The present invention relates to polypeptides expressed and processed in yeast, a DNA construct comprising a DNA sequence encoding such polypeptides, vectors carrying such DNA fragments and yeast cells transformed with the vectors, as well as a process of producing heterologous proteins in yeast.

The present invention relates to polypeptides produced in yeast, a DNA construct comprising a DNA sequence encoding such polypeptides, vectors carrying such DNA fragments and yeast cells transformed with the vectors, as well as a process of producing heterologous proteins in yeast.
Yeast organisms produce a number of proteins synthesized intracellularly, but having a function outside the cell. Such extracelluar proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European publication 088632A describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vector harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium. The signal peptide may be the desired protein""s heterologous signal peptide, or a hybrid of a homologous and a heterologous signal peptide.
A problem encountered with the use of signal peptides heterologous to yeast may be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
The Saccharomyces cerevisiae MFxcex11 (xcex1-factor) is synthesized as a pre-pro form of 165 amino acids comprising a 19 amino acids long signal- or pre-peptide followed by a 64 amino acids long xe2x80x9cleaderxe2x80x9d or pro-peptide, (Kurjan et al. (1982) Cell 30:933-943). Use of signal/leader peptides homologous to yeast is described in U.S. Pat. No. 4,546,082; EP publications 0116201A, 0123294A, 0123544A, 0163529A, 0123289A, EP No. 0100561B, and PCT Publication WO 95/02059.
In EP 0123289A utilization of the S. cerevisiae xcex1-factor precursor is described whereas EP 0100561 describes the utilization of the S. cerevisiae PHO5 signal and WO 95/02059 describes the utilization of YAP3 signal peptide for secretion of foreign proteins.
U.S. Pat. No. 4,546,082 and European Publication Nos. 0016201A, 0123294A, 0123544A and 0163529A describe processes by which the xcex1-factor signal-leader from S. cerevisiae (MFxcex11 or MFxcex12) is utilized in the secretion process of expressed heterologous proteins in yeast. Secretion and processing of the desired protein was demonstrated by fusing a DNA sequence encoding the S. cerevisiae MFxcex11 signal/leader peptide at the 5xe2x80x2 end of the gene for the desired protein.
EP 0206783 discloses a system for the secretion of polypeptides from S. cerevisiae whereby the xcex1-factor signal/leader sequence has been truncated to eliminate the four xcex1-factor peptides present on the native sequence so as to leave the signal/leader peptide itself fused to a heterologous polypeptide via the xcex1-factor processing site Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO:93). It is indicated that this construction leads to an efficient process for production of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native xcex1-factor leader sequence has been truncated to leave one or two xcex1-factor peptides between the leader peptide and the polypeptide.
A number of secreted proteins are routed so that the precursor is exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in S. cerevisiae encoded by the KEX 2 gene (Julius et al. (1984) Cell 37:1075). Processing of the product by the KEX 2 protease is needed for the secretion of active S. cerevisiae mating factor xcex11 (MFxcex11 or xcex1-factor) but is not involved in the secretion of active S. cerevisiae mating factor a.
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, however, the level of secretion is very low or there is no secretion, or the proteolytic processing may be incorrect or incomplete. As described in WO 90/10075, this is believed to be ascribable, to some extent, to an insufficient exposure of the processing site present between the C-terminal end of the leader peptide and the N-terminal end of the heterologous protein so as to render it inaccessible, or less accessible, to proteolytic cleavage, for example, by the KEX 2 protease.
WO 90/10075 describes a yeast expression system with improved processing of a heterologous polypeptide obtained by providing certain modifications near the processing site at the C-terminal end of the leader peptide and/or the N-terminal end of a heterologous polypeptide fused to the leader peptide.
The present invention describes modifications of the N-terminal end of the heterologous polypeptide designed as extensions which can be cleaved off either by naturally occurring yeast proteases before purification from the culture media or by in vitro proteolysis during or subsequently to purification of the product from the culture media.
In one aspect, the present invention is drawn to a DNA construct encoding a polypeptide having the structure:
signal peptide-leader peptide-X1-X2-X3-X4X5-X6-X7-heterologous protein
wherein
X1 is Lys or Arg;
X2 is Lys or Arg, X1 and X2 together defining a yeast processing site;
X3 is Glu or Asp;
X4 is a sequence of amino acids with the following structure
(A-B)n
wherein A is Glu or Asp, B is Ala, Val, Leu or Pro, and n is 0 or an integer from 1 to 5, and when nxe2x89xa72 each A and B is the same or different from the other A(s) and B(s); or
X4 is a sequence of amino acids with the following structure
(C)m
wherein C is Glu or Asp, and m is 0 or an integer from 1 to 5;
X5 is a peptide bond or is one or more amino acids which may be the same or different;
X6 is a peptide bond or an amino acid residue selected from the group consisting of Pro, Asp, Thr, Glu, Ala and Gly; and
X7 is Lys or Arg.
A specific embodiment of the present invention is drawn to a DNA construct encoding a polypeptide having the structure:
signal peptide-leader peptide-X1-X2-X3-X4-X5-X6-X7-heterologous protein
wherein X3-X4-X5-X6-X7 are the sequence Glu Glu Ala Glu Pro Lys (SEQ ID NO: 1).
The sequence Glu Glu Ala Glu Pro Lys (SEQ ID NO: 1) forms an extension at the N-terminal of the heterologous polypeptide. This extension not only increases the fermentation yield but is protected against dipeptidyl aminopeptidase (DPAP A) processing, resulting in a homogenous N-terminal of the polypeptide. The extension is constructed in such a way that it is resistant to proteolytic cleavage during fermentation so that the N-terminally extended heterologous protein product can be purified from the culture media for subsequent in vitro maturation, e.g. by trypsin or Achromobacter lyticus protease I. The desired in vitro removal of the N-terminal extension of SEQ ID NO:1 is readily achieved by either trypsin or Achromobacter lyticus protease I, presumably due to flexibility of the N-terminal extension peptide resulting in an improved yield of the matured heterologous protein.
Another specific embodiment of the present invention is drawn to a DNA construct encoding a polypeptide having the structure:
signal peptide-leader peptide-X1-X2-X3-X4-X5-X6-X7-heterologous protein
wherein X3-X4-X5-X6-X7 are the sequence Glu Glu Gly Glu Pro Lys (SEQ ID NO:2).
The sequence Glu Glu Gly Glu Pro Lys (SEQ ID NO:2) forms an extension at the N-terminal of the heterologous polypeptide. Without being bound by any specific theory, it is surprising shown that the location of a glycine (G) in the N-terminal extension compared to the repeated Glu Ala of the xcex1-factor leader results in improved heterologous protein yield which may reflect an improved translocation and/or secretion, since glycine with only a hydrogen atom as a side chain can adopt a much wider range of conformations than other amino acid residues, thus allowing unusual main chain conformations, and a possible more unstable precursor polypeptide and secretion process.
The term xe2x80x9csignal peptidexe2x80x9d is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of an extracellular protein expressed in yeast. The function of the signal peptide is to allow the heterologous protein to be secreted to enter the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. A preferred signal peptide in this invention is yeast aspartic protease 3 (YAP3) signal peptide or any functional analogue thereof. YAP 3 has been cloned and characterised by Egel-Mitani et al. (1990) YEAST 6:127-137.
The term xe2x80x9cleader peptidexe2x80x9d means a polypeptide sequence whose function is to allow the heterologous protein to be secreted to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the medium. Preferably the leader peptide used in the present invention is selected from the following group of leader peptides
Gln Pro Ile Asp Glu Asp Asn Asp Thr Ser Val Asn Leu Pro Ala (SEQ ID NO:3);
Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Pro Ala (SEQ ID NO:4);
Gln Pro Ile Asp Asp Glu Ser Asn Thr Thr Ser Val Asn Leu Pro Ala(SEQ ID NO:5);
Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Pro Val (SEQ ID NO:6);
Gln Pro Ile Asp Asp Thr Glu Asn Thr Thr Ser Val Asn Leu Pro Ala (SEQ ID NO:7);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro Ala (SEQ ID NO:8);
Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Met Ala (SEQ ID NO:9);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro Gly Ala (SEQ ID NO:10);
Gln Pro Ile Asp Asp Thr GIu Ser Asn Thr Thr Ser Val Asn Leu Met Ala (SEQ ID NO:11);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Val Pro Thr (SEQ ID NO:12;
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Leu Val Asn Val Pro Thr (SEQ ID NO:13;
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro Thr (SEQ ID NO:14);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Leu Val Asn Val Pro Gly Ala (SEQ ID NO:15);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Pro Ala Val Ala (SEQ ID NO:16);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Asp Leu Ala Val Gly Leu Pro Gly Ala (SEQ ID NO:17);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Pro Gly Ala (SEQ ID NO:18);
Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Pro Gly Ala (SEQ ID NO:19);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Leu Val Asn Leu Pro Gly Ala (SEQ ID NO:20);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val Asn Leu Pro Leu (SEQ ID NO:21);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Ala Asn Val Ala Met Ala (SEQ ID NO:22);
Gln Pro Ile Asp Asp Thr Glu Ser Ala Ile Asn Thr Thr Leu Val Asn Leu Pro Gly Ala (SEQ ID NO:23);
Gln Pro Ile Asp Asp Thr Glu Ser Phe Ala Thr Asn Thr Thr Leu Val Asn Leu Pro Gly Ala (SEQ ID NO:24);
Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val Asn Leu Pro Leu (SEQ ID NO:25);
Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Asp Val Val Asn Leu Pro Gly Ala (SEQ ID NO:26);
Gln Pro Ile Asp Asp Thr Glu Ser Ala Ala Ile Asn Thr Thr Leu Val Asn Leu Pro Gly Ala (SEQ ID NO:27);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val Asn Leu Ala Asn Val Ala Met Ala (SEQ ID NO:28);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Asp Val Val Asn Leu Ile Ser Met Ala (SEQ ID NO:29);
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asn Thr Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Asp Val Val Asn Leu Ile Ser Met Ala (SEQ ID NO:30);
identified in PCT/DK95/00249 and all C-terminally followed by a Lys-Arg sequence and any functional analogue thereof, and more preferably the leader peptide has an amino acid sequence of 43 or more amino acids, such as the leader peptide (LA19):
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Ala Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:31),
identified in PCT/DK95/00249 and which includes the C-terminal Lys-Arg processing site, or any functional analogue thereof. In the DNA construct of the present invention the leader peptide preferably contains an endopeptidase processing site at the C-terminal end, such as a Lys-Arg sequence.
Even more preferred leader peptides encoded by the DNA constructs of the invention are:
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Ala Gly Gly Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:32),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ala Phe Ala Thr Asn Thr Thr Leu Ala Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:33),
Ser Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Ala Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:34),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Asn Ser Gly Gly Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:35),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Ser Val Gly Gly Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:36),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Ala Gly Gly Leu Asp Val Val Gly Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:37),
Gln Pro Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ala Phe Ala Thr Asn Thr Thr Ser Val Gly Gly Leu Asp Val Val Gly Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:38),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ala Phe Ala Thr AsnThr Thr Leu Ala Gly Gly Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:39),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ala Phe Ala Thr Asn Thr Thr Asn Ser Gly Gly Leu Asp Val Val Asn Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:40),
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ala Phe Ala Thr Asn Thr Thr Leu Ala Gly Gly Leu Asp Val Val Gly Leu Ile Ser Met Ala Lys Arg (SEQ ID NO:41).
The term xe2x80x9cheterologous proteinxe2x80x9d means a protein or polypeptide which is not produced by the host yeast organism in nature.
In a still further aspect, the invention relates to a process for producing a heterologous protein in yeast, comprising cultivating the transformed yeast strain in a suitable medium to obtain expression and secretion of the heterologous protein, after which the protein is isolated from the medium.
The invention further relates to a recombinant expression vector which is capable of replicating in a eucaryotic cell, preferably a yeast cell, and which carries a DNA construct of the invention. Preferably, the DNA construct comprises a synthetic leader peptide, preferably the LA19 leader peptide. Besides, the invention relates to the DNA construct described in FIG. 2 herein. The invention also relates to a eucaryotic cell, preferably a yeast cell, which is capable of expressing a heterologous protein and which is transformed with a vector of the invention.

