality of long windows 61 or 71, each of which is radially longer than roller 80. These plurality of windows 61 and 71 provide roller retaining portions 64 and 74, which retain the plurality of needle rollers 80 in the vertically opposite directions. Each of roller retaining portions 64 and 74 of two retainers 60 and 70 has a radial length 1a shorter than a roller length 1. At least one of two retainers 60 and 70 is bent by appropriate working so that at least one of total thicknesses or vertical lengths t1 and t2 of radially outer and inner portions of retainers 60 and 70 is smaller than a total thickness t0 of roller retaining portions 64 and 74.
Radially outer plate portions 62 and 72 of two retainers 60 and 70 vertically are overlaid with each other, and end portions 67 and 77 of radially inner plate portions 63 and 73 of retainers 60 and 70 are vertically bent to overlap with each other, and are fixed together by caulking effected on end portion 67 of radially inner plate portion 63.
This structure improves the inflow and/or outflow properties of lubricant in at least one of the radially outer and inner portions, which have reduced thicknesses as compared with roller retaining portions 64 and 74, and thereby can increase the amount of lubricant passing through the bearing per time. Further, such a situation is suppressed that retainers 60 and 70 intercept the passing lubricant. Therefore, the lubricant does not remain, and increase in lubricant temperature can be suppressed so that durability of the bearing can be improved.
For ensuring intended strengths of retainers 60 and 70 as well as needle rollers 80 in the thrust needle roller bearing 50, two retainers 60 and 70 as well as needle rollers 80 are made of a cold-rolled steel plate (SPCC). Thus, thrust needle roller bearing 50 is primarily made of iron so that it suffers from a problem of a large weight. Since the weight of thrust needle roller bearing 50 is large, an inertia weight and thus inertia force are large so that a large torque loss occurs, e.g., in an automobile air conditioner or a compressor. This deteriorates the fuel consumption.
Since two retainers 60 and 70 as well as needle rollers 80 are primarily made of iron, large friction unpreferably occurs between needle rollers 80 and two retainers 60 and 70. The large friction between needle rollers 80 and two retainers 60 and 70 increases a required torque and noises during rolling of needle rollers 80.
Further, in the operation of the conventional thrust needle roller bearing 50, a peripheral speed of a raceway surface in contact with needle roller 80 increases as the position moves radially outward in thrust needle roller bearing 50. Therefore, a difference in peripheral speed between needle roller 80 and the raceway surface becomes maximum at the opposite end surfaces of needle roller 80. This tendency increases with increase in relative length of the needle roller with respect to the outer diameter. This peripheral speed difference causes differential sliding on needle roller 80. Thereby, heat is generated from contact portions of needle rollers 80 and the raceway surface so that surface damage (smearing) and/or surface-starting peeling occur. Since these phenomena are liable to occur in the conventional thrust needle roller bearing, it may also suffer from insufficient durability.


A structure for attaching a fluorescent tube includes a base, a holder supported by the base, and a pair of contacts. The holder holds an intermediate portion of the fluorescent tube while restraining the fluorescent tube from moving in a radial direction. To the pair of contacts, terminals of a pair of end portions of the fluorescent tube are respectively attachable in an attaching direction corresponding to the radial direction of the fluorescent tube. Each contact includes a pair of elastic pieces facing each other. The pair of elastic pieces have a pair of straight portions extending substantially straight along the attaching direction and facing each other. On the pair of straight portions, a pair of contact portions for clamping the corresponding terminal of the fluorescent tube therebetween are formed.

1. Field of the Invention
The present invention relates to a attaching structure for a long fluorescent tube.
2. Description of Related Arts
For example, as connecting structures for terminals of fluorescent tubes such as cold cathode fluorescent tubes to be used as a backlight of a liquid crystal display type television or monitor, the following documents 1 and 2 have been proposed.                Document 1: Japanese Unexamined Patent Publication No. 2001-250605 (Date of publication: Sep. 14, 2001)        Document 2: Japanese Unexamined Patent Publication No. 2004-259645 (Date of publication: Sep. 16, 2004)
In the documents 1 and 2, a connection fitting fixed to one end of an electric wire is soldered to a terminal (electrode) of a cold cathode fluorescent tube. The other end of the electric wire is connected to an inverter circuit via a connector. Electrical power from the inverter circuit is supplied to the cold cathode fluorescent tube via the connector and the electric wire.
However, in the documents 1 and 2, there is a risk that conducting failure may occur between the terminal and the connection fitting due to defective soldering. In addition, the soldering is troublesome. In particular, the cold cathode fluorescent tube has a thin shape with a diameter of several millimeters, and the terminal is even thinner than this, so that handling of the cold cathode fluorescent tube requires great care. Therefore, great labor is required for soldering.
Not only the case of the cold cathode fluorescent tube, but also cases using other fluorescent tubes such as an external electrode fluorescent tube have a similar problem.
An object of the invention is to provide a fluorescent tube attaching structure which realizes easy and reliable electrical connection with a fluorescent tube.


