text:
Anti-Viral Effect of an Extract of Ganoderma Lucidum
Compositions and methods for the treatment viral infections in organisms, particularly flu virus. The methods and compositions of the disclosure involve the administration to mammals and immune cells of a fucose-containing glycoprotein fraction from Reishi. An extract of G. lucidum induces immune responses that are shown to induce increase survivability in mice injected with lethal does of influenza virus. The effect is increased when the G. lucidum extract is dosed prior to injection of the lethal dose of influenza virus.
Yu, Alice (La Jolla, CA, US)
Wong, Chi-huey (Rancho Santa Fe, CA, US)
Jan, Jia Tsrong (Taipei, TW)
Cheng, -S Edmond Y. (Palo Alto, CA, US)
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KINSEY WHITE, NICOLE ERIN
Eckman Law Group (2225 East Bayshore Road Suite 200, Palo Alto, CA, 94303-3220, US)
1. A composition comprising: a fucose-containing glycoprotein extract of Ganoderma lucidum; wherein the extract is used to treat an organism having a virus.
2. The composition of claim 1, wherein the virus comprises an influenza virus.
3. The composition of claim 2, wherein the influenza virus comprises type A influenza.
4. The composition of claim 1, wherein the organism is a human.
5. The composition of claim 1, wherein the fucose-containing glycoprotein extract has a light absorbance of about 1.8 at O.D. 625
6. A composition comprising: at least a fucose-containing glycoprotein fraction of Ganoderma lucidum, wherein at least the fucose-containing glycoprotein fraction is used for the treatment of virus infections in an organism.
7. The composition of claim 6, further comprising a pharmaceutically acceptable carrier.
8. The method of claim 6, wherein the virus comprises an influenza virus.
9. The method of claim 8, wherein the influenza virus comprises type A influenza.
10. A method comprising: administering to an organism a pharmacologically effective amount of a fucose-containing glycoprotein extract of Ganoderma lucidum to treat virus infections in an organism.
11. The method of claim 11, wherein administration of the fucose-containing glycoprotein extract occurs at least prior to the infection of the organism by the virus.
12. The method of claim 11, wherein the virus is an influenza virus.
13. The method of claim 12, wherein the influenza virus is a type A influenza virus.
14. A kit comprising: at least a fucose-containing glycoprotein extract of Ganoderma lucidum.
RELATED APPLICATION
This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 60/846,675, filed Sep. 21, 2006.
This disclosure relates to compounds and methods for treating influenza virus in organisms with extracts of Ganoderma lucidum and Ganoderma tsugae, otherwise known as "Reishi" and/or (for purposes of this document) ("F3") provides medicinally active extracts and fractions, and methods of preparing the same, from components of
Compositions and methods for the treatment viral infections in organisms, particularly flu virus. The methods and compositions of the disclosure involve the administration to mammals and immune cells of a fucose-containing glycoprotein fraction from Ganoderma lucidum. The extract of G. lucidum induces immune responses that are shown to induce increase survivability in mice injected with lethal does of influenza virus. The effect is increased when the G. lucidum extract is dosed prior to injection of the lethal does of influenza virus.
According to a feature of the present disclosure, a composition is disclosed comprising a fucose-containing glycoprotein extract of Reishi wherein the extract is used to treat an organism having a virus.
According to a feature of the present disclosure, a composition is disclosed comprising at least a fucose-containing glycoprotein fraction of Reishi, wherein at least the fucose-containing glycoprotein fraction is used for the treatment of virus infections in an organism.
According to a feature of the present disclosure, a method is disclosed comprising administering to an organism a pharmacologically effective amount of a fucose-containing glycoprotein extract of Reishi to treat virus infections in an organism.
According to a feature of the present disclosure, a kit is disclosed comprising at least a fucose-containing glycoprotein extract of Reishi.
The above-mentioned features and objects of the present disclosure will become more apparent with reference to the following description taken in conjunction with the accompanying drawings wherein like reference numerals denote like elements and in which:
FIG. 1 is a graph of an embodiment illustrating the survival rate of mice injected with a lethal dose of H1N1 virus when treated with F3;
FIG. 3 is a graph of an embodiment illustrating the survival rate of mice injected with a lethal dose of H1N1 virus and various concentrations of F3;
FIGS. 4A and 4B is a graph of an embodiment illustrating the survival rate of mice injected with a lethal dose of H1N1 virus and various concentrations of F3;
FIG. 5 is a graph of an embodiment illustrating the survival rate of mice injected with a lethal dose of H1N1 virus and F3 obtained from various sources or F3 fragments;
FIG. 6 is a graph of an embodiment illustrating the survival rate of mice injected with a lethal dose of H5N1 virus and F3;
FIG. 8 are graphs of embodiments illustrating the serum concentrations of cytokines and chemokines at injection of F3 in mice;
FIG. 9 are graphs of embodiments illustrating the effect of F3 on various cell-types 72 hours after injection;
FIG. 10 is a graph of an embodiment illustrating the relative levels of granzyme B 72 hours after injection of F3;
FIG. 11 are graphs of embodiments illustrating the effect of F3 on various cell-types 72 hours after injection of F3 and a lethal dose of H1N1; and
FIG. 12 are graphs of an embodiment illustrating the effect of F3 on the ability of a virus to replicate in vivo.
In the following detailed description of embodiments of the invention, reference is made to the accompanying drawings in which like references indicate similar elements, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that logical, mechanical, biological, electrical, functional, and other changes may be made without departing from the scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims. As used in the present disclosure, the term "or" shall be understood to be defined as a logical disjunction and shall not indicate an exclusive disjunction unless expressly indicated as such or notated as "xor."
The bioactive polysaccharide fraction of G. lucidum is referred to as "Reishi-F3," "SF3," "Reishi polysaccharides-F3," or "EORP" and is described in WO 2006/044616, incorporated herein by reference in its entirety. F3 is a fucose-containing glycoprotein fraction. The present disclosure also hereby incorporates by reference U.S. Utility application Ser. No. 11/549,215 filed on 13 Oct. 2006, as if fully disclosed herein.
Botanical drugs could open up an unique opportunity for discovery or synthesis of safer and more cost-effective drugs. Reishi a popular traditional Chinese medicinal mushroom, has been used during the past two millennia as a home remedy for maintaining and improving health and for preventing and treating disease (1,2). The regular consumption of Reishi in the form of tea or mushroom powder was believed to preserve the human vitality and to promote longevity (1,3). In addition, numerous reports demonstrated that Reishi extensively modulates immune functions both in vivo and in vitro. Reishi has been used to treat various human diseases such as allergy, arthritis, bronchitis, gastric ulcer, hyperglycemia, hypertension, chronic hepatitis, hepatopathy, insomnia, nephritis, neurasthenia, scleroderma, inflammation, and cancer (1).
Reishi has been shown to be an anti-tumor agent. The potential clinical values has attracted intense interest to study its function and acting mechanism for pharmacological opportunity. A number of studies demonstrate that the anti-tumor activity of polysaccharide fraction of Reishi occurs not through direct induction of cell apoptosis or inhibition of proliferation, but mainly through the immunoenhancing activities (13,14).
The present inventors discovered that Reishi, in addition to its anti-tumor effects, is also an anti-influenza agent based on its ability to enhance the host's defense system. In one aspect, this disclosure provides a method for treating or preventing a viral infection in a mammal, the method comprising administering to the mammal the F3 extract of Ganoderma lucidum, or subfractions thereof that comprise fucose-containing glycoprotein(s).
Until recently, the immunomodulating activities of Reishi polysaccharide was demonstrated to through Toll-like receptor (TLR 2 and TLR4)-mediated signaling pathways, which in turn, activate the host immune response for B cell proliferation and cytokine production (10,15). However, more functions have been reported including promoting the function of antigen-presenting cells, mononuclear phygocyte system, humoral immunity and cellular immunity, and activation of the expression of and interleukin IL-1, IL-6, IL-12, IFN-γ, TNF-α, GM-CSF, G-CSF in splenocyte (16) induction of IL-1, IL-6 and TNF-α from activated macrophages. The above results obtained by various methodologies demonstrated that the immunomodulating effects of G. lucidum were extensive, diverse, and occur on many levels.
According to one aspect, a fucose-containing glycoproteic fraction of an extract of Reishi, is disclosed. The phrase "fucose-containing glycoprotein fraction" refers to a constituent part of the extract that includes at least one of a polysaccharide and a glycopeptide comprising fucose residues.
The term "glycoprotein" or "glycopeptide" refers to a protein of any length and dimensions with covalently attached sugar units, either bonded via the OH group of serine or threonine O glycosylated) or through the amide NH2 of asparagine (N-glycosylated) or portions thereof and/or proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide backbones.
The term "polysaccharide" refers to a polymers of any length and dimensions comprising monosaccharide residues linked glycosidically in branched or unbranched chains.
"Administering" means oral, or parenteral administration including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion as well as co-administered as a component of any medical device or object to be inserted (temporarily or permanently) into the body.