N-terminally extended proteins expressed in yeast


An image forming apparatus prevents link members for monochrome and for color from coming off while suppressing an increase in rotatory torque and a deficiency of clutch capacity for transmitting a driving force to a cam shaft. Coming-off protection sections for preventing the coming off of a second link member which is the link member for color is provided in a cam section for monochrome. In other words, it is not necessary to provide a coming-off protection section in a second cam section which is a cam section for color requiring a large rotatory torque. This eliminates the need for a coming-off protection section of the cam section for color to act on the link member for color when a cam is turned by a driving source.

A transfer device according to the intermediate transfer method is known that is installed in an image forming apparatus adopting the electrophotography method in which toner images are transferred in such a manner as to be superimposed sequentially from a plurality of image bearing members onto an intermediate transfer belt in the primary transfer and then the toner image is transferred from the intermediate transfer belt onto a paper sheet in the secondary transfer.
Among the image forming apparatus are ones that have operational modes such as monochromatic transfer mode, full-color transfer mode, and standby mode in which no image forming is performed. The intermediate transfer belt is in contact with only an image bearing member for black in the monochromatic transfer mode, is in contact with all the image bearing members in the full-color transfer mode, and is separate from all the image bearing members in the standby mode.
For example, transition between the intermediate transfer belt's contact with and separation from the image bearing members is achieved by causing a plurality of primary transfer rollers respectively facing each of the plurality of image bearing members across the intermediate transfer belt to be displaced in directions for the transition between the contact and the separation (for example, refer to Patent Literature 1).
The conventional transfer device as described in the Patent Literature 1 that is provided with a link member for monochrome to cause a primary transfer roller for monochrome that corresponds to a black toner image to be displaced in the contact-separation directions, a link member for color to cause primary transfer rollers for color that correspond to toner images of the three primary colors (cyan, magenta and yellow) to be displaced in the contact-separation directions, and a transfer member moving mechanism that includes a cam to move the link member for monochrome and the link member for color respectively and a driving source to turn the cam. The cam is formed in an eccentric cam made up of a cam section for monochrome and a cam section for color that are to be turned around an identical cam shaft, both sections being displaced from one another in a direction of the cam shaft and fixed to each other. The respective cam sections for monochrome and for color are in contact with one end of respective link members for monochrome and for color at a portion in each peripheral surface of the respective cam sections.
The peripheral surface of each cam section is made up of a circular arc part (hereinafter referred to as a pressed-contact generating face) at which a distance (radius) from a center of rotation of the cam becomes greatest, and a part at which the radius becomes shorter (hereinafter referred to as a release generating face) than the former. While the link member is in contact with the pressed-contact generating face of the cam section, the link member moves along its longitudinal direction in a direction to go farther from the cam shaft, and then the primary transfer roller is displaced in a direction to approach the image bearing member, thereby causing the intermediate transfer belt to be pressed against the image bearing member. On the other hand, while the link member is in contact with the release generating face of the cam section, the link member moves along its longitudinal direction in a direction to come closer to the cam shaft, and then the primary transfer roller is displaced in a direction to separate from the image bearing member, thereby causing the intermediate transfer belt to be released from the image bearing member. In this manner, the intermediate transfer belt is caused to be pressed against and be released from the image bearing member for monochrome by the link member for monochrome moving along its longitudinal direction; and the intermediate transfer belt is caused to be pressed against and be released from the image bearing members for color by the link member for color moving along its longitudinal direction.
In the monochromatic transfer mode, the link member for monochrome is in contact with the pressed-contact generating face of the cam section for monochrome while the link member for color is kept in a state of being in contact with the release generating face of the cam section for color, thereby causing the primary transfer roller for monochrome to be displaced in a direction to approach the image bearing member for monochrome. In the full-color transfer mode, the link members for monochrome and for color are both kept in a state of being in contact with corresponding pressed-contact generating faces of the cam sections, thereby causing all the primary transfer rollers for monochrome and for color to be displaced in directions for the pressed contact. In the standby mode, the link members for monochrome and for color are both kept in a state of being in contact with corresponding release generating faces of the cam sections, thereby causing all the primary transfer rollers for monochrome and for color to be displaced in directions for the separation.
In each of the modes, the driving source to turn the cam is stopped. Therefore, there is a risk that sliding occurs between the peripheral surface of the cam section and the link member, and that thereby the cam unintentionally turns and then the link member comes off the pressed-contact generating face to the release generating face of the cam section. Due to the occurrence of such coming off of the link member, the amount of movement of the link member, namely the amount of displacement of the primary transfer roller, becomes unstable; therefore, a state of the pressed contact of the intermediate transfer belt with the image bearing member varies, from which a problem arises that a harmful effect is exerted on the transfer of the toner image.

Transfer device of image forming device including monochromatic and full color transfer modes


Provided herein are small diameter inlays for correcting vision impairments by altering the shape of the anterior corneal surface. In an embodiment, inlays having diameters smaller than the pupil are provided for correcting presbyopia. To provide near vision, an inlay is implanted centrally in the cornea to induce an “effective” zone on the anterior corneal surface, within which diopter power is increased. Distance vision is provided by a region of the cornea peripheral to the “effect” zone. In another embodiment, small diameter inlays are provided that induce effective optical zones on the anterior corneal surface that are much larger in diameter than the inlays. The increase in the effective optical zone, due at least in part to a draping effect, allows an inlay to produce a much larger clinical effect on a patient's vision than the diameter of the inlay.

As is well known, abnormalities in the human eye can lead to vision impairment. Some typical abnormalities include variations in the shape of the eye, which can lead to myopia (near-sightedness), hyperopia (far-sightedness) and astigmatism as well as variations in the tissue present throughout the eye, such as a reduction in the elasticity of the lens, which can lead to presbyopia. A variety of technologies have been developed to try and address these abnormalities, including corneal implants.
Corneal implants can correct vision impairment by altering the shape of the cornea. Corneal implants can be classified as an onlay or an inlay. An onlay is an implant that is placed over the cornea such that the outer layer of the cornea, e.g., the epithelium, can grow over and encompass the implant. An inlay is an implant that is surgically implanted into the cornea beneath a portion of the corneal tissue by, for example, cutting a flap in the cornea and inserting the inlay beneath the flap. Both inlays and outlays can alter the refractive power of the cornea by changing the shape of the anterior cornea, by having a different index of refraction than the cornea, or both. Since the cornea is the strongest refracting optical element in the human ocular system, altering the cornea's anterior surface is a particularly useful method for correcting vision impairments caused by refractive errors. Inlays are also useful for correcting other visual impairments including presbyopia.