A building structure is supported above a foundation and above grade by a moment frame comprising a plurality of columns and beams. The columns are all generally similar and the beams are all generally similar, such that each column can be attached with up to four beams. The moment frame is secured to a foundation, which may include pilings extending downwardly into the ground. The foundation can include a concrete foundation member or a foundation framework comprising a plurality of attachment members secured together by beams. The pilings can include helical flighting.

Many building structures are supported above ground level, or above grade. For example, many homes in coastal areas and regions susceptible to flooding are supported above grade in order to position the home at a height sufficient to reduce the risk of damage from flooding. Recent destructive storms have prompted governmental agencies to focus on the risks associated with flooding for building structures. For example, following Hurricane Sandy in 2012, the United States Federal Emergency Management Agency (FEMA) updated its Advisory Base Flood Elevation (ABFE) information. The ABFE information identifies the minimum recommended elevation that a building structure must be above sea level.
Building codes have been updated to take account of FEMA's updated ABFE information, and impose construction requirements relating to the minimum elevation for a building structure. The National Flood Insurance Program (NFIP) has also updated its insurance requirements and rates based on FEMA's updated ABFE information. These changes to building codes and insurance programs impact existing building structures and new building structure construction. For example, an owner of an existing building structure may be required to elevate the building structure in order to qualify for or receive a lower rate for flood insurance under the NFIP. And a prospective owner of a new building structure will be required to have the building structure constructed so that it is at an appropriate elevation above sea level.
Various support structures have been used to elevate building structures, but they tend to suffer from various drawbacks, and these drawbacks can become magnified when trying to accommodate higher flood elevation requirements.

Systems for elevating a building structure above grade, and related methods


A rigid plastic enclosure made from flat blanks which is particularly well suited for use as an electrical or electronic component enclosure. The top, bottom, back, and side walls of the enclosure are parts of a unitary structure made by cutting and/or routing a single sheet of plastic which can be folded and locked into an open-faced, box-shaped structure. A cover or face plate can be fastened to the folded and locked structure to complete the enclosure. Because the enclosure is constructed from plastic, holes can be easily drilled into its walls, eliminating the need for providing knockout holes at predetermined locations. The invention is lighter than steel enclosures, and can be shipped and stored in a flat configuration, thereby saving space and allowing for lower shipping and handling costs.

The present invention relates to plastic enclosures which can be stored and shipped as flat sheets but are capable of being folded up along flexible fold lines and locked together to make rigid boxlike structures, and, more particularly, to such enclosures which are suitable for use as wall-mounted electrical component enclosures, such as pull boxes, junction boxes, splice boxes, and equipment cabinets.

Foldable electrical component enclosures


A sensor for a selected analyte in a test sample has (a) a semipermeable membrane with pores for retaining the analyte, where the membrane has been chemically modified by attachment of membrane modifiers; (b) immunoassay labels which have label binding ligands where these label binding ligands will have a binding affinity for the membrane modifiers in the presence of the analyte, and a measurably different binding affinity for the membrane modifiers in the absence of the analyte; and (c) a label detecting system, for detecting the presence of the labels on the membrane.