The term "extract" refers to a concentrated preparation obtained by removing active constituents from a given substance; when the active constituents are included in a solvent, the removal of the active constituents can be performed by evaporating all or nearly all the solvent and adjusting the residual mass or powder to a prescribed standard; extracts are usually prepared in three forms, semiliquid or of syrupy consistency, pilular or solid and as dry powder. The phrase "Ganoderma lucidum" refers to fungus Ganoderma lucidum or Reishi, any tissue, part or fraction therefrom or any preparation thereof including homogenates, suspensions, filtrates, filtration residues, and solutions. The term "preparation" refers to a composition processed, manufactured, or compounded starting from a given substance, the term "concentrated preparation" refers to a preparation with an increased ratio of the mass or volume of active constituents to the mass or volume of the non-active constituents or to the mass volume of the entire composition, compared with the same ratio in the given substance. The term "fraction" refers to one of the separable constituents of a substance.
The fucose-containing glycoprotein fraction of Ganoderma lucidum constitutes an active constituent of the extract of Ganoderma lucidum, in view of the extraction procedures described below. The activities and properties of the fucose-containing glycoprotein fraction as well as methods and compositions using or including the fucose-containing glycoprotein fraction shall not be limited in scope and application by the examples, compositions, and procedures described herein.
In some embodiments, the fucose-containing glycoprotein fraction is included in a fraction of Ganoderma lucidum (herein also denominated F3, Fraction 3, EORP, GL(PS)_Wu, or Wu) showing a light absorbance of about 1.8 O.D. at 625 nm identified and isolated from a water-soluble extract of Ganoderma lucidum (crude Reishi extract) by experimental procedures described below.
Fraction 3 includes a fucose-containing glycoprotein fraction, which comprises terminal fucose residues. The phrase "terminal fucose residues" identifies fucose residues of a chain of sugars located in a region proximate to a free end of a chain of sugars. The fucose-containing glycoprotein fraction of Fraction 3, also includes fucose residues bound with α1,2-fucosidic linkages and α3,4-fucosidic linkages.
In addition to fucose residue, fucose-containing glycoprotein fraction of Fraction 3 can also comprise glucose mannose, N-acetylglucosamine, xylose, and rhamnose (see in part Table I).
The fucose-containing glycoprotein fraction of Fraction 3 can also include an amino acidic component. The amino acidic component of Fraction 3, however, can be significantly modified without impairing the activities associated with the fucose-containing glycoprotein fraction of Fraction 3.
Fraction F3 can be obtained by a process comprising: homogenizing a plant tissue of Ganoderma lucidum or providing an homogenized plant tissue from Ganoderma lucidum; extracting the homogenized plant tissue of Ganoderma lucidum; and filtering the extracted homogenized plant tissue to form one or more fractions, the fractions comprising a saccharide component having fucose residues. The fractions formed in the above procedures can also be treated with protease.
The term "extracting" refers to any suitable procedure or protocol to provide an extract starting from a given substance; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the substance and the active constituents to be removed from the substance; exemplary procedures include treatment based on different solubility of the constituents of the substance in different solvents. Extracting the homogenized plant tissue can be performed by any suitable procedure or protocol to provide an extract of Ganoderma lucidum including a fucose-containing glycoprotein or fucose-containing polysaccharide constituents from the homogenized plant tissue, such as described below. For example, a suitable procedure includes treating the homogenized plant tissue with aqueous alkaline solution, for example 0.1 N NaOH, for a predetermined time to form a crude extract.
"Gel filtration" means separation of proteins, peptides, and oligonucleotides on the basis of size. Molecules move through a bed of porous beads, diffusing into the beads to greater or lesser degrees. Smaller molecules diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules enter less or not at all and thus move through the bed more quickly. Both molecular weight and three dimensional shape contribute to the degree of retention. Gel Filtration Chromatography may be used for analysis of molecular size, for separations of components in a mixture, or for salt removal or buffer exchange from a preparation of macromolecules.
Reishi" means the name for one form of the mushroom Ganoderma lucidum, and its close relative Ganoderma tsugae.
"Hematopoietic Cells" means blood forming stem cells. T cells and B cells arise from these cells.
The term "filtering" or "filtration" refers to any suitable procedure to separate a constituent of a substance, such as an active constituent, from other constituents of the substance, such as impurities; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the substance, the active constituents and the inactive constituents of the substance; exemplary filtration procedures include dialysis and gel filtration chromatography. Filtering the extracted homogenized plant tissue can be performed by subjecting the crude extract to filtration, such as gel filtration chromatography e.g. using a Sephacryl S-500 column, and eluting with an aqueous solution to form one or more fractions. In one embodiment the aqueous solution is buffered at about pH 7.0, for example a Tris buffer solution.
Specific embodiments of the above-mentioned process to obtain Fraction 3 described in detail below.
According to embodiments, the fucose-containing glycoprotein fraction is included in fractions of Fraction F3 (herein also collectively named Subfractions), herein identified as F3G1, F3G2, F3G3, and the F3G2 sub-fractions F3G2H1 and F3G2H2. The Subfractions are isolated from Fraction 3. Hence, the term "fucose-containing glycoprotein" refers not only to Fraction F3, but also to any Subfractions thereof, and any combination of F3 with the Subfractions, or any combination of the Subfractions.
The different Subfractions can be identified by the respective ability to absorb light. F3G1 shows a light absorbance of about 0.4 O.D. at 480 nm, F3G2 shows a light absorbance of about 0.1 O.D. at 480 nm; F3G2H1 shows a light absorbance of about 0.10 O.D. at 480 nm and F3G2H2 shows a light absorbance of about 0.5 O.D. at 480 nm.
F3 and the Subfractions are also herein collectively named Fractions.
The fucose-containing glycoprotein fraction comprised in the Subfractions can also include, in addition to the fucose residues, other sugars such as glucose and mannose, galactose, N-acetylglucosamine, and xylose (see in particular Table IV).
The Subfractions F3G1, FG2 and F3G3 can be obtained by partitioning Fraction 3. The term "partitioning" refers to any suitable procedure or protocol to divide a substance in two or more constituents thereof; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the substance, and the constituents to be partitioned. Partitioning Fraction 3 can be performed by filtering Fraction 3, (for example with an anion exchanger such as Diaion-WA30 anion exchanger or by gel filtration chromatography, e.g. on a TSK HW-75 column), and isolating the Subfractions F3G1, F3G2, and F3G3 from the filtered Fraction 3, (for example by elution with an alkaline solution, including for example at least one of NaCl).
"Cytokine" means any of a group of proteins and peptides that are used in organisms as signaling compounds. These chemical signals are similar to hormones and neurotransmitters and are used to allow one cell to communicate with another.
F3G2 subfractions F3G2H1 ad F3G2H2 can be obtained by subjecting F3G2 to further partitioning. For example, partitioning of F3G2 can be performed by filtering Sub-fraction F3G2 e.g. by gel filtration chromatography e.g. on a TSK HW-75 column, and isolating the Subfractions F3G2H1 and F3G2H2 from the filtered Subfraction F3G2 e.g., by eluting the filtered Subfraction F3G2 with an aqueous solution.
According to embodiments, a method to mediate a biological event associated with activated expression of a cytokine in a mammalian cell, is disclosed. The phrase "biological event" refers to an occurrence being of or relating to biology or to life and living processes. The term "mediate" used interchangeably with "regulate" and "modulate" refers to specifically controlling, or influencing an item identified thereafter, the item including a molecule, pathway, event or function, wherein "mediate" can include regulation by activation, stimulation, inhibition, alteration or modification of such molecule, pathway event or function.
The term "express" or "expression" of a cytokine refers to a process by which the gene's coded information is converted into the cytokine. The term "activate" refers to initiating or enhancing of a process such as expression of a cytokine, or to converting a compound, such as protein nucleic acid lipid, ion or other compound, from an inactive into an active form or into a different compound, the active form or different compound having a particular biological action. The term "cytokines" refers to proteins or biological factors that are released by cells, such as normal macrophages, fibroblasts, keratinocytes, and a variety of transformed cell lines, have specific effects on cell-cell interaction, communication, and behavior of other cells, which participate in regulating immunological and inflammatory processes and can contribute to repair processes and to the regulation of normal cell growth and differentiation; cytokines include interleukins, lymphokines and several related signaling molecules such as TNF-α and interferons.
The phrase "mammalian cell" indicates a cell of mammalian origin, in particular a human or murine, which can be located inside or outside the mammal. The mammal cells can be a human or a murine cell, such as T lymphocytes, human and murine macrophage and murine spleen cells.
The cytokine can be IL-1, IL6, IFN-γ, TNF-α IL-12, GM-CSF, G-CSF or M-CSF, and the mammalian cell can be a human or a mouse cell, such as T lymphocytes, human and murine macrophages and monocytes, and murine spleen cells. In certain specific embodiments, the cytokine is IL-1, and the mammalian cell is a macrophage or a monocyte.
The method to mediate a biological event associated with activated expression of a cytokine in a mammalian cell comprises administering to the mammalian cell an effective amount of the fucose-containing glycoprotein fraction.
The term "effective amount" of a compound is at least the minimum amount of the compound that is necessary to minimally achieve the desired effect. An effective amount of fucose-containing glycoprotein fraction for use in a given method can be readily determined by one skilled in the art without undue experimentation, depending upon the particular circumstances encountered (e.g. concentrations, cell type and number, etc.) upon reading of the present disclosure and in particular the Examples section.