Method of using small diameter intracorneal inlays to treat visual impairment


A valve case includes an attaching portion and a sleeve. The valve case is attached to a cylinder head cover main body formed of resin. The attaching portion is formed of resin and is a portion of the valve case to which an oil control valve is to be attached. The sleeve is embedded in the attaching portion, and has an interior space that permits the oil control valve to be accommodated therein and oil holes. Each oil hole is selectively connected to one of ports of the oil control valve accommodated in the interior space of the sleeve. The sleeve is formed of material having higher rigidity than the resin forming the attaching portion. Therefore, the valve case prevents deformation of the attaching portion.

The present invention relates to a valve case to which an oil control valve (OCV) is attached that controls supply and drainage of hydraulic pressure to and from a variable valve actuation mechanism of an internal combustion engine, and to a resin cylinder head cover having such a valve case.
In a case where a hydraulically operated variable valve actuation mechanism is provided for a timing sprocket or a timing pulley of an internal combustion engine, hydraulic pressure supplying/draining oil passages from the oil control valve to the variable valve actuation mechanism are normally formed through a camshaft.
For such a case, for example, a configuration has been proposed in which an oil control valve is attached to the inner surface of the cylinder head cover, and the oil control valve controls supply and drainage of the hydraulic pressure through oil passages in a cam cap and the oil passages in the camshaft.
However, according to the configuration in which the oil control valve is accommodated inside the cylinder head cover as described above, the height of the cylinder head cover is increased by a space necessary for accommodating the oil control valve. This undesirably increases the size of the internal combustion engine. Therefore, a technique has been proposed in which an oil control valve is accommodated in a valve case attached to the cylinder head cover to cover an opening formed in the upper wall of the cylinder head cover (for example, see Japanese Patent No. 3525709).
If the cylinder head cover and the valve case configured as described above are formed with resin to achieve the weight reduction, since the rigidity of resin is generally lower than that of metal, the cylinder head cover may be distorted or deformed when attaching the cylinder head cover to a cylinder head. This may, in turn, affect the valve case.
The dimensional accuracy of the attaching portion to which the oil control valve is attached may be decreased due to such distortion or deformation. Thus, the oil control valve may not be properly attached to the attaching portion or the oil control valve may malfunction.

Valve case and resin cylinder head cover


According to one embodiment, the present invention relates to a method and a system for reimbursing credit card transactions for health care services and/or products. An exemplary method and system may involve registering a user for reimbursement wherein the user identifies one or more payment mechanisms and one or more reimbursement methods; identifying a user transaction with the one or more payment mechanisms at a merchant wherein the user transaction comprises one or more purchases; receiving merchant data regarding the user transaction; determining reimbursement eligibility of the one or more purchases of the user transaction based at least in part on the merchant data; and reimbursing the user for eligible purchases via the one or more reimbursement methods.

As health care expenses continue to rise, consumers are taking on a bigger role in paying and managing health care costs. This increase in consumer responsibility ranges from greater cost-sharing for medical services to having to choose among increasing health care financing options. One important tool for consumer participation in health care is the use of consumer-directed accounts. Consumer-directed accounts are dedicated funding pools used for health care expenses at the discretion of an individual. These accounts can be procured either individually or through an employer, and may or may not involve tax advantages to the user of the account. A key requirement in the use of consumer-directed accounts is that they finance only those goods and services that are authorized by the Internal Revenue Service (IRS) as eligible medical expenses. This creates the need for close oversight of consumer-directed account programs, which involves high paper volumes, manual intervention, and substantial administrative expense to ensure compliance. In addition, the current process often requires participants to pay for goods and services and then file for reimbursement out of the funds contributed to the consumer-directed account. This requires participants to make the payment up-front even though the funds may have already been set aside and to handle a variety of paper documents during filing and reimbursement. These challenges significantly undermine the efficiency of consumer-directed account programs, resulting in dissatisfied participants and program sponsors. Given these difficulties, there is a clear need for an electronic solution that automates the administration and use of consumer-directed accounts, while maintaining compliance with the rules and regulations under which consumer-directed accounts are governed.
The trend to consumer-directed health care has been supported by three major legislative innovations within the past thirty years; namely, the Revenue Act of 1978, the Health Insurance Portability and Accountability Act of 1996 (HIPAA) and a ruling in 2002 by the IRS establishing Health Reimbursement Arrangements. Each of these initiatives created the model for a consumer-directed account in use today. The Revenue Act of 1978 created Flexible Spending Accounts (FSA), which are employer-sponsored accounts funded by employees' pre-tax contributions from payroll. Flexible Spending Accounts can be established and used for a variety of purposes, such as the payment of health care, dependent care, or public transportation expenses. Because Flexible Spending Accounts are funded with pre-tax dollars, participants in an FSA program can realize substantial savings on eligible expenses. Employers value FSA programs because these accounts provide benefits to employees and reduce employees' taxable income on which an employer must pay Federal Insurance Contributions Act (FICA) taxes. Currently, there are more than 8 million Health Flexible Spending Accounts in the US, with over 9 billion dollars in annual contributions. Health FSA contributions are expected to grow significantly over the next several years, as consumers take on more of the burden for health care costs and seek tax-advantaged tools to finance those expenses.
Two other types of consumer-directed accounts—the Medical Savings Account (MSA) and the Health Reimbursement Arrangement (HRA)—were introduced within the past decade. The MSA was established with the passage of HIPAA in 1996. The MSA provides for a tax-advantaged account reserved for individuals who are self-employed or employed at a small business, and who use a qualifying high-deductible health plan. The MSA is designed to provide tax benefits on payments towards a deductible portion of a high-deductible health plan. A key advantage of the MSA to participants is the ability to carry over unused funds in the account from one plan year to the next. This differs from FSA programs that require unused funds to be forfeited by the employee and turned over to the employer health plan, a requirement commonly known as the FSA "use it or lose it" provision. The IRS established the HRA in June 2002, drawing on the strengths of both the FSA and the MSA. For example, employers of all sizes may implement an HRA program (e.g., FSA). In addition, unused funds contributed to an HRA may roll over from one plan year to the next, like MSA. Due to its flexibility and attractiveness to employees, the HRA is expected to quickly become prevalent throughout the employee benefits marketplace.
A result of these initiatives is the proliferation of consumer-directed accounts used to provide tax advantages for health care spending. Although FSA, MSA, and HRA are the most common forms of consumer-directed accounts, others may also exist. As consumer-directed health care evolves, there will be continued innovation in the field of consumer-directed accounts. This will likely result in new account structures being introduced over time.
Because consumer-directed accounts are legal structures, there are a wide variety of associated rules and regulations. Some important rules include provisions on the types of expenses that can be financed through a consumer-directed account, as determined by the IRS. In general, consumer-directed accounts can only pay for expenses that are medically necessary (such as prescription eye care, prescription drugs, hearing devices, or any other medically necessary expenses), whereas expenses that are not medically necessary (typically "cosmetic" goods and services such as non-prescription sunglasses, cosmetic surgery, or any other non-medically necessary expenses) are deemed ineligible. Because consumer-directed accounts confer tax advantages to participants, the IRS requires strict compliance from employers and their agents to ensure that only eligible medical expenses are financed through a consumer-directed account. If ineligible medical expenses are paid for through a consumer-directed account, tax advantages are being made available inappropriately. As a result, the IRS can assign and enforce penalties for non-compliance.
Most consumer-directed accounts offered through an employer benefits plan are managed and administered by an entity other than the employer itself, usually a Third Party Administrator (TPA). A TPA can provide a variety of services to employers, such as managing claim submissions from employees and generating reports on plan usage. The requirements to comply with IRS guidelines for expense eligibility create a heavy burden on the TPA in managing a consumer-directed account program. Whenever an employee files for reimbursement out of a consumer-directed account, the TPA must audit and validate the claim to ensure that it is for a valid medical expense. Typically, the employee will provide proof of eligibility in the form of a paper receipt or invoice documenting the nature of the expense, which the TPA reviews to confirm compliance. This validation process is extremely labor-intensive and generally requires TPA personnel to review a given claim manually. Further complicating the TPA's task of ensuring compliance is the ability of employers to customize expense eligibility rules to their particular program. That is, although the IRS governs what is potentially allowed, employers have the option to limit further the specific types of expenses that their particular consumer-directed account program will authorize. A TPA must therefore consult the plan rules for a given program before granting approval to an employee's claim. After the claim has been deemed eligible and sufficient funds have been confirmed within the employee's consumer-directed account, the TPA will remit funds, usually by paper check, with a paper explanation of payment to the employee.
Because of the challenges associated with the current manual processing and administration of consumer-directed accounts, industry participants have attempted to develop a variety of mechanisms to streamline activities and improve automation. As consumer-directed accounts grow in importance and usage, these efforts will become increasingly relevant to the ways in which health care expenses are paid and managed.
Efforts to innovate within the administration and use of consumer-directed accounts have historically proceeded in two key ways. First, many Third Party Administrators have attempted to manage the existing paper process better without actually reducing the submissions of paper claim forms. An example of this type of innovation is the use of scanning and character-recognition technology to store an electronic copy of paper claims as they are received and to adjudicate claims based on the information contained in the electronic image. Using this type of technology reduces the amount of paper that a TPA has to keep on file and enables better management of claim documents than if such documents were to remain in paper form. Although this approach may realize certain cost savings, the key problems associated with the current process for managing consumer-directed accounts still remain. That is, participants still have to prepare and file paper claims and front payment for goods and services, and Third Party Administrators must still handle paper documents and manually review the expense eligibility of submitted claims.
The second way industry participants have sought to improve the administration and use of consumer-directed accounts is to attempt to reduce the number of paper claim forms submitted by participants. Manual mechanisms for obviating claim form submission have existed for several years. For example, in some cases, a TPA may be able to make a direct payment from a consumer-directed account to a provider of eligible goods or services without the participant having to file a claim form or initiate a payment. This method is usually applied for payments that are recurring in nature (such as elder care or regular prescription drug purchases), because it requires notifying the provider to change their process and forward an invoice for payment to the TPA directly rather than to the employee. In addition, such payments are only possible when payment is not required at the time services are rendered. For prescription drug transactions within a pharmacy, for example, payment is expected at the point of sale, precluding a post-hoc payment from the TPA to the provider. Moreover, although the participant does not have to file a paper claim form, the TPA must still employ a manual process for adjudicating and paying claims.
Another manual mechanism for reducing the number of paper claim submissions by savings account participants is to automatically apply consumer-directed account funds towards deductibles or co-payments determined under an employer-sponsored health plan. This option has historically been possible only when the TPA manages claims for both the consumer-directed account and the health plan for a given employer. In this scenario, an employee obtains goods or services from a medical provider, causing the provider to generate and submit a claim to the employee's health plan. Once the TPA has adjudicated the claim and determined the employee's payment responsibility, the TPA remits funds from the consumer-directed account directly to the provider, eliminating the need for the employee to pay the provider up-front and then prepare and file a claim with the TPA for reimbursement from the consumer-directed account. However, instances when an individual TPA manages both the consumer-directed accounts and the health plan for a given employer are limited, making this option available to relatively few consumer-directed account participants.
An alternative mechanism for reducing paper claims submissions and improving efficiencies involves enabling electronic claim submission and payment through the use of a stored value program. A stored value program typically includes supplying each participant with a payment card that is linked to a consumer-directed account. A participant then presents the physical card or the card number for payment wherever medical expenses are incurred. The health care provider initiates a payment transaction, that transfers funds from the consumer-directed account to the provider's bank account, eliminating the need for the participant to provide up-front payment and then file for later reimbursement through the TPA. Likewise, the TPA benefits by not having to manage and process paper forms and claims associated with the card-initiated transaction. Because of the wide acceptance of payment cards, participants are able to use consumer-directed account funds in a variety of venues, such as in a retail location, through the mail, or over the phone and other types of transactions.
In stored value arrangements, payments are made directly from the FSA without being substantiated first as eligible expenses. This forces the TPA to solicit receipts from the employee and make restitution to the FSA for non-compliant claims. Often, the employee is lax with submission and sometimes may fail to submit receipts at all.
Although stored value programs have the potential to create substantial efficiencies and benefits, they also have the potential for substantial abuse. In particular, current programs risk exposing employers and Third Party Administrators to non-compliance with IRS guidelines for consumer-directed account expense eligibility. Unlike the current manual process that allows a TPA to audit detailed transaction information in the form of a submitted receipt, card transactions generally communicate only the payment amount requested and the type of merchant that is initiating the transaction (e.g., a pharmacy, a toy store, etc.). Many stored value programs thus authorize the use of card payments to merchants that are considered "health care providers" (such as doctors, optometrists, pharmacies, etc.) and deny card transactions at non-health care merchants (such as a toy store or apparel retailer). This type of filtering is inadequate because many putative health care merchants may still sell goods or services that do not meet the IRS guidelines for payment from a consumer-directed account. For example, a consumer-directed account participant receiving services from a physician's office may be receiving cosmetic enhancements that are not medically necessary. Alternatively, a participant may present their card at a pharmacy for payment toward a wide variety of non-eligible goods (e.g., magazines, office supplies, etc.). Unless a stored value program analyzes detailed, individual transaction data to determine expense eligibility, ineligible expenses may be inappropriately authorized and funded by the consumer-directed account.
Because of the risk of non-compliance associated with card programs, many Third Party Administrators review card transactions post-payment and audit them for inappropriate activity. According to IRS guidelines, claims do not need to be confirmed as eligible expenses prior to payment as long as 100% of all claims are reviewed post-payment. In cases where expenses paid by a consumer-directed account are later deemed ineligible, participants must be made to restore misappropriated funds to the savings account, forcing the TPA into the costly and awkward role of collections officer. Due to the risk of fraudulent payment, a TPA may even require that a participant substantiate every card transaction with paper documentation, which then fails to relieve employees and Third-Party Administrators of the traditional challenges associated with participating in and managing a consumer-directed account. Frequent TPA audits and collection activities can significantly undermine the efficiencies that stored value programs were designed to achieve.
Because the IRS has the ability to levy substantial fines for non-compliance, many employers are unwilling to accept the risks associated with current stored value programs, opting instead for the less efficient but potentially more compliant traditional, manual process.