1. Field of the Invention
The present invention relates generally to assays and more specifically to binding assays, such as antibody/hapten or DNA interactions, using nanoporous membranes for increasing the local concentration of the analyte, to improve the overall sensitivity of the assay. This technique can be used with a wide range of labeling schemes, including radio labeling and labeling with magnetic beads.
2. Description of the Related Art
Binding assays, for example immunoassays, are widely used in the food, medical, and pharmaceutical industries as diagnostic tests for a wide range of target molecules. Many binding assays have been produced and marketed since the principle was first developed.
Immunoassays typically exploit the binding capabilities of antibodies. Antibodies are protein molecules which are frequently considered fighters of infections. They fight infections by binding to the infectious material in a specific manner, forming a complex. This is a signal to the organism to reject that complex. Antibodies may also be produced to specifically bind to a wide range of compounds, as a key fits a lock. However other molecules (e.g., chelators, strands of polynucleic acids, receptors including cellular receptors) that are capable of recognizing and selectively binding other molecules may be employed to detect a wide range of species, such as polynucleic acids (DNA or RNA), polypeptides, glycolipids, hormones, polymers, metal ions, and certain low molecular weight organic species including a number of illegal drugs. To be useful in an assay, this recognition event must generate a signal that is macroscopically observable. The method employed to generate such a signal is one way of distinguishing the various types of immunoassays.
The first immunoassay used radioactive labeling. This radioimmunoassay (RIA) is quite sensitive and widely used, but the hazards, expense, and restrictions associated with handling radioactive material makes alternative immunoassays desirable. Recently, enzyme and fluorescence assays have replaced radioimmunoassays. The present inventors and others have developed techniques using magnetic beads as labels for immunoassays. Other known immunoassay labeling techniques use colloids or fluorescent dyes.
An ongoing goal of immunoassay development is improving the lower limit of detection (LLD). Likewise, it is an ongoing goal of immunoassay development to shorten processing time. This is particularly true in order to counter threats of biological warfare and terrorism, as well as other field applications.
Solid supports are used in many immunoassays, typically as adsorbent layers. Many of these, such as nylon and nitrocellulose membranes have pore sizes greater than 25 nm, to amplify the signal by increasing the surface area of the assay.
Some microbiological assays use membranes to separate and concentrate bacteria. These membranes are typically on the order of 200 nm pore size.
Viruses have been identified with aluminum ultrafiltration membranes with 20 nm pores. Organisms immobilized on these membranes have been identified using both specific and nonspecific dyes.
Chemically selective membranes are used in some chemical sensors to pass the analyte through the membrane. Larger molecules are not allowed to pass through the membrane into the internal sensing solution.

The United States of America as represented by the Secretary of the Navy


This disclosure presents methods, systems, computer-readable media, and apparatuses for optically tracking the location of one or more objects. The techniques may involve accumulation of initial image data, establishment of a dataset library containing image features, and tracking using a plurality of modules or trackers, for example an optical flow module, decision forest module, and color tracking module. Tracking outputs from the optical flow, decision forest and/or color tracking modules are synthesized to provide a final tracking output. The dataset library may be updated in the process.

This disclosure generally relates to the field of image processing, and more specifically, to image processing for the optical tracking of objects.
A variety of computing and camera applications require optical tracking of the position or movement of an object. For example, certain computer interfaces enable a user to control a cursor through hand movement sensed by a camera. These interfaces require the hand to be optically tracked with a high degree of accuracy, and without requiring an excessive use of computational resources.
Despite the need for reliable object tracking, known trackers have certain deficiencies. A variety of individual object tracking algorithms are available which have performance characteristics which vary depending on image conditions. There is a need for enhanced optical tracking performance and efficiency.

Robust and efficient learning object tracker


A method of measuring flux density and run out to accommodate rotors of different diameters, evaluate intrinsic properties of magnet material and the magnetization process. Circular run out measurement capability is also used to evaluate bearing journal “ovality.” The method includes the use of a scan tool, or a DLA Rotor Flux Density Scan Fixture, which evaluates the electromagnetic field strength (gauss), combined with surface run out and presents the data in a scalable pictorial format. The scan tool includes a probe which measures a magnetic field strength and circular run out of the perimeter of the magnet. Simultaneously, a non-contact measurement sensor is used to measure the rotor surface for subtle variations. The resulting sine wave gauss data and the surface dimension data are manipulated into a scalable “radar” plot. The radar plot correlates magnetic pole field strength and surface circular run out variation to the index position.