The term "administering" refers to any process or protocol suitable to put a compound, and in particular the fucose-containing glycoprotein fraction, in contact with the cell, wherein the term "contact" or the phrase "put in contact" mean to place the compound and in particular the fucose-containing glycoprotein fraction and the cell, in a mutual spatial relationship such that a biological interaction between the compound and the cell is feasible; the phrase "biological interaction" refers to the process by which a compound and in particular the fucose-containing glycoprotein fraction controls, influences or otherwise affects the normal functioning or survival of the cell; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the type of cell, whether the contact occurs in vitro, in vivo, or ex vivo. Acceptable protocols to administer the fucose-containing glycoprotein fraction include individual dose size, number of doses, frequency of dose administration, and mode of administration, such as topical administration, local administration, or oral administration in vivo, incubation and assays in vitro, or ex vivo administration, e.g., to isolated hematopoietic cells, which can be identified by a person skilled in the art upon reading of the present disclosure and, in particular, the Examples section.
In particular, the effective amount of fucose-containing glycoprotein fraction to mediate a biological event, and suitable modes of administration identifiable by a person skilled in the art in view of the biological event to be mediated and the cell where the cytokine expression is activated upon reading of the present disclosure, and in particular the Examples section.
In some embodiments the biological event is hematopoiesis, the mammalian cell is a hematopoietic cell and the cytokines are GM-CSF, G-CSF, and M-CSF. The term "hematopoiesis" refers to the formation of blood cells in a living body, (especially in the bone marrow of mammals). The term "hematopoietic cells" refers to a cell involved in the hematopoietic process, such as a B cell, a macrophage, a dendritic cell, and a natural killer cell.
In some embodiments, the biological event is hematopoiesis, the fucose-containing glycoprotein fraction is included in F3, F3G2, F3G2H1 or F3G2H1, possibly in combination with F3G1 and the effective amount to mediate hematopoiesis and suitable modes of administration can be identified by a person skilled in the art upon reading of the present disclosure.
In embodiments where the fucose-containing glycoprotein fraction is included in F3, the effective amount to mediate hematopoiesis and suitable modes of administration, are identifiable by a person skilled in the art upon reading of the present disclosure.
Preparation and Analysis of Reishi Extracts F1, F2, F3, F4, and F5
Crude Reishi extract (prepared via alkaline extraction (0.1 N NaOH), neutralization and ethanol precipitation) was obtained from Pharmanex Co. (CA, USA). Twenty-eight mg of the crude extract were dissolved in 2 mL of Tris buffer (pH 7.0, 0.1 N) and centrifuged to remove the insoluble materials (7 mg). The supernatant was purified by gel filtration chromatography using a Sephacryl S-500 column (100×1.6 cm) with 0.1 N Tris buffer (pH 7.0) as the eluent. The flow rate was set at 0.5 mL/min, and the elute (7.5 mL per tube) was collected. Five fractions were collected (fractions 1-5), each dialyzed to remove excessive salt and lyophilized to give 1.0 mg, 6.2 mg, 5.3 mg, 2.1 mg, and less than 1 mg, respectively.
The main fraction having a light absorbance of about 1.8 at O.D. 625 was designated as Fraction 3. After the chromatography, the crude extract and each of the isolated fraction were subjected to anthrone analysis (Somani et al 1987; Jarmyn 1975; Halhoul and I. Kleinberg 1972) to detect sugar components.
Sugar Composition Analysis: Anthrone Colorimetric Method
Each 1.5 mL of anthrone (9,10-dihydro-9-oxoanthracene) solution (0.2 g anthrone dissolved in 100 mL of concd sulfuric acid) in a series of test tubes immersed in an ice water bath was carefully overlayed with 1.5 mL of sample
(20-40 μg/mL of -glucose or equivalent). After all additions had been made, the tubes were shaken rapidly and then replaced in an ice water bath. The tubes were heated for 5 min in a boiling water bath and then cooled; the optical densities were read within an hour at 625 nm against distilled water. Standards, reagent blanks and unknowns were run in triplicate because of likely contamination by other carbohydrate sources. Calculations made on the basis that the optical densities are directly proportional to the carbohydrate concentration.
Sugar Composition Analysis—TMS Method
For monosaccharide analysis, the polysaccharide extracts/fractions were methanolyzed with 0.5 M methanolic-HCl (Supelco) at 80° C. for 16 h, re-N-acetylated with 500 μL of methanol, 10 μL of pyridine, and 50 μL of acetic anhydride, and then treated with the Sylon HTP® trimethylsilylating reagent (Supelco) for 20 min at room temperature, dried and redissolved in hexane. GC-MS analysis of the trimethylsilylated derivatives was carried out using a Hewlett-Packard (HP) Gas Chromatograph 6890 connected to a HP 5973 Mass Selective Detector. Samples were dissolved in hexane prior to splitless injection into a HP-5MS fused silica capillary column (30 m×0.25 mm I.D., HP). The column head pressure was maintained at around 8.2 psi to give a constant flow rate of 1 mL/min using helium as carrier gas. Initial oven temperature was held at 60° C. for 1 min, increased to 140° C. at 25 C/min, to 250° C. at 5° C./min, and then increased to 300° C. at 10° C./min.
The carbohydrate composition of crude extract is reported in Table I, the carbohydrate composition of Fraction 3 is reported in Table II, below.
TABLE I
Carbohydrate compositions of crude Reishi extract
Sugar components Percentage (%)
D-Glucose 58.0
D-Mannose 15.5
L-Fucose 9.7
D-Galactose 9.3
D-Xylose 5.4
D-GlcNAc 1.0
L-Rhamnose 0.5
TABLE II
Carbohydrate compositions of Fraction 3
D-Galactose 13.5
High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) analysis, confirmed that F3 includes a glycoprotein or polysaccharide comprising fucose residues.
Also H2SO4/phenol analysis showed that overall polysaccharides concentration in F3 (85%) is higher than crude extract (60%).
Amino Acid Composition Analysis
The analysis was carried out based on a well-established method (Spachman et al 1958; Lo et al 1990). A sample of crude Reishi extract (6 mg) was dissolved in 1 mL solution of 6 M HCl and TFA (4/1), and heated at 140° C. for 3 h. The mixture was concentrated to give a dry residue and dissolved in 100 μL citrate buffer. A small aliquot (4 μL) was withdrawn and subjected to composition analysis by amino acid analyzer (Jeol JLC-6AH).
The resulting amino acid composition of Reishi Crude extract is shown in Table III below.
TABLE III
Amino acid analysis of Reishi extract
Amino acid Relative abundance
Asp 117
Thr 66
Ser 54
Glu 120
Gly 108
Ala 100
Mel 6
Ile 36
Leu 55
Tyr 16
Phe 28
His 12
Lys 21
Arg 22
Analysis directed to investigate protein concentration in Reishi crude extract and in Fraction 3 showed a F3 (˜10%), crude extract (˜20%) in Lowey method with BSA as a standard.
Further indications concerning F3 composition, differences with composition of crude Reishi extract and procedures to obtain F3 can be found in Chen et al. 2004 herein incorporated in its entirety.
Alternative Preparation of Reishi Extract Fraction 3
A crude G. Lucidum PS extract prepared via alkaline extraction with 0.1 N of NaOH, followed by neutralization and ethanol precipitation, was obtained from Pharmanex (CA). The crude G. Lucidum extract (100 g) was dissolved in 3 L of double-distilled H2O and stirred at 4° C. for 24 h. The solution was centrifuged (16,000 g) at 4° C. for 1 h, and the supernatant was concentrated at 35° C. The slurry product was then lyophilized to obtain 70 g of water-soluble dark brown G. Lucidum extract. The extract (2.5 g) was fractionated on Sephacryl S-500 column (95 2.6 cm) with 0.1 N of Tris buffer (pH 7.0) as the eluent. The flow rate was set at 0.6 ml/min, and fractions were collected with 7.5 ml per tube. Five fractions were collected, and each was dialyzed to remove excessive salts and lyophilized to give fractions 1-5; each fraction was characterized, as described above. The fucose-containing glycoprotein fraction (20˜30% yield), i.e., Fraction 3 or F3, was isolated.
To avoid LPS contamination the crude G. lucidum materials and PS extracts were prepared, from growth to harvest, as GMP grade from Pharmanex and the possible bacterial contamination was carefully monitored to meet the United States Food and Drug Administration standard. The reagents and utensils for preparation of F3 were either endotoxin-free grade or washed with PBS containing 50 μg/ml polymyxin B (PMB), then rinsed with PBS. F3 contained <1 ng of LPS/25 μg, as measured by LAL assay (Sigma-Aldrich). In addition, certain reagents were routinely checked by LAL for examination of LPS contamination.
An additional procedure was performed as described in Wang et al 2002 herein incorporated by reference in its entirety. According to embodiments, a modified version of the procedure described in Wang et al. comprises direct centrifugation, isolated polysaccharide from water soluble Reishi sample which showed components as well as F3.
The procedure as below: The water soluble polysaccharide from crude powder of Reishi 1 g was centrifuged (5000 r.p.m., 2800 g) at 4° C. for 1 h to separate polysaccharide by centrifugal filter with MWCO: 100K, the polysaccharide fraction was collected and lyophilized to give F3 (F3>100K) 172 mg (17%). This portion of polysaccharide shown familiar HPLC profile with F3 and its bio-function assay was analyzed, such as effect of proliferation and cytokines release to mice splenocyte, as well as previously F3 function.
Preparation and Purification of Reishi Extracts F3-F3G1 FRG2, F3 GH1, and F3 GH2
Crude Reishi extract (prepared via alkaline extraction (0.1 N NaOH), neutralization and ethanol precipitation) was obtained from Pharmanex Co., (CA, USA). All the chemicals and reagents were from Sigma Co., (St. Louis, Mo., USA) unless indicated.
Crude Reishi extract (100 g) was dissolved in 3 L of double distilled water, stirred at 4° C. for 24 h, and centrifuged for 1 h to remove the insoluble. The resulting solution was concentrated at 35° C. to give a small volume and lyophilized to generate 70 g powder of dark-brown color, 2.5 g of which were dissolved in a small volume of Tris buffer (pH 7.0, 0.1 N), and purified by gel filtration chromatography using a Sephacryl S-500 column (95×2.6 cm) with 0.1 N Tris buffer (pH 7.0) as the eluent. The flow rate was set at 0.6 mL/min, and 7.5 mL per tube was collected. After the chromatography, each fraction was subjected to anthrone analysis or the phenol-sulfuric acid method as described above, to detect sugar components. Five fractions were collected (fractions 1-5), each dialyzed to remove excessive salt and lyophilized, to give 450 mg of each fraction and in particular of F3.