Method and system for credit card reimbursements for health care transactions


Methods are provided for treating liquid hazardous waste containing anionic radioactive or heavy metal materials by binding the hazardous waste to hydroxyapatite powder, drying and then cold or hot pressing the hydroxyapatite powder into a solid mass for storage or disposal. The methods are useful for treatment and storage of radioactive waste, anions, and heavy metals. Methods are also provided for treating high concentration liquid hazardous waste and liquid hazardous waste which does not contain materials known to decompose at high temperatures.

As a result of research and production of nuclear weapons, large amounts of radioactive and heavy metal waste have been generated. This waste, termed "mixed waste" when it contains both radioactivity and other hazardous waste products, can have a tremendous impact on the environment and pose serious health risks to the general population. Hazardous waste products are also generated in hospitals, industries and other commercial facilities through a number of processes such as medical diagnostic testing, pharmaceutical and biotechnology research and pesticide research. In 1990, the United States produced over 4000 m.sup.3 of low level mixed waste including 2,840 m.sup.3 of liquid waste, 720 m.sup.3 of organic solvents such as chlorofluorocarbons (CFC's), corrosive organics and waste oil and 120 m.sup.3 of toxic metals. The Department of Energy (DOE) estimates that nuclear sites in 22 states will produce over 226,000 m.sup.3 of nuclear waste over the next two decades and that it will cost over $60 billion to treat or store this waste over a 75 year period. Thus, treatment and disposal of these wastes poses tremendous technical and environmental problems. To further complicate the situation, there is currently no available treatment method for technetium (Tc), a major waste product of nuclear facilities in the form of TcO.sub.4.sup.-.
The DOE reports that 399,000 m.sup.3 of high level radioactive waste are currently stored in large tanks at four locations: Hanford, Washington; Idaho National Engineering Laboratories (INEL), Idaho; Savannah River Site (SRS), South Carolina; and the West Valley Demonstration Project, New York. DOE is proceeding with plans to treat high level waste by processing it into a solid form (e.g. borosilicate glass) that would not be readily dispersable into the air or leachate into the ground or surface water. This treatment process is called vitrification. The vitrification process will generate approximately 29,000 canisters to be disposed of in a geologic repository.
Hazardous waste can easily contaminate its environment. For instance, over the past decade, several incidents have occurred in which radioactive material used in industrial devices has been mixed with scrap metal that was being recycled for steel production. This radioactive material (usually cesium) cannot always be detected because it is shielded by the container or scrap metal. Consequently, the radioactive dust can contaminate the steel facility's emission control system and the emissions dust. Steel producers in the U.S. are currently storing more than 10,000 tons of contaminated dust. In most cases, this waste contains both radioactivity and other hazardous materials such as lead, cadmium and chromium.
Disposal options for these materials have been limited, principally because of their mixed waste classification and the cost associated with disposal of large volumes of mixed or radioactive waste. Thus, because of high cesium concentrations, hazardous waste facilities may not provide suitable treatment protocols. Similarly, licensed low-level radioactive waste disposal facilities can only dispose of the cesium after the other hazardous waste has been removed. One procedure involves immobilizing radionuclides on solid matrixes and building barriers around the matrixes to minimize the spread or immobilization. However, hazardous waste can leach out of the barriers and permeate the surrounding environment.
Jeanjean et al. (1995) J. Radioanal. Nucl. Chem. 201(6)529-539 describe how the cations uranium, cadmium and lead can be sorbed to hydroxyapatite (HA). These authors postulate that these positively-charged elements are immobilized by occupying empty Ca2.sup.+ sites on the HA.
Thus, there remains a need for a safe and inexpensive method of treating hazardous waste, including materials such as Technetium, mixed waste and heavy metals.

Methods of treating nuclear hydroxyapatite materials


An brain scanning apparatus having a scanner device and a host computer. The scanner radiation detectors detect radiation emitted from a desired portion, or slice, of a brain source. The scanner device contains microprocessors and code which control the movement of the radiation detectors and performs the data acquisition over a number of slices and transmits this data to the host computer. The host computer initiates a scan by sending desired setup parameters to the scanner device and instructing the scanner to begin collecting data. During the scan, acquired data is sent to the host computer and spooled to a hard disk. The computer can be instructed to perform a slice by slice reconstruction of the source brain while the scan is taking place in order to produce a 2-dimensional reconstruction of the mapped brain. A complete 3-dimensional reconstruction of all the compiled slices is performed and visually displayed after acquisition of all the required slices.