Digital linear actuators (DLAs) and stepper motors typically incorporate a rotor in the form of a magnetic cylinder. During calibration of the rotor, the flux density is measured (in Gauss), to determine the capacity of the rotor. Typical methods used to determine the flux density involve spinning the rotor at a predetermined high velocity (such as 1800 rpm), in a generated voltage fixture device required for screw-in family rotors. A separate tool is required for other types of rotors. These are only applicable for measuring flux density during the production process because the fixture is not suitable to fit nonproduction stepper motor rotors, or rotors which have bearings attached.
Another diagnostic method involves spinning the rotor assembly at high velocity, such as 1800 rpm, while assembled within the DLA. This method is an intrusive test method and degrades functionality of the DLA. The rotational speed required to develop generated voltage from the motor is sufficient to predicate degradation of internal bearings or lubricants. Insertion of the threaded shaft adaptor may damage the internal lead screw thread. The flux density in millitesla (mT) is plotted as a sine wave as shown in FIG. 1. While the plot shown in FIG. 1 shows the different flux density measurements, the plot in FIG. 1 is difficult to interpret, and provides no correlation between the flux density, and variations in the dimensions of the rotor.
Therefore, there exists a need for a method of measuring the flux density of a rotor used for a DLA or stepper motor during the development stages, which measures not only flux density, but also is capable of measuring surface irregularities of the rotor.

DLA rotor flux density scan method and tool


An electronic endoscope system comprises a video-scope, an imaging device, and a black balance processor. The imaging device, which is provided on the video-scope, is exposed at a normal shutter speed so as to generate an image signal corresponding to an optical image that is formed thereon. The black balance processor generates a black balance value for adjusting the black balance of the image signal, based on a black image signal corresponding to a black image. The black image signal is generated by exposing the imaging device at a high shutter speed that is faster than the normal shutter speed.

1. Field of the Invention
The present invention relates to an electronic endoscope system that adjusts a black balance of an image signal.
2. Description of the Related Art
An endoscope system is composed of a video-processor, and a video-scope and a monitor that are connected to the video-processor. The video-scope is provided with an imaging device (for example, a CCD) on a tip portion thereof. The imaging device generates image signals corresponding to an optical image that is formed at the tip portion, and the image signals are output on the monitor as the moving image, after image processing at the video-processor.
The endoscope can be attached to or removed from the video-processor easily, and several types of the video-scope can be attached to the video-processor. The characteristic of the imaging device is different according to the type of the video-scope; therefore, the black balance of the image signals that are input to the video-processor is different according to which type of the video-scope is attached thereto.
Conventionally, the black balance adjustment is conducted in the video-processor using a black balance value in order to maintain an appropriate black balance of the image signals. Because the characteristics of the image device are different according to the type of the video-scope, the black balance value needs to be recomputed whenever the type of the attached video-scope is changed.
However, the tip portion of the video-scope needs to be blacked out by a shading instrument when the black balance value is calculated. Therefore, it leaves the complicated work to the user when the type of the attached video-scope is changed.
In order to eliminate the complicated work, the black balance values regarding the several types of video-scope are stored in the memory in the video-processor, and the black balance value is read from the memory according to the type of the attached video-scope, as shown in Japanese Unexamined Patent Publication (KOKAI) NO. 11-197103. In this system, the black balance value is automatically set up according to the type of the attached video-scope.
However, the black balance of the image signal may be different according to the temperature in the operation room where the video-scope is used, or the degree of deterioration of the light source for illuminating an object, even if the same type of video-scope is attached to the same video-processor. Therefore, the black balance cannot be correctly adjusted if the black balance value is set only based on the type of the video-scope.
Recently, the auto fluorescent endoscope system has been put to practical use. In this system, a lesion, such as one of cancer, in an organ is identified by the auto-fluorescence, which the tissue emits when excitation light is illuminated thereto. The auto-fluorescence which the tissue emits is very weak; therefore, the image signal based on the auto-fluorescence needs to be amplified before it is displayed on the monitor. Due to amplification, the auto-fluorescent image that is displayed on the monitor is greatly influenced by a small change in black balance. Accordingly, it is necessary more precisely to adjust the black balance of the auto-fluorescent image than the black balance of a normal image.