F3 was further subjected to a column of Diaion-WA30 anion exchanger (Cl-form, 40×3.5 cm) eluted with 0.2 and 0.8 M NaCl at a flow rate of 0.5 mL/min, and two fractions were designated as F3G1 (11% yield based on F3) and F3G2 (10% yield based on F3), respectively, as shown in FIG. 14. Another fraction (F3G3, 11% yield based on F3) was generated when the column was further eluted with 2 M NaOH.
The carbohydrate composition of the F3G1, F3G2 and F3G3 was determined by anthrone colorimetric method and TMS method. The results are shown in Table IV below.
TABLE IV
Carbohydrate compositions of F3, F3G1, F3G2, and F3G3
Percentage (%)
L- D-
Fuc Xyl D-Man D-Gal D-GlcNAc D-Glc Unknown
F3 7.1 3.1 15.1 13.5 1.20 58.1 1.90
F3G1 8.0 5.7 10.2 12.6 0.25 63.2 0.05
F3G2 6.2 4.5 18.3 5.3 0.78 64.9 0.02
The results show that both Fraction 3 and the subfractions F3G1, F3G2 and F3G3 comprise glucose and mannose as major components together with smaller amounts of other sugars, including fucose N-acetylglucosamine, xylose and rhamnose, The percentage of galactose is apparently less in F3G2 and F3G3 than in other fractions.
Gel-filtration chromatography of F3G2 was carried out on a TSK HW-75 column (130×2.6 cm) eluted with double distilled water at a flow rate of 0.5 mL/min. Two fractions were collected; F3G2H1 (19% yield based on F3G2) and F3G2H2 (69% yield based on F3G2), as shown in FIG. 15.
Further indications concerning the composition of F3G1, F3G2, F3G3, F3G2H1 and F3G2H2 and procedures to obtain the Subfractions can be found in Chen et al. 2004, herein incorporated in its entirety.
In one implementation, F3 (or subfractions thereof that comprise fucose-containing glycoprotein(s)) is administered to a mammal or a bird to prevent or treat infection with influenza virus, including but not limited to influenza type A virus strains. Type A strains include H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7. The inventors of this disclosure discovered that treatment of mice with F3 of Reishi shortly after infection by a lethal dose of influenza virus can prolong their survival. Additionally, the inventors discovered that when F3 treatment of mice is started before challenge with a lethal dose of influenza virus, the incidence of lethality is drastically reduced.
When administered to a mammal (e.g., a human or a mouse) or a bird, therapeutically effective amounts of F3 (or subfractions thereof that comprise fucose-containing glycoprotein(s)) may be in the form of a pharmaceutical compositions which may also comprise one or more pharmaceutically acceptable carriers, diluents, or excipients. The administration of the pharmaceutical composition for the prevention or treatment of viral infections, such as influenza infections, may occur via any route known in the art, for example via topical administration, via enteral administration (e.g. orally or rectally), or via parenteral administration (e.g. intravenously, intra-muscularly, or subcutaneously).
The following examples are non-limiting and are provided solely to further illustrate the methods and compositions of the disclosure.
Use of F3 to Treat H1N1 Influenza Virus Infection
Thirty minutes after infection with a lethal dose of H1N1, BALB/c mice were treated with 10 μg or 100 μg of F3 (administered via intra-peritoneal injection). F3 treatment was then repeated every other day for 12 days or until death. As shown in FIG. 1, 10/10 control mice died by day 8. Survival curve analysis showed that mice treated with either 10 or 100 μg F3 had significantly prolonged survival with p=0.0015 and 0.0094, respectively. One of the 10 mice treated with 10 μg F3 survived the infection.
Pretreatment with F3 Prior to Infection with H1N1 Influenza Virus
In another study, BALB/c mice were pretreated twice per week for two weeks with 10 μg of F3 (administered via intra-peritoneal injection) and then challenged with 10 LD50 of H1N1. F3 treatment was then continued every other day for 12 days or until death. As shown in FIG. 2, only 3/20 (15%) of the mice in the control group survived, whereas 16/20 (80%) F3 treated mice survived (p=0.000044).
Pretreatment with Various Dosages of F3 Prior to Infection with Influenza Virus
BALB/c mice were pretreated twice per week for two weeks with 1-30 μg of F3 (administered via intra-peritoneal injection) and then challenged with 10 LD50 of H1N1. F3 treatment was then continued every other day for 6 days or until death. As shown in FIG. 3, 100% of the mice in the control group died by day 7, with median survival of 5 days, whereas mice treated with 10 and 30 μg had significantly prolonged survival, with median survival of 6 days (p=0.049, and 0.026, respectively). Four of the 20 mice treated with 10 μg F3 survived the infection.
Pretreatment with Various Dosages of F3 Prior to Infection with Different Viral Titers of Influenza Virus
BALB/c mice were pretreated twice per week for two weeks with 30-100 μg of F3 (administered via intra-peritoneal injection) and then challenged with 10 LD50 or 1 LD50 of H1N1 (FIGS. 4A, and 4B, respectively). F3 treatment was then continued every other day until death. As shown in FIG. 4A, 60% of the mice in the control group died by day 9, whereas mice treated with 30 and 100 μg had no significantly prolonged survival (p=0.7185, and 0.6023, respectively). However, mice treated with 50 μg appeared to be worse than control on survival rate (p=0.046). Ten, two, and five of the 20 mice treated with 10, 50, and 100 μg F3, respectively, survived the infection. We than repeated the same experiment with a lower virus titer at 1LD50. As shown in FIG. 4B, 30% of the mice in the control group died by day 11, whereas mice treated with 30, 50, and 100 μg had no significantly prolonged survival (p=0.2185, 0.1257, and 0.0882, respectively). Ten, ten, and nine of the 20 mice treated with 10, 50, and 100 μg F3, respectively, survived the infection.
Pretreatment with Different Batch of F3 and 1-3Beta Glucan Prior to Infection with Influenza Virus
BALB/c mice were pretreated twice per week for two weeks with 10 μg F3 from Wyntek company, F3 from GRC, and the backbone of F3, 1-3 beta glucan (administered via intra-peritoneal injection), and then challenged with 10 LD50 of H1N1. F3 treatment was then continued every other day for 6 days or until death. As shown in FIG. 5, 100% of the mice in the control group died by day 7, whereas mice treated with 10 μg F3 from Wyntek Inc. and GRC had significantly prolonged survival, (p=0.016, and 0.026, respectively). Although one of the 20 mice treated with 10 μg 1-3 beta glucan survived the infection, the survival of the entire group did not differ significantly from the control group (p=0.08).
BALB/c mice were pretreated twice per week for two weeks with 10 μg of F3 (administered via intra-peritoneal injection) and then challenged with 20 LD50 of H5N1 (NIBRG-14). F3 treatment was then continued every other day for 12 days or until death. As shown in FIG. 6, only 13/22 (59%) of the mice in the control group survived, whereas 20/22 (90%) F3 treated mice survived the infection (p=0.0145).
Pretreatment with F3 Prior to Infection with Lower Titer of H5N1 Influenza Virus
In another study, BALB/c mice were pretreated twice per week for two weeks with 10 μg of F3 (administered via intra-peritoneal injection) and then challenged with 2 LD50 of H5N1 (NIBRG-14). F3 treatment was then continued every other day for 12 days or until death. As shown in FIG. 6, 4/22 (18%) of the mice in the control group survived, whereas 22/22 (100%) F3 treated mice died by day 7 and shown no statistically difference in comparison to control (p=0.4684).
Kinetics of Serum Cytokine/Chemokine Profile in Response to F3
BALB/c mouse serum samples (N=3) were collected at 0, 2, 18, 48, and 72 hr after intra-peritoneal injection of 10 μg F3. The serum concentrations of 21 cytokines/chemokines were measured by Beadlyte Mouse 21-plex Cytokine Detection System (cat #48-012, Upstate, NY), and read by a Luminex 100™ system. IL-1RA is measured by another ELISA kit. As shown in FIG. 8, most cytokines/chemokines, such as IL-1β (A), IL-10 (B), IL-12 p40 (C), IL-12 p70 (D), IL-13 (E), IL-17 (F), IL-6 (G), IL-9 (H), IL-1RA (I), TNF-α (J), KC (N), MCP-1 (0), MIP-β (P), RANTES (Q), have similar kinetics that reached their peaks at 2 hr and declined at 18 hr post-injection. In comparison, IL-2 (K) increased at 48 hr and reached its peak at 72 hr post-injection. However, IFN-γ (L) and IL-4 (M) as well as IL-1α, IL3, IL-5, GM-CSF, and VEGF (data not shown) had no significant increase during 72 hr.
Expansion/Activation of Splenocytes after Single Injection of F3
Spleens from BALB/c mice (N=3) were harvested 72 h after injection of F3 intraperitoneally. The increase of the total number of splenic nucleated cells was shown as (A). The induction of the subpopulation of immune cells by F3, including mature dendritic cells (CD11C+/CD80+/CD86+), activated B cells (CD45R+/CD23+/CD69+) and plasma cell (CD45R/CD138), activated NK cells (U5A2-13Ag+/CD3−/CD69+), activated NKT cells (U5A2-13Ag+/CD3+/CD69+), CD4 (CD3+/CD4+/CD8−), CD8 (CD3+/CD4−/CD8+), and Treg (CD4+/CD25+/FoxP3+) were shown in (B-E, respectively). Quantitative data are shown as the mean ±s.d. *, p<0.05, compared with saline control.
Expression of Granzyme B in Splenocytes after Injection of F3
Spleens from BALB/c mice (N=3) were harvested 72 h after injection of F3 intraperitoneally, and then cultured in vitro for 48 hr to determine the production of Granzyme B by ELISPOT assay. Briefly, 5×104 splenocytes were cultured in 96-well culture plate pre-coated with anti-Granzyme B Ab, and then treated with 10 ug/ml F3 or control saline and incubated for another 48 hr. After that, cells were washed and the number of Granzyme B secreting-cells was detected as the color spots, using Ab and substrates, following manufacture's protocol. Data were obtained from six repeats and shown as the mean ±s.d. *, p<0.05, compared with saline control. As shown in FIG. 10, F3 increased the number of granzyeme B-secreting cells in spleen.
Expansion/Activation of Splenocytes after Influenza Virus Infection and F3 Treatment