In the last quarter century the use of brain imaging for the treatment and understanding of diseases and genetic flaws has grown dramatically following the introduction of Tomographic X-Ray (CT) in 1972 followed in 1982 by magnetic resonance in the gene (MRI). The reason for this growth and importance in brain imaging is that neurologists, psychotherapist, and neuro-scientists utilize and attached substantial importance to high resolution, three dimensional anatomical images of the brain. The development of functional brain imaging which seeks to map the distribution of brain activity has closely followed the development of structural imaging which maps some physical property of the brain such as tissue density. While SPECT is playing an important role in functional brain imaging, it has been limited in many applications by its low spatial resolution. The tiny structures of the brain where thinking takes place are much smaller than the resolution of the best SPECT scanners and therefore are not seen. Only in the situations where gross functional changes or small changes over a large population of subjects have occurred is SPECT useful.
U.S. Pat. No. 4,209,700 to Stoddart discloses a first generation nuclear transverse sectional brain function imager. Stoddart discloses an imaging apparatus having a transverse radio nuclide scanfield and a method for using highly focused collimators in an array surrounding the scanfield. This allows the scanner to concentrate its information gathering capability on a single cross-section of the head as opposed to the rotating gamma camera whose sensitivity is distributed over the entire volume of the head. In the situations where only part of the brain is of interest, this is a huge advantage, especially for dynamic studies where one needs to make rapid repetitive scans of the same area. The scanner is not limited to single sections. By moving the patient through the scanner, a stack of sections may be obtained which cover the entire volume of the head.
In general, the typical clinical resolution of the best SPECT rotating gamma-cameras is about 7 mm. This is inferior to both PET and fMRI which provide 5 mm and 3 mm resolution, respectively. The two avenues of improvement used to bring rotating gamma-cameras to their state-of-the-art are: 1) increasing the number of camera heads (now 3) and 2) modifying the original parallel hole collimator design to the higher performance mildly converging tapered hole designs' while increasing camera area in order to maintain a sufficiently large field-of-view (FOV). Further improvement is difficult since the cameras of 3-headed systems now totally encircle the patient with little room left for more or larger versions.
Collimators are simply blocks of lead with holes drilled through them (or cast with holes in them) to allow gamma rays to pass through which are traveling in a specific direction. The longer or narrower the holes, the more precise that direction becomes. This is good for geometrical resolution but bad for sensitivity and one needs both. Tapered holes are vastly superior than straight holes in that they provide both better geometrical resolution and sensitivity at the same time. While rotation gamma-cameras benefitted from mildly tapered holes, they cannot take advantage of highly tapered holes since the resulting FOV would not cover the entire head. The present scanning system overcomes this problem by sweeping the narrow FOV.

High resolution photon emission computed tomographic imaging tool


An image forming apparatus comprises a developer including a latent image carrying member, a toner carrying member and a toner container for developing an electrostatic latent image formed on the latent image carrying member, and a transfer member for transferring to a transfer medium a toner image formed on the latent image carrying member. The latent image carrying member and the toner carrying member are disposed within a given gap. The toner container holds a magnetic toner and the magnetic toner is fed onto the toner carrying member. The magnetic toner comprises a binder resin and a magnetic power and has a volume average particle diameter of from 7 .mu.m to 10 .mu.m, and the number distribution and quantity of triboelectricity of magnetic toner particles satisfy the following expressions: EQU 0.1.times.A+2.ltoreq.-Q.ltoreq.0.1.times.A+16. where A represents a real number from 25 to 45 calculated as a coefficient of variation of number distribution, (S/D.sub.1)x 100, wherein S represents a standard deviation of the number distribution of magnetic toner particles and D.sub.1 represents a number average particle diameter (.mu.m), and where Q represents a value of the quantity of triboelectricity (.mu.c/g) of the magnetic toner produced by friction with an iron powder.

1. Field of the Invention
The present invention relates to an image forming method, and an image forming apparatus, in which an electrostatic latent image formed in an image forming process such as electrophotography, electrostatic printing and electrostatic recording is developed with a magnetic toner.
2. Related Background Art
As a developing method making use of a one-component magnetic toner, the developing method using a conductive magnetic toner as disclosed in U.S. Pat. No. 3,909,258, etc. is known in the art.
In such a developing method, however, toners are required to be substantially conductive, and, in the case of conductive toners, it has been difficult to transfer a toner image formed on a latent image carrying member to a final image supporting member, e.g., plain paper, by utilizing an electric field.
A proposal has been made on a novel developing method that can solve problems involved in developing methods making use of conventional one-component magnetic toners (for example, Japanese Patent Applications Laid-open No. 55-18656 and No. 55-18659). According to this method, an insulating magnetic toner is uniformly applied to a cylindrical toner carrying member having a magnet in its inner part, and the toner carrying member is opposed to a latent image carrying member without being in contact therewith, in the state of which development is carried out. As a method of forming a magnetic toner layer on the toner carrying member, there is a method in which a coating blade is used, provided at an outlet of a toner container. For example, the image forming apparatus illustrated in FIG. 1 comprises a toner carrying member 2, and a blade 1a comprised of a magnetic material, provided at the position opposed to a magnetic pole N1 of a fixed magnet built in the toner carrying member 2. A magnetic toner is caused to rise along the magnetic line of force between the magnetic pole and the magnetic material blade, and the toner having risen is cut with an edge at the tip of the blade so that the thickness of the magnetic toner layer can be controlled by utilizing the action of the magnetic force (see, for example, Japanese Patent Application Laid-open No. 54-43037).
At the time of development, a low-frequency alternating voltage is applied between the toner carrying member and a conducting base body of the latent image carrying member so that the magnetic toner may reciprocate between the toner carrying member and the latent image carrying member. Desired good development can be thus carried out. In this developing method, the magnetic toner has insulation properties and hence can be readily electrostatically transferred.
The image forming apparatus illustrated in FIG. 2 is equipped with a developing device 7 holding therein a toner 10, and a latent image carrying member 9 such as a photosensitive drum used in electrophotography or an insulating drum used in electrostatic recording (hereinafter "photosensitive member" or "photosensitive drum").
In the developing method making use of such apparatus, very important are the subject (A): to uniformly apply the magnetic toner to the toner carrying member and the subject (B): to prevent or decrease contamination on the surface of the toner carrying member, caused by some constituents in the magnetic toner. The subject (A) and the subject (B), however, have the relation that they conflict with each other, and it is difficult to settle both of them at the same time.
In the subject (A), as a method of uniformly applying the magnetic toner to the toner carrying member, a proposal is made on a developing device capable of forming a toner coat layer on a toner carrying member in a stable state for a period long enough from a practical viewpoint (Japanese Patent Application Laid-open No. 57-66455). This developing device is a superior developing device, which is comprised of a toner carrying member made to have a rough surface with a specific irregularity by sand-blasting using amorphous particles and thus can maintain on the surface of the toner carrying member the state of a toner coat that is uniform, free from uneveness and always good for a long period of time. As shown in FIG. 10, a cylindrical toner carrying member has an embodiment in which its surface is provided over the entire surface with numberless cuts or protuberances formed in random directions.
In the developing device comprised of a toner carrying member having such a specific surface state, some of magnetic toners to be applied tend to cause adhesion of a toner or constituents in a toner to the surface of the toner carrying member. Hence the surface of the toner carrying member may be contaminated, consequently tending to cause a lowering of image density at the initial stage and, when it has become more contaminated as a result of copying on a large number of sheets, cause image blank areas to occur in every period of rotation of the toner carrying member. This is due to the fact that the constituents in a toner adhere to the slopes of projections and the hollows or concavities, of the surface of the toner carrying member to cause poor static charge of magnetic toner particles, resulting in a lowering of the quantity of static charge in a magnetic toner layer.
In general, the constituents of a magnetic toner are formed of materials such as a binder resin, a magnetic material, a charge control agent and a release agent. Materials are selected so that the contamination on the surface of the toner carrying member can be prevented. Hence, under present circumstances, the materials are limited.
In the subject (B), as a method of preventing or decreasing contamination on the surface of the toner carrying member, it has been clear that it is advantageous to make smoother the surface of a toner carrying member. Such a method, however, is experimentally found to tend to bring about a non-uniform toner coat when a magnetic toner has a volume average particle diameter of 12 .mu.m or more, to cause uneveness in toner images, often resulting in no formation of good toner image. The phenomenon in which this non-uniform toner coat is formed was observed in detail by carrying out running of a developing device without image-copying to reveal the following.
At the initial stage of the running without image-copying, the toner coat layer becomes excessively thick when the surface of the toner carrying member is smooth, although its cause is unclear. When the toner thickness is controlled using the blade 1a, the toner gradually comes to bulge out on the photosensitive member 9 side of the blade 1a (the part A in FIG. 2). As shown in FIG. 3 as a partially enlarge cross section, the toner forms a heap 10a at the part A. When this toner heap reaches a certain limit quantity, it moves to the surface of a sleeve 2 on the outside of the toner container because of the transporting force of the sleeve 2 to give coat uneveness 3a. When the coat uneveness 3a (a toner uneveness 3a. When the coat uneveness 38 (a toner mass) is present on the toner layer 3 formed in a uniform coating, this uneveness comes out as an uneveness on a toner image. The latter uneveness corresponds to density uneveness and fog. The toner coat uneveness 3a is found to have the shape of a rectangular spot, a wavelike spot, a wavelike pattern, or the like.
As discussed above, it has been very difficult for the conventional developing method to settle both the subject (A) and the subject (B) at the same time. This tendency becomes more remarkable under conditions of a low humidity or in a developing device designed to have a toner carrying member that rotates at a higher peripheral speed.
With an intent to improve image quality, European Patent Application Publication No. 0314459 proposes a magnetic toner having a volume average particle diameter of from 4 .mu.m to 9 .mu.m and also having a specific particle size distribution, and an image forming apparatus making use of such a magnetic toner.
European Patent Application Publication No. 0331425 also proposes an image forming method in which a magnetic toner having a volume average particle diameter of from 4 .mu.m to 9 .mu.m and also having a specific particle size distribution is fed to a toner carrying member having the surface with an uneveness comprising sphere-traced concavities and thus an electrostatic latent image is developed.
It, however, has been sought to provide a more improved image forming method or apparatus that has been adapted to the developing speed made increasingly higher.