A switching regulator includes a first switch circuit, a second switch circuit, and a protection circuit. The first switch circuit has a first connection node coupled to a first reference voltage, and a second connection node coupled to one end of an inductor. The second switch circuit has a first connection node coupled to a second reference voltage, and a second connection node coupled to the one end of the inductor. The protection circuit senses a voltage level at the first connection node of the first switch circuit, and selectively enables an auxiliary current path in response to the voltage level at the first connection node of the first switch circuit, wherein the auxiliary current path and at least the first switch circuit are arranged in a parallel connection fashion.

The present invention relates to a voltage regulator design, and more particularly, to a switching regulator using a protection circuit for avoiding voltage stress and an associated power management integrated circuit (PMIC).
Switching regulators are widely used in various electronic apparatuses to supply appropriate voltages to electronic circuits used inside the electronic apparatuses. For example, a switching regulator is a voltage regulator that uses switching elements to transform the incoming power supply into a pulsed voltage, which is then smoothed using passive components such as inductors, capacitors, and/or resistors. The switching elements may be implemented by metal-oxide-semiconductor (MOS) transistors. A safe operating area (SOA) is the voltage and current conditions over which a MOS transistor operates without permanent damage or degradation. The MOS transistor must not be exposed to conditions outside the safe operating area even for an instant. However, there are parasitic resistors, inductors, and capacitors in the power path. During switching, the MOS transistor implemented in the switching regulator may suffer from SOA violation due to voltage bouncing resulting from the parasitic resistors, inductors, and capacitors in the power path. One typical solution may protect the switching regulator from SOA violation at the expense of power efficiency. Furthermore, the typical solution requires a complex hardware detector, and is hard to determine an optimum control setting for protecting the switching element. Thus, there is a need for an innovative switching regulator which has a simple detector design and can maintain the power efficiency while obeying the SOA requirement.

Switching regulator using protection circuit for avoiding voltage stress and associated power management integrated circuit


A novel polypeptide that functions as an IL-18 receptor is disclosed. The receptor is multimeric and includes at least one AcPL polypeptide, or fragment thereof, and at least one IL-1Rrp1 polypeptide, or fraction thereof. The receptor binds IL-18 and finds use in inhibiting biological activities mediated by IL-18.

1. Field of the Invention
The present invention relates to proteins that are members of the IL-1 receptor family. More particularly, the present invention relates to IL-1Rp1 and AcPL receptor complexes that mediate high affinity IL-18 binding and activity as well as inhibit IL-18 mediated activity.
2. Description of Related Art
The type I interleukin-1 receptor (IL-1RI) mediates the biological effects of interleukin-1, a pro-inflammatory cytokine (Sims et al., Science 241:585-589, 1988; Curtis et al., Proc. Natl. Acad. Sci. USA 86:3045-3049, 1989). A second interleukin-1 receptor (designated type II IL-1R or IL-1RII) binds IL-1, but does not appear to mediate signal transduction (McMahan et al., EMBO J. 10:2821, 1991; Sims et al., Proc. Natl. Acad. Sci. USA 90:6155-6159, 1993). IL-1RI and IL-1RII each bind IL-1□ and IL-1β. IL-1 has been implicated in chronic inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. There is increasing evidence that IL-1 plays a role in osteoporosis. All of these activities are initiated by the signaling function of the cytoplasmic portion of the Type I IL-1R. IL-1ra inhibits the activities of IL-1 by binding to the type I IL-1 receptor, thereby blocking access to IL-1α and IL-1β while eliciting no biological response of its own.
IL-1RI and IL-1RII belong to a family of proteins that exhibit significant sequence homology. One such protein is IL-1R accessory protein (IL-1R AcP), described in Greenfeder et al. (J. Biol. Chem. 270: 13757-13765, 1995). This protein, by itself, is not capable of binding IL-1, but does form a complex with IL-1RI and IL-1α and IL-1β. When co-expressed with IL-1RI, recombinant IL-1R AcP increases the binding affinity of IL-1RI for IL-1β (Greenfeder et al., supra).
Another protein exhibiting sequence homology to the IL-1RI and IL-1RII family is the IL-1 receptor related protein 1 (IL-1Rrp1) (See Parnet et al. J. Biol Chem 271:3967, 1996, and Torigoe et al., J. Biol Chem 272:25737, 1997). Still another such protein is AcPL.
IL-18 is a homologue of IL-1α and IL-1β and is known to activate many of the same responses activated by IL-1. For example, cells stimulated with IL-18 activate NFκB and produce known inflammatory mediators. IL-18 acts as a stimulator of Th1 cell growth and differentiation and is a potent inducer of γ-interferon production from Th1 cells. The Th1 class of helper T cells are known to mediate inflammatory reactions. IL-18 enhances NK cell killing activity and has been implicated in septic shock, liver destruction, inflammatory bowel disease and diabetes.
Recently it was shown that IL-1Rrp1 binds IL-18 and mediates IL-18 signaling in transfected cells. However, the IL-1Rrp1 binding affinity for IL-18 is very low and it is likely that one or more additional receptors or receptor subunits are involved with IL-18 binding and signaling.
Thus, the identification of additional receptors of for IL-18 is desirable. Such receptor proteins can be studied to determine whether or not they bind IL-18 and, if so, whether the receptors play a role in mediating signal transduction. Furthermore, soluble forms of such receptors may be used to inhibit IL-18 activity and ameliorate any inflammatory and/or autoimmune diseases attributable to IL-18 signaling. The possible existence of additional affinity-converting subunits for IL-18 can be explored, as well.