BALB/c mice were pretreated twice per week for two weeks with 10 μg of F3 (administered via intra-peritoneal injection) and then challenged with 10 LD50 of H1N1. Spleens from BALB/c mice (N=3) were harvested 72 h after virus infection. Among the immune effector cells, activated CD4 (CD3+/CD4+/CD69+), CD8 (CD3+//CD8+/CD69+) T cells increased after virus infection, while treatment of F3 did not show significant difference from the control (A and B, respectively). However, an increase in activated B cells (CD45R+/CD23+/CD69+), dendritic cells (CD11C+/CD80+/CD86+), and CD4+NKT cells (U5A2-13Ag+/CD3+/CD4+) were noted only in F3 treated mice after virus infection (C, D, and F, respectively). The number of NK cells (U5A2-13Ag+/CD3−) significantly decreased after virus infection in both F3 and control mice (E), whereas NKT cells (U5A2-13Ag+/CD3+) significantly increased after virus infection in both F3 and control subjects Quantitative data are shown as the mean ±s.d. *, p<0.05, compared with normal control; #, p<0.05, compared with infected control.
Effect of F3 on Entry/Replication of H1N1 Virus In Vivo/In Vitro
BALB/c mice were pretreated twice per week for two weeks with 10 μg of F3 (administered via intra-peritoneal injection) and then challenged with 10 LD50 of H1N1. Lungs from infected BALB/c mice (N=2) were harvested 72 h post-infection for assessment of the surviving virus titer. The total virus in each lung extract were individually calculated as tissue culture infection dose (TCID50) and converted the data to virus titer/lung as shown in (A). In this experiment, it appears that F3 did not inhibit the virus replications in vivo.
In order to determine whether F3 directly interferes with the ability of H1N1 virus to infect target cells, MDCK cells (2×105) were cultured in 96-wells plate and pretreated with F3 (10 μg/ml) (B) or saline for 2 hrs. After that, MDCK cells were infected by H1N1 virus at the titer of 1×TCID50, and then cultured for another 48 hrs. The cytopathetic effects (CPE) on MDCK cells were observed by microscope. In comparison to control, there was no significant direct effect of F3 on virus entry/replication.
According to another aspect, the fucose-containing glycoprotein fraction can be included in a pharmaceutical composition together with additional active agents, carriers, vehicles, excipients, or auxiliary agents identifiable by a person skilled in the art upon reading of the present disclosure.
The pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the fucose-containing glycoprotein forms the "active compound," also referred to as the "active agent." As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
Subject as used herein refers to humans and non-human primates (e.g. guerilla, macaque, marmoset), livestock animals (e.g. sheep, cow, horse, donkey, pig), companion animals (e.g. dog, cat), laboratory test animals (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animals (e.g. fox, deer), and any other organisms who can benefit from the agents of the present disclosure. There is no limitation on the type of animal that could benefit from the presently described agents. A subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, animal, host, or recipient.
Pharmaceutical compositions suitable for an injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. According to embodiments, isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition are added. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation are prepared by vacuum drying or freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
According to embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
As defined herein, a therapeutically effective amount of the active compound (i.e., an effective dosage) may range from about 0.001 to 30 mg/kg body weight. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
According to another aspect, one or more kits of parts can be envisioned by the person skilled in the art, the kits of parts to perform at least one of the methods herein disclosed, the kit of parts comprising two or more compositions, the compositions comprising alone or in combination an effective amount of fucose-containing glycoprotein fraction according to the at least one of the above mentioned methods.
The kits possibly include also compositions comprising active agents other than the fucose-containing glycoprotein fraction, identifiers of a biological event, or other compounds identifiable by a person skilled upon reading of the present disclosure. The term "identifier" refers to a molecule, metabolite or other compound, such as antibodies, DNA or RNA oligonucleotides, able to discover or determine the existence, presence, or fact of or otherwise detect a biological event under procedures identifiable by a person skilled in the art; exemplary identifiers are antibodies, exemplary procedures are western blot, nitrite assay and RT-PCR, as described in the Examples. Exemplary biological events are cytokine expression or other immunomodulating events; an exemplary active agent other than the fucose-containing glycoprotein fraction is LPS.
The kit can also comprise at least one composition comprising an effective amount of fucose-containing glycoprotein fraction or a cell line. The compositions and the cell line of the kits of parts to be used to perform the at least one method herein disclosed according to procedure identifiable by a person skilled in the art.
While the apparatus and method have been described in terms of what are presently considered to be the most practical and preferred embodiments, it is to be understood that the disclosure need not be limited to the disclosed embodiments. It is intended to cover various modifications and similar arrangements included within the spirit and scope of the claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures. The present disclosure includes any and all embodiments of the following claims.
The following references are each incorporated herein by reference in their entirety. The citation of these references is not an admission that any of them forms part of the body of the prior art in any country.
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text:
Home/1980s/NCAA Football – 1985 – SWC Game Of Week – TCU Hornd Frogs Vs Texas Longhons
1980sAustinFootballNCAANCAAFSWCTexas LonghornsTV AdsUT AustinUT Football
NCAA Football – 1985 – SWC Game Of Week – TCU Hornd Frogs Vs Texas Longhons
DOG COMMENTARY:
The 1985 TCU Horned Frogs football team represented Texas Christian University (TCU) in the 1985 NCAA Division I-A football season….as the Horned Frogs finished the season 3 – 8 overall….while posting 0 – 8 in the Southwest Conference. The team was coached by Jim Wacker….in his third year as head coach….as the Frogs played their home games in Amon G. Carter Stadium….which is located on campus in Fort Worth, Texas.
The 1985 Texas Longhorns football team represented the University of Texas at Austin in the 1985 NCAA Division I-A football season….as the Longhorns finished the regular season with an 8–3 record….while posting a 6 – 2 record in the SWC…. and then lost to the Air Force Academy by a score of 24 – 16 in the Bluebonnet Bowl.
This game was played on November 16, 1985 at Texas Memorial Stadium in Austin, TX in front of a crowd of 66,397….which the Longhorns won by a score of 20 – 0…in a hard hitting Southwest Conference match-up….as dang near every SWC opponent was a longtime rival.
TCU Head Coach Jim Wacker was the father of Mike Wacker….who was a 6'9″ F/C for the University of Texas Longhorn basketball team from 1981 to 1984….when in his freshman year he team up LaSalle Thompson "down on the block" during the 1981 season under Head Coach Abe Lemons….as Wacker and Thompson led the Horns to a 14 – 0 record….with a # 4 in the nation ranking….as Wacker averaged 15.2 points and 9.3 rebounds….while Thompson average 18.6 points and 13.5 rebounds per game for the season….and what team wouldn't like to have that kid of production in the key and down under.
During the 1981 -1982 season….Coach Wacker, who was at that time coaching just down the road from Austin at Southwest Texas State (now Texas State)…..where he had just finished leading the Bobcats football team to their 2nd NCAA Division II National Championship in the past two years….and that's when Coach Wacker took a Thursday off from football to drive up to Waco, TX to see his son take on the Baylor Bear….which was at the end of the 1st half of the Texas basketball season….only to make a surprise appearance while at Bone Daddy's bar…. Madison Square Garden, The Sports Place at 302 E. 6th Street in Austin….when he walked through the doors with UT Head Basketball Coach Abe Lemons….who would be speaking at the weekly Abe Lemons Luncheon every Thursday during the entire men's basketball season….for we are pretty sure that the two coaches got to know each other during the recruiting process of his son, Mike.
As Bone Daddy puts it…."I got to spend some time with a coach who not only had won 2 x NAIA Division Ii National Football Championships….along with winning 2 x NCAA Division II National Championships….plus the man who had sired one-half of the best "Twin Tower Tandem" in collegiate basketball that season….as his son Mike and LaSalle Thompson had teamed up to lead the Horns to a 14 – 0 record….and # 4 national ranking on the very day that these two Texas coaching icons came to The Garden to hear Abe speak.
That night in a game against Baylor University….Texas F/C Mike Wacker suffered a serious career threatening knee injury that required a year and a half to heal well enough for him to return to competitive basketball again. The injury to Wacker was devastating to the Longhorn basketball team….who after being undefeated through 14 games….went on to finish the remaining 13 games of the season with a 2 – 11 record….which caused the other half of the best "Twin Tower Tandem"….a two-time 1st team All-SWC in his freshman and sophmore year…LaSalle Thompson to not return to Texas for his senior year….while opting to begin his 14 year NBA career by becoming 5th pick overall by the Kansas City Kings.
The most devastating aspect of Mike Wacker's injury was the fact that it led to the eventual firing of Coach Abe Lemons at Texas….which ended a wonderful 5 year business relation between Bone Daddy and Coach Abe….while Coach Jim Wacker would leave Southwest Texas State to coach the TCU Horned Frogs for the next 8 years. Any way you cut the pie….this video does for Bone Daddy what 1000's of our videos do here at ImaSportsphile for 10,000's of viewers…it bring back fond memories of days gone by.
NFL - 1970s - Film - Son Of Football Follies - Narrated By Elmer Fudd + Daffy Duck + Yosemite Sam
NFL - 1979 - CBS Special - Passport To Pasadena - Preview Of The 1979 Upcoming Season
Comedy – 1978 – Franken & Davis – Khomeini The Magnificient – Iran's Mystical Leader
Boxing – WBC Heavyweight Title – Mike Tyson VS Frank Bruno
Music – 1980 – Best Of ACL – Roy Clark & Gatemouth Brown + John Hartford + Jimmy Buffet
Phil Collins – My Life is So Lonely & I'm Getting Stronger By the Minute &
Boxing – 10 Rnd Middleweight Bout – Thomas Hearns VS Ernie Singletary