A meat product is prepared by mixing frozen particles of salami in a brine, a pickle, or a marinade medium to obtain a suspension of the frozen particles in the medium and then the suspension is incorporated into a chilled portioned piece of meat to obtain a meat product containing the suspension.

The present invention relates to incorporation of substances into meat.
Meat trimmings are obtained by removal from the meat during the standard preparation of whole cuts of meat in the meat industry. The trimmings are usually, but not always, of low quality and usually contain some fat and some muscle tissue. It is possible, by using technology introduced onto the market in recent years, to incorporate suspensions made of meat trimmings into whole cuts of like meat to increase the weight using a multi-needle injector. By controlling parameters such as the amount of trimmings injected, the meat/fat ratio and the quality of the meat, this technology enables the production of cooked ham or marinated meat products without affecting the standard quality with regard to flavour or shelf-life and without visibility of the suspension, and a way to improve binding and yield is described in U.S. Pat. No. 4,960,599. The cost saving of injecting trimmings is considerable when the trimmings are of low value compared to whole cuts of meat.
In order to impart a specificity to the flavour and to improve microbiological stability, it has been proposed to ferment raw meat by using a starter culture in a brine or marinade prior to cooking to produce bacteriocins. However, since the raw meat can under no circumstances be allowed to ferment at a temperature higher than about +8.degree. C. prior to cooking, the biggest problem is to find a starter culture that can produce bacteriocins and a specific flavour at low temperature. We have tested some commercially available cultures, but the effect on the final quality of the product regarding flavour and microbiological stability is minimal.
In addition, the production time before the cooking step must be prolonged considerably.

Preparation of portioned meat pieces having salami incorporated therein


The present application relates to psilocin derivatives of Formula (I), to processes for their preparation, to compositions comprising them and to their use in activation of a serotonin receptor in a cell, as well as to treating diseases, disorders or conditions by activation of a serotonin receptor in a cell.