Disclosed are stable microcapsule compositions each containing a microcapsule dispersed in an aqueous phase and a stabilizing agent. The microcapsule compositions are stable for at least 4 weeks when storing at 45° C., and the microcapsule composition is considered stable when (i) the composition has a viscosity of 3000 cP or less and (ii) 20% or less water by volume of the composition is separated from the composition. Also disclosed are consumer products having such a stable microcapsule composition.

Microcapsules are useful in a variety of applications where there is a need to deliver, apply, or release a fragrance or other active material in a time-delayed and controlled manner.
Conventional microcapsule compositions contain microcapsules dispersed in an aqueous phase. They tend to form gel, separate into layers, release the active material prematurely, etc. See US 2014/0287008 and WO 2015/023961. Certain microcapsule compositions have been developed to improve the stability via engineering a more robust microcapsule wall. See US 20140044760, WO2014011860, and US20130337023. However, these microcapsule compositions still face stability issues including short storage lifetime, gel formation during transportation in a harsh temperature, and instability in consumer products.
There is a need to develop a stable microcapsule composition that can be stored for a extended period of time and provide a long shelf life in consumer products.


The invention relates to a tool for cleaning and/or drying a cavity in a workpiece, such as a hole, in particular a threaded bore. The workpiece has a workpiece adapter. The workpiece adapter includes a workpiece contact surface and has a connector for feeding cleaning medium and/or drying medium. There is an opening in the connection adapter for discharging cleaning medium and/or drying medium containing dirt particles. A nozzle having a nozzle chamber is formed in the workpiece adapter. The connector opens into the nozzle chamber such that a cyclonic flow of the fed cleaning medium and/or drying medium can form in the nozzle and exits the nozzle. The cyclonic flow transports dirt particles from the cavity in the workpiece to the opening for discharging dirt particles and cleaning medium and/or drying medium.

A tool and a process of the type mentioned in the introduction are known from DE 103 18 238 B4. The document describes a tool for cleaning the inner surfaces of threaded bores in a workpiece which has a lance-shaped blowing insert. This blowing insert can be inserted into a threaded bore. A supply channel for cleaning medium is formed in the blowing insert. The supply channel has an opening slot which extends in the longitudinal direction over the blowing insert. The opening slot serves for directly applying a flow of cleaning medium tangentially to the wall of a threaded bore. Dirt particles can thereby be removed from the thread turns in the threaded bore. In order to achieve a satisfactory cleaning action with this cleaning tool, it is necessary to adapt the length of the blowing insert to the depth of a threaded bore to be cleaned. The cleaning tool therefore has a technically complex design. A rotatory movement of the cleaning tool is additionally required for the defined cleaning of portions of a threaded bore which lie far away from the opening slot in a workpiece.
For machining workpieces, for instance engine components, for example, cylinder heads, cooling lubricants are used and chips are formed. As a result, workpieces are contaminated. These contaminants can cause disruptions in subsequent installation processes and impair the technical functionality of systems which are produced from corresponding workpieces. Instances of contamination with cooling lubricants and chips in cylinder head bores and injection nozzles can cause irreparable damage to the engine in the case of internal combustion engines.

Tool for cleaning and/or drying a cavity