redpajama_set_name: RedPajamaCommonCrawl


text:
Orthodox Missionaries in Guatemala
Father David and Matushka Rozanne Rucker have been mission specialists for many years with the Orthodox Christian Mission Center (OCMC), which has missionaries around the world under the umbrella of the Assembly of Bishops. Guatemala has seen an explosive growth of Orthodox Christianity among thousands of indigenous Mayans, so now the Ruckers will be going there to live, to train catechists and clergy and to help establish the first Orthodox seminary.
Father David and Matushka Rozanne Rucker with Metropolitan Tikhon
While Father David spends much of his life on the road, he is a priest of the Orthodox Church in America and is formally attached to Saint Sergius Chapel here in Syosset. Last weekend the Ruckers were here to meet with Metropolitan Tikhon. Father David served the Divine Liturgy with His Beatitude and was awarded a gold cross for his years of service.
You need to know that OCMC missionaries have to raise their own funds for their work. Incredibly, there are scores of faithful Orthodox missionaries like the Ruckers who take up this life and do this willingly and joyfully. Please consider helping them. You can learn more about Father David and Matushka Rozanne and donate through the Orthodox Christian Missions Center here.


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Q: timeout not working as expected I am trying to get the button to change from the default 'this Will Change' to then change what it says to 'New message' and back to 'someButton' every 2 seconds and then stop it with a click.
So far, I have this code:

var timeouts = [];

var someArray = {button: 'new Message', button: 'Some Btn'};

$(document).ready(function(){
var i = 2000;
$.each(someArray, function(index, value){
    timeouts.push(setTimeout(function(){
          $(index).html(value);
     },i));
     i = i + 2000;
});

$('#stop').click(function(){
    $.each(timeouts, function (_, id) {
       clearTimeout(id);
    });
    timeouts = [];
    $(button).html('Some Btn');
});
});

<button type='button'>
This will change
</button>
Stop

It seems to ignore the first setTimeout() or that timeout is not run on the New message index.. Then when I press the click button, no timeouts get cleared?
Any help would be greatly appreciated to where I'm going wrong.
Update: I'd like to keep this as jQuery oriented as possible, thanks!

A: The reason why its skipping new Message is because of this:
var someArray = {button: 'new Message', button: 'Some Btn'};

Your someArray is an object. So if you have same property name, it will get overriden and it will be parsed as
var someArray = {button: 'Some Btn'};

Just use a proper structure and it should work

var timeouts = [];

var someArray = [{
  type: "button",
  message: 'new Message',
}, {
  type: "button",
  message: 'Some Btn'
}];

$(document).ready(function() {
  var i = 2000;
  $.each(someArray, function(index, value) {
    timeouts.push(setTimeout(function() {
      $(value.type).html(value.message);
    }, i));
    i = i + 2000;
  });

  $('#stop').click(function() {
    $.each(timeouts, function(_, id) {
      clearTimeout(id);
    });
    timeouts = [];
    $(button).html('Some Btn');
  });
});

<button type='button'>
  This will change
</button>
Stop

Updated code
This is how I would write. Few basic changes,

*

*Use $(document).ready as start of your code flow. Not as wrapper for your code. It should call to necessary function.

*You should use Array.map, Array.forEach instead of $.each(). These functions are very powerful and handy.

*Variables defined outside any function will be part of global scope. Its a bad practice to pollute global scope.

*Use small functions. This way there is a higher possibility you can reuse your code. Any code that can be use somewhere else, can be used as a function.

$(document).ready(function() {
  var someArray = [
    { type: "button", message: 'new Message', },
    { type: "button", message: 'Some Btn' }
  ];

  var timeouts = registerTimeouts(someArray);
  registerEvents(timeouts)
});

function registerTimeouts(array) {
  return array.map(function(value, index) {
    return setTimeout(function() {
      $(value.type).html(value.message);
    }, (index + 1) * 2000);
  });
}

function registerEvents(timeouts) {
  $('#stop').click(function() {
    timeouts.forEach(function(id) {
      clearTimeout(id);
    });
    timeouts = [];
    $(button).html('Some Btn');
  });
}

<button type='button'>
  This will change
</button>
Stop

A: Your use case describes exactly what a setInterval is meant for.

"Repeatedly calls a function or executes a code snippet, with a fixed time delay between each call. Returns an intervalID."

the setInterval function returns an ID, which can be passed into clearInterval to stop the code from executing every X seconds.
I've posted a code snippet to what I think you're trying to achieve.
Plnkr Link

var strings = ["This will change", "Some button"];
var intervalID;
var button;
var index = 0;

$(document).ready(function() {
  button = $("#myButton");
  intervalID = window.setInterval(function() {
    console.log("hi");
    button.html(strings[index]);
    index = (index == 1 ? 0 : 1);
  }, 2000);
  $("#stop").click(function() {
    window.clearInterval(intervalID);
  });
});

<button id="myButton" type='button'>
  This will change
</button>
Stop

A: Here's a clean, functional way. Just for fun.
The selected answer is spot on. I'm just offering another option, using a slightly different style.
It starts, toggles and stops.

// "constant" array of messages
var messagesArray = ['new Message', 'Some Btn'];

// original button text
var btnOrigText = $('button').text()

// reverse an array
var _arrReverse = function(arr) {
    return arr.reverse()
}

// insert text from array 0th index into an element
var _insertTextArrayZero = function(el, messages) {
    // inserts an array's 0th index as HTML to an element
    return $(el).html(messages[0]);
}

// clears a timeout when given an id
var _clearIntvlAt = function(intvlId) {
    return clearTimeout(intvlId);
}

$(document).ready(function(){
    // set interval
    var intvl = 2000;
    // keep our original array intact
    var messagesCloned = messagesArray.slice(0)
    // run interval
    var toggleTextIntvl = setInterval(function() {
     // insert reversed array from [0] index of array into HTML element
        // NOTE: this only works for arrays of 2 items which need to toggle
     return _insertTextArrayZero('button', _arrReverse(messagesCloned))
    }, intvl);

    // initiate "stopping" of toggle text
    $('#stop').click(function() {
        // stop toggleTextIntvl
        _clearIntvlAt(toggleTextIntvl)
        // set markup back to default
        $('button').html(btnOrigText);
    });
});

<button type='button'>
This will change
</button>
Stop

redpajama_set_name: RedPajamaStackExchange


Virginia State Police recently conducted a special traffic safety enforcement operation on Route 340 in Page County.
From 6 a.m. to 4 p.m. on March 16, state troopers issued a total 89 traffic citations on Route 340. Troopers stopped 44 speeders and three reckless drivers. The average speed of the violators was 68 mph in the posted 55 mph zone.
Four drivers were cited for failing to wear a seat belt plus one child safety seat violation was cited. Troopers also cited 22 vehicle equipment violations.
In 2017, Page County experienced 247 traffic crashes with three of them resulting in a fatality and 199 involving injuries.* Of those 247 traffic crashes, speed was a causative factor in 73 of the crashes and two of the fatalities. Twenty-six of the traffic crashes were the result of a drunken driver; and 26 of the traffic crashes involved individuals who were not buckled up.
The Virginia State Police Area 14 Office is part of the Culpeper Division and encompasses the Page and Shenandoah counties.


temp sound solutions was formed in october of 1997 out of a penchant for several loved things; video games, hard electronic music, tracking (namely the demoscene), and the ability to fluidly meld all of the above with anything else via a doppelganging mutagen found resident in the bloodstream of all imperfect humans. it wasnt until years later, that shawn phase, the only remaining survivor of the once-vastly overwhelming clan that comprised TSS in its earliest incarnations was able to realize and harness the stregnth, the courage, the willpower and fortitude to survive in this world and progress to the point of spreading this knowledge of all of the elements above to the world. in august of 1999, the first performance took place at a normal club called 'the void' in new york city, in front of a crowd of 40 young people gathering around a laptop and two pairs of laptop speakers; unknowing survivors of other clans had destroyed any available powar, and a very wary but unaverted shawn phase brought the experience to the table by performing an energy laiden, yet downtempo set. the return trip to TSSBAY04 proved to be almost fatal, as shawn phase almost fell ill and expired due to overexposure in a hotel room in the famed waldorf astoria. the rest is history... temp sound solutions is shawn phase, house the grate, spookmeister c, kirby pufocia, cacomistile, and most importantly, you.


text:
Oblast' di Saratov – oblast' della Russia
 Gubernija di Saratov – governatorato dell'Impero russo

redpajama_set_name: RedPajamaWikipedia


Supervises management of the Bulldog Shop and leverages beverage and food services, the gift shop, and other resources in serving both students and conference attendees/patrons.
Master's degree in a relevant field or a bachelors with considerable related experience.
This job frequently requires a non-traditional work schedule including nights and weekends.