Mental health disorders, or mental illness, refer to a wide range of disorders that include, but are not limited to, depressive disorders, anxiety and panic disorders, schizophrenia, eating disorders, substance misuse disorders, post-traumatic stress disorder, attention deficit/hyperactivity disorder and obsessive compulsive disorder. The severity of symptoms varies such that some individuals experience debilitating disease that precludes normal social function, while others suffer with intermittent repeated episodes across their lifespan. Although the presentation and diagnostic criteria among mental illness conditions are distinct in part, there are common endophenotypes of note across the diseases, and often comorbidities exist. Specifically, there exist phenotypic endophenotypes associated with alterations in mood, cognition and behavior. Interestingly, many of these endophenotypes extend to neurological conditions as well. For example, attentional deficits are reported in patients with attention deficit disorder, attention deficit hyperactivity disorder, eating disorders, substance use disorders, schizophrenia, depression, obsessive compulsive disorder, traumatic brain injury, Fragile X, Alzheimer's disease, Parkinson's disease and frontotemporal dementia.
Many mental health disorders, as well as neurological disorders, are impacted by alterations, dysfunction, degeneration, and/or damage to the brain's serotonergic system, which may explain, in part, common endophenotypes and comorbidities among neuropsychiatric and neurological diseases. Many therapeutic agents that modulate serotonergic function are commercially available, including serotonin reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, monoamine oxidase inhibitors, and, while primarily developed for depressive disorders, many of these therapeutics are used across multiple medical indications including, but not limited to, depression in Alzheimer's disease and other neurodegenerative disease, chronic pain, existential pain, bipolar disorder, obsessive compulsive disorder, anxiety disorders and smoking cessation. However, in many cases, the marketed drugs show limited benefit compared to placebo, can take six weeks to work and for some patients, and are associated with several side effects including trouble sleeping, drowsiness, fatigue, weakness, changes in blood pressure, memory problems, digestive problems, weight gain and sexual problems.
The field of psychedelic neuroscience has witnessed a recent renaissance following decades of restricted research due to their legal status. Psychedelics are one of the oldest classes of psychopharmacological agents known to man and cannot be fully understood without reference to various fields of research, including anthropology, ethnopharmacology, psychiatry, psychology, sociology, and others. Psychedelics (serotonergic hallucinogens) are powerful psychoactive substances that alter perception and mood and affect numerous cognitive processes. They are generally considered physiologically safe and do not lead to dependence or addiction. Their origin predates written history, and they were employed by early cultures in many sociocultural and ritual contexts. After the virtually contemporaneous discovery of (5R,8R)-(+)-lysergic acid-N,N-diethylamide (LSD) and the identification of serotonin in the brain, early research focused intensively on the possibility that LSD and other psychedelics had a serotonergic basis for their action. Today there is a consensus that psychedelics are agonists or partial agonists at brain serotonin 5-hydroxytryptamine 2A (5-HT2A) receptors, with particular importance on those expressed on apical dendrites of neocortical pyramidal cells in layer V, but also may bind with lower affinity to other receptors such as the sigma-1 receptor. Several useful rodent models have been developed over the years to help unravel the neurochemical correlates of serotonin 5-HT2A receptor activation in the brain, and a variety of imaging techniques have been employed to identify key brain areas that are directly affected by psychedelics.
Psychedelics have both rapid onset and persisting effects long after their acute effects, which includes changes in mood and brain function. Long lasting effects may result from their unique receptor affinities, which affect neurotransmission via neuromodulatory systems that serve to modulate brain activity, i.e., neuroplasticity, and promote cell survival, are neuroprotective, and modulate brain neuroimmune systems. The mechanisms which lead to these long-term neuromodulatory changes are linked to epigenetic modifications, gene expression changes and modulation of pre- and post-synaptic receptor densities. These, previously under-researched, psychedelic drugs may potentially provide the next-generation of neurotherapeutics, where treatment resistant psychiatric and neurological diseases, e.g., depression, post-traumatic stress disorder, dementia and addiction, may become treatable with attenuated pharmacological risk profiles.
Although there is a general perception that psychedelic drugs are dangerous, from a physiologic safety standpoint, they are one of the safest known classes of CNS drugs. They do not cause addiction, and no overdose deaths have occurred after ingestion of typical doses of classical psychotic agents, such as LSD, psilocybin, or mescaline (Scheme 1). Preliminary data show that psychedelic administration in humans results in a unique profile of effects and potential adverse reactions that need to be appropriately addressed to maximize safety. The primary safety concerns are largely psychologic, rather than physiologic, in nature. Somatic effects vary but are relatively insignificant, even at doses that elicit powerful psychologic effects. Psilocybin, when administered in a controlled setting, has frequently been reported to cause transient, delayed headache, with incidence, duration, and severity increased in a dose-related manner [Johnson et al., Drug Alcohol Depend (2012) 123(1-3):132-140]. It has been found that repeated administration of psychedelics leads to a very rapid development of tolerance known as tachyphylaxis, a phenomenon believed to be mediated, in part, by 5-HT2A receptors. In fact, several studies have shown that rapid tolerance to psychedelics correlates with downregulation of 5-HT2A receptors. For example, daily LSD administration selectively decreased 5-HT2 receptor density in the rat brain [Buckholtz et al., Eur. J. Pharmacol. 1990, 109:421-425. 1985; Buckholtz et al., Life Sci. 1985, 42:2439-2445].
Scheme 1: Chemical Structures of or Mescaline (i), LSD (ii), Psilocybin (iii) and Psilocin (iv)
Classic psychedelics and dissociative psychedelics are known to have rapid onset antidepressant and anti-addictive effects, unlike any currently available treatment. Randomized clinical control studies have confirmed antidepressant and anxiolytic effects of classic psychedelics in humans. Ketamine also has well established antidepressant and anti-addictive effects in humans mainly through its action as an NMDA antagonist. Ibogaine has demonstrated potent anti-addictive potential in pre-clinical studies and is in the early stages of clinical trials to determine efficacy in robust human studies [Barsuglia et al., Prog Brain Res, 2018, 242:121-158; Corkery, Prog Brain Res, 2018, 242:217-257].
Psilocybin (4-phosphoryloxy-N,N-dimethyltrypatmine (iii, Scheme 1) has the chemical formula C12H17N2O4P. It is a tryptamine and is one of the major psychoactive constituents in mushrooms of the psilocybe species. It was first isolated from psilocybe mushrooms by Hofmann in 1957, and later synthesized by him in 1958 [Passie et al. Addict Biol., 2002, 7(4):357-364], and was used in psychiatric and psychological research and in psychotherapy during the early to mid-1960s up until its controlled drug scheduling in 1970 in the US, and up until the 1980s in Germany [Passie 2005; Passie et al. Addict Biol., 2002, 7(4):357-364]. Research into the effects of psilocybin resumed in the mid-1990s, and it is currently the preferred compound for use in studies of the effects of serotonergic hallucinogens [Carter et al. J. Cogn. Neurosci., 2005 17(10):1497-1508; Gouzoulis-Mayfrank et al. Neuropsychopharmacology 1999, 20(6):565-581; Hasler et al, Psychopharmacology (Berl) 2004, 172(2):145-156], likely because it has a shorter duration of action and suffers from less notoriety than LSD. Like other members of this class, psilocybin induces sometimes profound changes in perception, cognition and emotion, including emotional lability.
In humans as well as other mammals, psilocybin is transformed into the active metabolite psilocin, or 4-hydroxy-N,N-dimethyltryptamine (iv, Scheme 1). It is likely that psilocin partially or wholly produces most of the subjective and physiological effects of psilocybin in humans and non-human animals. Recently, human psilocybin research confirms the 5HT2A activity of psilocybin and psilocin, and provides some support for indirect effects on dopamine through 5HT2A activity and possible activity at other serotonin receptors. In fact, the most consistent finding for involvement of other receptors in the actions of psychedelics is the 5-HT1A receptor. That is particularly true for tryptamines and LSD, which generally have significant affinity and functional potency at this receptor. It is known that 5-HT1A receptors are colocalized with 5-HT2A receptors on cortical pyramidal cells [Martin-Ruiz et al. J Neurosci. 2001, 21(24):9856-986], where the two receptor types have opposing functional effects [Araneda et al. Neuroscience 1991, 40(2):399-412].
Although the exact role of the 5-HT2A receptor, and other 5-HT2 receptor family members, is not well understood with respect to the amygdala, it is evident that the 5-HT2A receptor plays an important role in emotional responses and is an important target to be considered in the actions of 5-HT2A agonist psychedelics. In fact, a majority of known 5HT2A agonists produce hallucinogenic effects in humans, and rodents generalize from one 5HT2A agonist to others, as between psilocybin and LSD [Aghajanian et al., Eur J Pharmacol., 1999, 367(2-3):197-206; Nichols at al., J Neurochem., 2004, 90(3):576-584]. Psilocybin has a stronger affinity for the human 5HT2A receptor than for the rat receptor and it has a lower K(i) for both 5HT2A and 5HT2C receptors than LSD. Moreover, results from a series of drug-discrimination studies in rats found that 5HT2A antagonists, and not 5HT1A antagonists, prevented rats from recognizing psilocybin [Winter et al., Pharmacol Biochem Behav., 2007, 87(4):472-480]. Daily doses of LSD and psilocybin reduce 5HT2 receptor density in rat brain.
Clinical studies in the 1960s and 1970s showed that psilocybin produces an altered state of consciousness with subjective symptoms such as "marked alterations in perception, mood, and thought, changes in experience of time, space, and self." Psilocybin was used in experimental research for the understanding of etiopathogenesis of selective mental disorders and showed psychotherapeutic potential [Rucker et al., Psychopharmacol., 2016, 30(12):1220-1229]. Psilocybin became increasingly popular as a hallucinogenic recreational drug and was eventually classed as a Schedule I controlled drug in 1970. Fear of psychedelic abuse led to a significant reduction in research being done in this area until the 1990s when human research of psilocybin was revived when conditions for safe administration were established [Johnson et al., Psychopharmacol., 2008, 22(6):603-620]. Today, psilocybin is one of the most widely used psychedelics in human studies due to its relative safety, moderately long active duration, and good absorption in subjects. There remains strong research and therapeutic potential for psilocybin as recent studies have shown varying degrees of success in neurotic disorders, alcoholism, depression in terminally ill cancer patients, obsessive compulsive disorder, addiction, anxiety, post-traumatic stress disorder and even cluster headaches. It could also be useful as a psychosis model for the development of new treatments for psychotic disorders. [Dubovyk and Monahan-Vaughn, ACS Chem. Neurosci. (2018), 9(9):2241-2251].
Recent developments in the field have occurred in clinical research, where several double-blind placebo-controlled phase 2 studies of psilocybin-assisted psychotherapy in patients with treatment resistant, major depressive disorder and cancer-related psychosocial distress have demonstrated unprecedented positive relief of anxiety and depression. Two recent small pilot studies of psilocybin assisted psychotherapy also have shown positive benefit in treating both alcohol and nicotine addiction. Recently, blood oxygen level-dependent functional magnetic resonance imaging and magnetoencephalography have been employed for in vivo brain imaging in humans after administration of a psychedelic, and results indicate that intravenously administered psilocybin and LSD produce decreases in oscillatory power in areas of the brain's default mode network [Nichols D E. Pharmacol Rev., 2016, 68(2):264-355].
Preliminary studies using positron emission tomography (PET) showed that psilocybin ingestion (15 or 20 mg orally) increased absolute metabolic rate of glucose in frontal, and to a lesser extent in other, cortical regions as well as in striatal and limbic subcortical structures in healthy participants, suggesting that some of the key behavioral effects of psilocybin involve the frontal cortex [Gouzoulis-Mayfrank et al., Neuropsychopharmacology, 1999, 20(6):565-581; Vollenweider et al., Brain Res. Bull. 2001, 56(5):495-507]. Although 5HT2A agonism is widely recognized as the primary action of classic psychedelic agents, psilocybin has lesser affinity for a wide range of other pre- and post-synaptic serotonin and dopamine receptors, as well as the serotonin reuptake transporter [Tyls et al., Eur. Neuropsychopharmacol., 2014, 24(3):342-356]. Psilocybin activates 5HT1A receptors, which may contribute to antidepressant/anti-anxiety effects.
Depression and anxiety are two of the most common psychiatric disorders worldwide. Depression is a multifaceted condition characterized by episodes of mood disturbances alongside other symptoms such as anhedonia, psychomotor complaints, feelings of guilt, attentional deficits and suicidal tendencies, all of which can range in severity. According to the World Health Organization, the discovery of mainstream antidepressants has largely revolutionized the management of depression, yet up to 60% of patients remain inadequately treated. This is often due to the drugs' delayed therapeutic effect (generally 6 weeks from treatment onset), side effects leading to non-compliance, or inherent non-responsiveness to them. Similarly, anxiety disorders are a collective of etiologically complex disorders characterized by intense psychosocial distress and other symptoms depending on the subtype. Anxiety associated with life-threatening disease is the only anxiety subtype that has been studied in terms of psychedelic-assisted therapy. This form of anxiety affects up to 40% of individuals diagnosed with life-threatening diseases like cancer. It manifests as apprehension regarding future danger or misfortune accompanied by feelings of dysphoria or somatic symptoms of tension, and often coexists with depression. It is associated with decreased quality of life, reduced treatment adherence, prolonged hospitalization, increased disability, and hopelessness, which overall contribute to decreased survival rates. Pharmacological and psychosocial interventions are commonly used to manage this type of anxiety, but their efficacy is mixed and limited such that they often fail to provide satisfactory emotional relief. Recent interest into the use of psychedelic-assisted therapy may represent a promising alternative for patients with depression and anxiety that are ineffectively managed by conventional methods.
Generally, the psychedelic treatment model consists of administering the orally-active drug to induce a mystical experience lasting 4-9 h depending on the psychedelic [Halberstadt, Behav Brain Res., 2015, 277:99-120; Nichols, Pharmacol Rev., 2016, 68(2): 264-355]. This enables participants to work through and integrate difficult feelings and situations, leading to enduring anti-depressant and anxiolytic effects. Classical psychedelics like psilocybin and LSD are being studied as potential candidates. In one study with classical psychedelics for the treatment of depression and anxiety associated with life-threatening disease, it was found that, in a supportive setting, psilocybin, and LSD consistently produced significant and sustained anti-depressant and anxiolytic effects.
Psychedelic treatment is generally well-tolerated with no persisting adverse effects. Regarding their mechanisms of action, they mediate their main therapeutic effects biochemically via serotonin receptor agonism, and psychologically by generating meaningful psycho-spiritual experiences that contribute to mental flexibility. Given the limited success rates of current treatments for anxiety and mood disorders, and considering the high morbidity associated with these conditions, there is potential for psychedelics to provide symptom relief in patients inadequately managed by conventional methods.
Further emerging clinical research and evidence suggest psychedelic-assisted therapy, also shows potential as an alternative treatment for refractory substance use disorders and mental health conditions, and thus may be an important tool in a crisis where existing approaches have yielded limited success. A recent systematic review of clinical trials published over the last 25 years summarizes some of the anti-depressive, anxiolytic, and anti-addictive effects of classic psychedelics. Among these, are encouraging findings from a meta-analysis of randomized controlled trials of LSD therapy and a recent pilot study of psilocybin-assisted therapy for treating alcohol use disorder [dos Santos et al., Ther Adv Psychopharmacol., 2016, 6(3):193-213]. Similarly encouraging, are findings from a recent pilot study of psilocybin-assisted therapy for tobacco use disorder, demonstrating abstinence rates of 80% at six months follow-up and 67% at 12 months follow-up [Johnson et al., J Drug Alcohol Abuse, 2017, 43(1):55-60; Johnson et al., Psychopharmacol. 2014, 28(11):983-992], such rates are considerably higher than any documented in the tobacco cessation literature. Notably, mystical-type experiences generated from the psilocybin sessions were significantly correlated with positive treatment outcomes. These results coincide with bourgeoning evidence from recent clinical trials lending support to the effectiveness of psilocybin-assisted therapy for treatment-resistant depression and end-of-life anxiety [Carhart-Harris et al. Neuropsychopharmacology, 2017, 42(11):2105-2113]. Research on the potential benefits of psychedelic-assisted therapy for opioid use disorder (OUD) is beginning to emerge, and accumulating evidence supports a need to advance this line of investigation. Available evidence from earlier randomized clinical trials suggests a promising role for treating OUD: higher rates of abstinence were observed among participants receiving high dose LSD and ketamine-assisted therapies for heroin addiction compared to controls at long-term follow-ups. Recently, a large United States population study among 44,000 individuals found that psychedelic use was associated with 40% reduced risk of opioid abuse and 27% reduced risk of opioid dependence in the following year, as defined by DSM-IV criteria [Pisano et al., J Psychopharmacol., 2017, 31(5):606-613]. Similarly, a protective moderating effect of psychedelic use was found on the relationship between prescription opioid use and suicide risk among marginalized women [Argento et al., J Psychopharmacol., 2018, 32(12):1385-1391]. Despite the promise of these preliminary findings with classical psychedelic agents, further research is warranted to determine what it may contribute to the opioid crisis response given their potential toxicity. Meanwhile, growing evidence on the safety and efficacy of psilocybin for the treatment of mental and substance use disorders should help to motivate further clinical investigation into its use as a novel intervention for OUD.
Regular doses of psychedelics also ameliorate sleep disturbances, which are highly prevalent in depressive patients with more than 80% of them having complaints of poor sleep quality. The sleep symptoms are often unresolved by first-line treatment and are associated with a greater risk of relapse and recurrence. Interestingly, sleep problems often appear before other depression symptoms, and subjective sleep quality worsens before the onset of an episode in recurrent depression. Brain areas showing increased functional connectivity with poor sleep scores and higher depressive symptomatology scores included prefrontal and limbic areas, areas involved in the processing of emotions. Sleep disruption in healthy participants has demonstrated that sleep is indeed involved in mood, emotion evaluation processes and brain reactivity to emotional stimuli. An increase in negative mood and a mood-independent mislabeling of neutral stimuli as negative was for example shown by one study while another demonstrated an amplified reactivity in limbic brain regions in response to both negative and positive stimuli. Two other studies assessing electroencephalographic (EEG) brain activity during sleep showed that psychedelics, such as LSD, positively affect sleep patterns. Moreover, it has been shown that partial or a full night of sleep deprivation can alleviate symptoms of depression suggested by resetting circadian rhythms via modification of clock gene expression. It further was suggested that a single dose of a psychedelic causes a reset of the biological clock underlying sleep/wake cycles and thereby enhances cognitive-emotional processes in depressed people but also improving feelings of well-being and enhances mood in healthy individuals [Kuypers, Medical Hypotheses, 2019, 125:21-24].
In a systematic meta-analysis of clinical trials from 1960-2018 researching the therapeutic use of psychedelic treatment in patients with serious or terminal illnesses and related psychiatric illness, it was found that psychedelic therapy (mostly with LSD) may improve cancer-related depression, anxiety, and fear of death. Four randomized controlled clinical trials were published between 2011 and 2016, mostly with psilocybin treatment, that demonstrated psychedelic-assisted treatment can produce rapid, robust, and sustained improvements in cancer-related psychological and existential distress. [Ross S, Int Rev Psychiatry, 2018, 30(4):317-330]. Thus, the use of psychedelics in the fields of oncology and palliative care is intriguing for several reasons. First, many patients facing cancer or other life-threatening illnesses experience significant existential distress related to loss of meaning or purpose in life, which can be associated with hopelessness, demoralization, powerlessness, perceived burdensomeness, and a desire for hastened death. Those features are also often at the core of clinically significant anxiety and depression, and they can substantially diminish quality of life in this patient population. The alleviation of those forms of suffering should be among the central aims of palliative care. Accordingly, several manualized psychotherapies for cancer-related existential distress have been developed in recent years, with an emphasis on dignity and meaning-making. However, there are currently no pharmacologic interventions for existential distress per se, and available pharmacologic treatments for depressive symptoms in patients with cancer have not demonstrated superiority over placebo. There remains a need for additional effective treatments for those conditions [Rosenbaum et al., Curr. Oncol., 2019, 26(4): 225-226].
Recently, there has been growing interest in a new dosing paradigm for psychedelics such as psilocybin and LSD referred to colloquially as microdosing. Under this paradigm, sub-perceptive doses of the serotonergic hallucinogens, approximately 10% or less of the full dose, are taken on a more consistent basis of once each day, every other day, or every three days, and so on. Not only is this dosing paradigm more consistent with current standards in pharmacological care, but may be particularly beneficial for certain conditions, such as Alzheimer's disease and other neurodegenerative diseases, attention deficit disorder, attention deficit hyperactivity disorder, and for certain patient populations such as elderly, juvenile and patients that are fearful of or opposed to psychedelic assisted therapy. Moreover, this approach may be particularly well suited for managing cognitive deficits and preventing neurodegeneration. For example, subpopulations of low attentive and low motivated rats demonstrate improved performance on 5 choice serial reaction time and progressive ratio tasks, respectively, following doses of psilocybin below the threshold for eliciting the classical wet dog shake behavioral response associated with hallucinogenic doses (Blumstock et al., WO 2020/157569 A1). Similarly, treatment of patients with hallucinogenic doses of 5HT2A agonists is associated with increased BDNF and activation of the mTOR pathway, which are thought to promote neuroplasticity and are hypothesized to serve as molecular targets for the treatment of dementias and other neurodegenerative disorders (Ly et al. Cell Rep., 2018, 23(11):3170-3182). Additionally, several groups have demonstrated that low, non-hallucinogenic and non-psychomimetic, doses of 5HT2A agonists also show similar neuroprotective and increased neuroplasticity effects (neuroplastogens) and reduced neuroinflammation, which could be beneficial in both neurodegenerative and neurodevelopmental diseases and chronic disorders (Manfredi et al., WO 2020/181194, Flanagan et al., Int. Rev. Psychiatry, 2018, 13:1-13; Nichols et al., 2016, Psychedelics as medicines; an emerging new paradigm). This repeated, lower, dose paradigm may extend the utility of these compounds to additional indications and may prove useful for wellness applications.
Psychosis is often referred to as an abnormal state of mind that is characterized by hallucinatory experiences, delusional thinking, and disordered thoughts. Moreover, this state is accompanied by impairments in social cognition, inappropriate emotional expressions, and bizarre behavior. Most often, psychosis develops as part of a psychiatric disorder, of which, it represents an integral part of schizophrenia. It corresponds to the most florid phase of the illness. The very first manifestation of psychosis in a patient is referred to as first-episode psychosis. It reflects a critical transitional stage toward the chronic establishment of the disease, that is presumably mediated by progressive structural and functional abnormalities seen in diagnosed patients. [ACS Chem. Neurosci., 2018, 9, 2241-2251]. Anecdotal evidence suggests that low, non-hallucinogenic, doses (microdosing) of psychedelics that are administered regularly can reduce symptoms of schizophrenia and psychosis.