text:
'use strict';

var allocCluster = require('./lib/alloc-cluster.js');

allocCluster.test('request() without hasNoParent', {
    numPeers: 2
}, function t(cluster, assert) {
    var one = remoteService(cluster.channels[0]);
    var two = cluster.channels[1];

    var subTwo = two.makeSubChannel({
        serviceName: 'server',
        peers: [one.hostPort]
    });

    subTwo.waitForIdentified({
        host: one.hostPort
    }, function onIdentified(err) {
        assert.ifError(err);

        assert.throws(doRequest, /For the call to server/);

        assert.end();
    });

    function doRequest() {
        subTwo.request({
            serviceName: 'server'
        }).send('echo', 'a', 'b');
    }
});

allocCluster.test('request() without as header', {
    numPeers: 2
}, function t(cluster, assert) {
    var one = remoteService(cluster.channels[0]);
    var two = cluster.channels[1];

    var subTwo = two.makeSubChannel({
        serviceName: 'server',
        peers: [one.hostPort]
    });

    subTwo.waitForIdentified({
        host: one.hostPort
    }, function onIdentified(err) {
        assert.ifError(err);

        doRequest();
    });

    function doRequest() {
        subTwo.request({
            serviceName: 'server',
            hasNoParent: true
        }).send('echo', 'a', 'b', onResponse);
    }

    function onResponse(err, res) {
        assert.equal(err && err.type,
                     'tchannel.handler.outgoing-req-as-header-required',
                     'expected error type');
        assert.notOk(res, 'expected no response');

        assert.end();
    }
});

allocCluster.test('request() without cn header', {
    numPeers: 2
}, function t(cluster, assert) {
    var one = remoteService(cluster.channels[0]);
    var two = cluster.channels[1];

    var subTwo = two.makeSubChannel({
        serviceName: 'server',
        peers: [one.hostPort]
    });

    subTwo.waitForIdentified({
        host: one.hostPort
    }, function onIdentified(err) {
        assert.ifError(err);

        doRequest();
    });

    function doRequest() {
        subTwo.request({
            serviceName: 'server',
            hasNoParent: true,
            headers: {
                as: 'raw'
            }
        }).send('echo', 'a', 'b', onResponse);
    }

    function onResponse(err, res) {
        assert.equal(err && err.type,
                     'tchannel.handler.outgoing-req-cn-header-required',
                     'expected error type');
        assert.notOk(res, 'expected no response');

        assert.end();
    }
});

function remoteService(chan) {
    chan.makeSubChannel({
        serviceName: 'server'
    }).register('echo', function echo(req, res, arg2, arg3) {
        res.headers.as = 'raw';
        res.sendOk(arg2, arg3);
    });

    return chan;
}

redpajama_set_name: RedPajamaGithub


The Emergency Medical Technician program is designed to train the lay person in emergency medical care in the pre-hospital environment. The program instructs the student in medical practices available to pre-hospital personnel, precise patient assessment and management of trauma and medical emergencies, airway management, resuscitation and pharmacological interventions. This program will develop the student's confidence, knowledge and skills in performing critical assessment and management of patient's with medical and trauma related life threats. It also focuses on development of the EMT's critical role as a team member during patient crisis situations.
Interested ? Fill out form below and someone from our training center will call you back.
Why do you want to become an Emt ?


Registration cost includes materials, snacks and lunch. The deadline for registration is 5:00PM on September 6, 2018. The event begins with registration and refreshments from 8:45 to 9:15am on Thursday, September 13, 2018. Sessions will conclude at 4:30pm. Attendees are invited to attend a post session dinner to network with their colleagues.
The next Advanced Director Workshop is scheduled for the following date and will be held in the Lansing or East Lansing area: Friday, September 14, 2018. Registration will open in June 2018.
Registration cost includes materials, snacks and lunch. The deadline to register is is 5:00PM on Wednesday, September 6, 2018. The event begins with registration and refreshments from 8:45 to 9:15am. Sessions will conclude at 4:30pm.
Check the Library of Michigan Continuing Education Page in June for Registration Links.


text:
Q: Chart bind to non-primitive type I have a C#.NET Windows Forms application using a chart with a number of line series. I have a physical device that stores readings on it, and as I'm downloading the readings I want the graph to fill in the detail.
I have created types in the software for quantifiable values (such as temperature) that may use different units depending on location, allowing me to have a user setting for display units, and just deal with everything in the background without caring, and when it's displayed to the user it's in the correct unit.
The C#.NET Charting.Series class is very strict and when attempting to draw using the bound array it throws an ArgumentExeption with the statement: "Series data points do not support values of type Units.Temperature only values of these types can be used: Double, Decimal, Single, int, long, uint, ulong, String, DateTime, short, ushort".
The Temperature type has a property called Value which is a double, and also is implicitly convertible to double, but this doesn't seem to matter. I'm hoping there's a way to bind this so that it converts whenever it requests the value from the results array so it can draw the graph correctly, anybody know how this
 might be done?
I'm hoping to avoid the need to the LogItem[] and then a secondary double[] just for the conversion.
private LogItem[] results;

private async void GraphPage_Load(object sender, EventArgs e) {
    var count = await device.LogCount.Read(CancelToken);
    results = new LogItem[count];
    graph.DataSource = results;
    graph.Series[0].XValueMembers = nameof(LogItem.DateTime); //Type is System.DateTime
    graph.Series[0].YValueMembers = nameof(LogItem.Temperature); //Type my library, Units.Temperature

    var prog = new Progress<double>();
    prog.ProgressChanged += (sender2, e2) => {
        BeginInvoke(new Action(() => {
            progBar.Value = results.Count(log => !Equals(log, LogItem.Empty));
            CheckEmptyPoints(); //Sets or clears Points[x].IsEmpty;
            graph.Invalidate(); //Force graph to draw new results, maybe a better way of doing this?
        }));
    };
    await device.DownloadLogs(CancelToken, results, prog); //Takes output buffer and IProgress to report more data has been downloaded.
}

A: You can shape the result to a chart-friendly shape using linq. For example:
var list = results.Select(x => new { X = x.DateTime, Y = x.Temprature.Value }).ToList();
this.chart1.DataSource = list;
this.chart1.Series[0].XValueMember = "X";
this.chart1.Series[0].YValueMembers = "Y";

X and Y are just place holders and you can use DateTime and Tempratue or any suitable label.

A: Many thanks to Reza Aghaei for the help.
The Linq statements do hold the answer to providing a conversion function, but just with a little tweaking so it's not creating a new array and rebinding with every refresh.
The solution is to use Charting.DataPointsCollection.DataBind(IEnumerable dataSource, string xField, string yField, string otherFields) to bind to the data. But to use Linq statements to create an IEnumerable object to be used in the databind, and on every call of MoveNext, this IEnumerable will fetch the data from the array and convert for the chart.
// IEnumerable<LogItem>.Select(...) creates an IEnumerable which references the array,
//   and performs the selection and conversion on each call to MoveNext().

graph.Series[0].Points.DataBind(
    results.Select(l => new { X = l.DateTime, Y = l.OilCondition.Value }),
    "X",
    "Y",
    string.Empty);

EDIT: The above does work correctly, but according to the documentation on Points.DataBind(,,,), the evaluation is only performed once, so the DataBind(,,,) method does need to be called each time.


text:
Tag: extinction events
Sorry Hollywood, it's Going to Take a Lot More to Destroy an Asteroid
It's become something of an action movie cliche: an asteroid is hurling towards Earth, its impact will cause a mass extinction, and the only hope for humanity is a ragtag group of astronauts and average Joes who will fly to the asteroid and blow it to pieces using nukes. The idea has been explored so many times by Hollywood that it seems like this is actually something space agencies have planned.
And in truth, they are, though the execution may be a little more sophisticated. For decades, space agencies have considered various methods for destroying asteroids that threaten Earth. But according to a new study led by researchers from John Hopkins University, incoming asteroids may be harder to break apart than we thought.
Continue reading "Sorry Hollywood, it's Going to Take a Lot More to Destroy an Asteroid"
A Rapid Rise in Temperature Led to the Worst Extinction in our Planet's History
Everyone knows about the extinction of the dinosaurs. A cataclysmic asteroid strike about 66 million years ago (mya) caused the Death of the Dinosaurs. But there've been several mass extinctions in the Earth's history, and they didn't involve killer asteroids. The worst extinction was caused by a rapid rise in temperature.
Earth's most severe extinction occurred long before the killer asteroid impact that wiped out the dinosaurs. It happened some 252 mya, and it marked the end of what's called the Permian Period. The extinction is known as the Permian-Triassic Extinction Event, the End-Permian Extinction, or more simply, "The Great Dying." Up to 70% of terrestrial vertebrates and up to 96% of all marine species were extinguished during The Great Dying.
How did it happen? Could it happen again?
Continue reading "A Rapid Rise in Temperature Led to the Worst Extinction in our Planet's History"
New Estimate Puts the Supernova Killzone Within 50 Light-Years of Earth
There are a lot of ways that life on Earth could come to an end: an asteroid strike, global climate catastrophe, or nuclear war are among them. But perhaps the most haunting would be death by supernova, because there's absolutely nothing we could do about it. We'd be sitting ducks.
New research suggest that a supernova's kill zone is bigger than we thought; about 25 light years bigger, to be exact.
Iron in the Ocean
In 2016, researchers confirmed that Earth has been hit with the effects from multiple supernovae. The presence of iron 60 in the seabed confirms it. Iron 60 is an isotope of iron produced in supernova explosions, and it was found in fossilized bacteria in sediments on the ocean floor. Those iron 60 remnants suggest that two supernovae exploded near our solar system, one between 6.5 to 8.7 million years ago, and another as recently as 2 million years ago.
Iron 60 is extremely rare here on Earth because it has a short half life of 2.6 million years. Any of the iron 60 created at the time of Earth's formation would have decayed into something else by now. So when researchers found the iron 60 on the ocean floor, they reasoned that it must have another source, and that logical source is a supernova.
This evidence was the smoking gun for the idea that Earth has been struck by supernovae. But the questions it begs are, what effect did that supernova have on life on Earth? And how far away do we have to be from a supernova to be safe?
"…we can look for events in the history of the Earth that might be connected to them (supernova events)." – Dr. Adrian Melott, Astrophysicist, University of Kansas.
In a press release from the University of Kansas, astrophysicist Adrian Melott talked about recent research into supernovae and the effects they can have on Earth. "This research essentially proves that certain events happened in the not-too-distant past," said Melott, a KU professor of physics and astronomy. "They make it clear approximately when they happened and how far away they were. Knowing that, we can consider what the effect may have been with definite numbers. Then we can look for events in the history of the Earth that might be connected to them."
Earlier work suggested that a supernova kill zone is about 25-30 light years. If a supernova exploded that close to Earth, it would trigger a mass extinction. Bye-bye humanity. But new work suggests that 25 light years is an under-estimation, and that a supernova 50 light years away would be powerful enough to cause a mass extinction.
Supernovae: A Force Driving Evolution?
But extinction is just one effect a supernova could have on Earth. Supernovae can have other effects, and they might not all be negative. It's possible that a supernovae about 2.6 million years ago even drove human evolution.
"Our local research group is working on figuring out what the effects were likely to have been," Melott said. "We really don't know. The events weren't close enough to cause a big mass extinction or severe effects, but not so far away that we can ignore them either. We're trying to decide if we should expect to have seen any effects on the ground on the Earth."
Melott and his colleagues have written a new paper that focuses on the effects a supernova might have on Earth. In a new paper titled "A SUPERNOVA AT 50 PC: EFFECTS ON THE EARTH'S ATMOSPHERE AND BIOTA", Melott and a team of researchers tried to shed light on Earth-supernova interactions.
The Local Bubble
There are a number of variables that come into play when trying to determine the effects of a supernova, and one of them is the idea of the Local Bubble. The Local Bubble itself is the result of one or more supernova explosion that occurred as long as 20 million years ago. The Local Bubble is a 300 light year diameter bubble of expanding gas in our arm of the Milky Way galaxy, where our Solar System currently resides. We've been travelling through it for the last five to ten million years. Inside this bubble, the magnetic field is weak and disordered.
Melott's paper focused on the effects that a supernova about 2.6 million years ago would have on Earth in two instances: while both were within the Local Bubble, and while both were outside the Local Bubble.
The disrupted magnetic field inside the Local Bubble can in essence magnify the effects a supernova can have on Earth. It can increase the cosmic rays that reach Earth by a factor of a few hundred. This can increase the ionization in the Earth's troposphere, which mean that life on Earth would be hit with more radiation.
Outside the Local Bubble, the magnetic field is more ordered, so the effect depends on the orientation of the magnetic field. The ordered magnetic field can either aim more radiation at Earth, or it could in a sense deflect it, much like our magnetosphere does now.
Focusing on the Pleistocene
Melott's paper looks into the connection between the supernova and the global cooling that took place during the Pleistocene epoch about 2.6 million years ago. There was no mass extinction at that time, but there was an elevated extinction rate.
According to the paper, it's possible that increased radiation from a supernova could have changed cloud formation, which would help explain a number of things that happened at the beginning of the Pleistocene. There was increased glaciation, increased species extinction, and Africa grew cooler and changed from predominantly forests to semi-arid grasslands.
Cancer and Mutation
As the paper concludes, it is difficult to know exactly what happened to Earth 2.6 million years ago when a supernova exploded in our vicinity. And it's difficult to pinpoint an exact distance at which life on Earth would be in trouble.
But high levels of radiation from a supernova could increase the cancer rate, which could contribute to extinction. It could also increase the mutation rate, another contributor to extinction. At the highest levels modeled in this study, the radiation could even reach one kilometer deep into the ocean.
There is no real record of increased cancer in the fossil record, so this study is hampered in that sense. But overall, it's a fascinating look at the possible interplay between cosmic events and how we and the rest of life on Earth evolved.
A supernova at 50 pc: Effects on the Earth's atmosphere and biota
Recent near-Earth supernovae probed by global deposition of interstellar radioactive 60Fe
The locations of recent supernovae near the Sun from modelling 60Fe transport
Proof that ancient supernovae zapped Earth sparks hunt for after effects
It Looks Like an Asteroid Strike Can't Cause a Worldwide, Dinosaur-Killing Firestorm
For decades, scientists have debated the cause of the mass extinction that wiped out the dinosaurs and other life 65 million years ago. While the majority of researchers agree that a massive asteroid impact at Chicxulub, Mexico is the culprit, there have been some dissenters. Now, new research is questioning just a portion of the asteroid/Cretaceous-Paleogene extinction scenario. While the scientists involved in the study don't doubt that such an asteroid impact actually happened, their research shows it is just not possible that vast global firestorms could have ravaged our planet and be the main cause of the extinction.
Researchers from the University of Exeter, University of Edinburgh and Imperial College London recreated the vast energy released from a 15-km wide asteroid slamming into Earth, which occurred around the time that dinosaurs became extinct.
They found that close to the impact site — a 180 km wide crater in Mexico — the heat pulse would have lasted for less than a minute. This intense but short-lived heat, the team says, could not have ignited live plants, challenging the idea that the impact led to global firestorms.
However, they did find that the effects of the impact would actually be worse on the other side of the planet, where less intense but longer periods of heat could have ignited live plant matter.
"By combining computer simulations of the impact with methods from engineering we have been able to recreate the enormous heat of the impact in the laboratory," said Dr. Claire Belcher from the University of Exeter. "This has shown us that the heat was more likely to severely affect ecosystems a long distance away, such that forests in New Zealand would have had more chance of suffering major wildfires than forests in North America that were close to the impact. This flips our understanding of the effects of the impact on its head and means that palaeontologists may need to look for new clues from fossils found a long way from the impact to better understand the mass extinction event."
The Cretaceous-Paleogene extinction was one of the biggest in Earth's history and geologic evidence of the impact has been discovered in rock layers around the world from this time period. Some critics of the asteroid impact theory as a cause of the extinction have pointed to some of the microfossils from the Gulf of Mexico that show the impact occurred well before the extinction and could not have been its primary cause. Others point to volcanism that produced the Deccan traps of India around this time as a possible cause of the extinction.
But multiple models have showed such an impact would have instantly caused devastating shock waves, tsunamis, and the release of large amounts of dust, debris and gases that would have led to a low light levels and a prolonged cooling of Earth's surface. The darkness and a global winter would have decimated the planet life and the dependent animals.
So while fire and brimstone may not have played a big role in the Cretaceous-Paleogene extinction, there was plenty of destruction and mayhem for the resulting extinction of more than 70% of known species.
Here's a video from the researchers that shows their findings that close to the impact site, the heat pulse was too short to ignite live plant material.
Their research is published in the Journal of the Geological Society.
Source: University Exeter
Posted on January 12, 2015 March 1, 2017
Are Gamma Ray Bursts Dangerous?
Gamma ray bursts are the most energetic explosions in the Universe, outshining the rest of their entire galaxy for a moment. So, it stands to reason you wouldn't want to be close when one of these goes off.
If comics have taught me anything, it's that gamma powered superheroes and villains are some of the most formidable around.
Coincidentally, Gamma Ray bursts, astronomers say, are the most powerful explosions in the Universe. In a split second, a star with many times the mass of our Sun collapses into a black hole, and its outer layers are ejected away from the core. Twin beams blast out of the star. They're so bright we can see them for billions of light-years away. In a split second, a gamma ray burst can release more energy than the Sun will emit in its entire lifetime. It's a super-supernova.
You're thinking "Heck, if the gamma exposure worked for Banner, surely a super-supernova will make me even more powerful than the Hulk." That's not exactly how this plays out.
For any world caught within the death beam from a gamma ray burst, the effects are devastating. One side of the world is blasted with lethal levels of radiation. Our ozone layer would be depleted, or completely stripped away, and any life on that world would experience an extinction level event on the scale of the asteroid that wiped out the dinosaurs.
Astronomers believe that gamma ray bursts might explain some of the mass extinctions that happened on Earth. The most devastating was probably one that occurred 450 million years ago causing the Ordovician–Silurian extinction event. Creatures that lived near the surface of the ocean were hit much harder than deep sea animals, and this evidence matches what would happen from a powerful gamma ray burst event. Considering that, are we in danger from a gamma ray burst and why didn't we get at least one Tyrannosaurus Hulk out of the deal?
This artist's impression of a gamma-ray burst shows the two intense beams of relativistic matter emitted by the black hole. To be visible from Earth, the beams must be pointing directly towards us. (Image: NASA/Swift/Mary Pat Hrybyk-Keith and John Jones)
There's no question gamma ray bursts are terrifying. In fact, astronomers predict that the lethal destruction from a gamma ray burst would stretch for thousands of light years. So if a gamma ray burst went off within about 5000-8000 light years, we'd be in a world of trouble.
Astronomers figure that gamma ray bursts happen about once every few hundred thousand years in a galaxy the size of the Milky Way. And although they can be devastating, you actually need to be pretty close to be affected. It has been calculated that every 5 million years or so, a gamma ray burst goes off close enough to affect life on Earth. In other words, there have been around 1,000 events since the Earth formed 4.6 billion years ago. So the odds of a nearby gamma ray burst aren't zero, but they're low enough that you really don't have to worry about them. Unless you're planning on living about 5 million years in some kind of gamma powered superbody.
We might have evidence of a recent gamma ray burst that struck the Earth around the year 774. Tree rings from that year contain about 20 times the level of carbon-14 than normal. One theory is that a gamma ray burst from a star located within 13,000 light-years of Earth struck the planet 1,200 years ago, generating all that carbon-14.
Clearly humanity survived without incident, but it shows that even if you're halfway across the galaxy, a gamma ray burst can reach out and affect you. So don't worry. The chances of a gamma ray burst hitting Earth are minimal. In fact, astronomers have observed all the nearby gamma ray burst candidates, and none seem to be close enough or oriented to point their death beams at our planet. You'll need to worry about your exercise and diet after all.
So what do you think? What existential crisis makes you most concerned, and how do gamma ray bursts compare?
Podcast (audio): Download (Duration: 4:15 — 3.9MB)
Podcast (video): Download (Duration: 4:38 — 55.2MB)