Psilocin derivatives as serotonergic psychedelic agents for the treatment of CNS disorders


An ignition coil for an internal combustion engine includes a wire-wound resistor. The wire-wound resistor includes a core material, a conductor winding, a pair of metal caps disposed at both ends of the core material, the pair of metal caps in contact with the conductor winding, and a resin coating material disposed so as to be in close contact with the core material and the conductor winding. The resin coating material is filled between the conductive winding and the resin coating material is formed to cover the conductor winding from an outer peripheral side of the conductor winding The metal cap includes a bottom portion and a cylindrical side portion. An inner peripheral side of the side portion is provide with ridges projecting internally formed along a direction crossing a winding direction of the conductor winding.

There is known an ignition coil for an internal combustion engine that has a coil body formed by accommodating a primary coil and a secondary coil within an insulating body case, and a high-voltage tower formed by disposing a high-voltage output terminal that is electrically connected to the secondary coil as well as projecting from the coil body towards a tip side thereof inside the high-voltage tower.
An ignition coil that has a resistive element inserted and disposed in a space at a tip side of the high-voltage output terminal in the high-voltage tower is disclosed in the Japanese Patent Application Laid-Open Publication No. 2011-35019.
As the resistive element, a wire-wound resistor can be used.
The wire-wound resistor has an insulating core material, a conductor winding wound spirally on an outer periphery of the core material, and metal caps disposed at both ends of the core material in an axial direction.
However, the following problems may occur in the ignition coil with the wire-wound resistor as described above.
That is, in the wire-wound resistor, the metal caps are required to be disposed at both ends of the core material in the axial direction as conduction terminals so as to contact the conductor windings from an outer peripheral side thereof.
Further, in order to prevent the conductor windings from becoming displaced in the axial direction relative to the core material, resin is required to be filled between parts of the conductor windings that are adjacent in the axial direction in an outer peripheral surface of the core material.
Here, the conductor windings are required to be exposed from the resin at the outer periphery thereof so that the conductor windings and the metal caps can be contacted.
However, since it is difficult to dispose the resin reliably between the parts of the conductor windings that are adjacent in the axial direction while exposing the entire outer peripheral surface of the conductor windings, in practice, a state is likely to occur where the parts of the outer peripheral surface in the conductor windings that are exposed and the parts that do not are not exposed coexist.
In such a state, creeping discharge may occur to the exposed conductor windings from the metal caps via a surface of the resin.
If creeping discharge occurs, this creeping discharge may become electromagnetic noise and is likely to affect peripheral devices.
Moreover, in the long term, there is also a concern the breakage of the conductor winding may occur due to the creeping discharge due to a deterioration of the resin between the conductive windings.
Here, it is conceivable to prevent the creeping discharge and damage of the conductor windings from occurring by preventing the exposure of the conductor windings by covering the entire conductor windings from the outer periphery thereof by the resin.
However, in this case, a problem that electrical conduction between the conductor windings and the metal caps become difficult may occur.

Ignition coil for internal combustion engine