text:
|Publication number||US4322338 A|
|Application number||US 06/181,450|
|Publication date||Mar 30, 1982|
|Filing date||Aug 25, 1980|
|Priority date||Dec 13, 1979|
|Publication number||06181450, 181450, US 4322338 A, US 4322338A, US-A-4322338, US4322338 A, US4322338A|
|Inventors||Bernard J. Abbott, David S. Fukuda|
|Original Assignee||Eli Lilly And Company|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (12), Non-Patent Citations (13), Referenced by (18), Classifications (16), Legal Events (1)|
|External Links: USPTO, USPTO Assignment, Espacenet|
CH3 (CH2)n CH═CH(CH2)m --
This application is a continuation-in-part of application Ser. No. 103,314, filed Dec. 13, 1979, now abandoned.
This invention relates to novel semi-synthetic antifungal compounds which are prepared by the acylation of the cyclic peptide nucleus produced by the enzymatic deacylation of antibiotic A30912 factor B.
Antibiotic A-30912 factor B is an antifungal cyclic peptide having the formula: ##STR2## wherein R is the linoleoyl group ##STR3## Throughout this application, the cyclic peptide formulas, such as formula I, assume that the amino acids represented are in the L-configuration. The factor is isolated from the A30912 complex which contains other factors arbitrarily designated factors A, C, D, E, F, and G. The A-30912 complex and the individual factors A through G are disclosed by M. Hoehn and K. Michel in U.S. Pat. No. 4,024,245. Antibiotic A-30912 factor A is identical to antibiotic A-22802 which is described by C. Higgins and K. Michel in U.S. Pat. No. 4,024,246. Factor B has also been found to be identical to antibiotic echinocandin C [see R. Traber et al., Helv. Chim. Acta, 62, 1252 (1979)] and to antibiotic SL 7810/F-II [See Belgium Pat. No. 834,289, Derwent Abstract 30159X].
Antibiotic A-30912 factor B is prepared by fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113 (see U.S. Pat. No. 4,024,245); (b) Aspergillus nidulans var. echinulatus A-32204, NRRL 3860, as described in Swiss Pat. No. 568,386; (c) Aspergillus rugulosus NRRL 8039 (see Belgian Pat. No. 834,289); or (d) Aspergillus nidulans var. roseus NRRL 11440 (see co-pending application of L. Boeck and R. Kastner, METHOD OF PRODUCING THE A-30912 ANTIBIOTICS, Ser. No. 126,078, filed March 3, 1980, which is a continuation-in-part of application Ser. No. 46,744, filed June 8, 1979, (now abandoned), the entire disclosure of which is incorporated herein by reference.
A subculture of A. nidulans var. roseus has been deposited and made a part of the permanent culture collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Ill. 61604, from which it is available to the public under the number NRRL 11440.
When a strain of A nidulans var. roseus NRRL 11440 is used to produce A-30912 factor B, a complex of factors is obtained which for convenience is called the A-42355 antibiotic complex. A-30912 factor A is the major factor of the A-42355 antibiotic complex, while factors B, D and H are minor factors. Examples 5, 6, and 7 herein, illustrate the preparation of the A-42355 complex and the isolation and purification of A-30912 factor B therefrom. A-30912 factor H is further described in a co-pending application of Karl H. Michel entitled ANTIBIOTIC A-30912 FACTOR H, Ser. No. 117,739, filed Feb. 1, 1980, which is a continuation-in-part of application Serial No. 46,875, filed June 8, 1979 (now abandoned).
In the antibiotic A-30912 factor B molecule (Formula I), the linoleoyl side chain (R) is attached at the cyclic peptide nucleus at the α-amino group of the dihydroxyornithine residue. Surprisingly, it has been found that the linoleoyl side chain can be cleaved from the nucleus by an enzyme without affecting the chemical intregity of the nucleus. The enzyme employed to effect the deacylation reaction is produced by a microorganism of the family Actinoplanaceae, preferably the microorganism Actinoplanes utahensis NRRL 12052, or a variant thereof. To accomplish deacylation, antibiotic A30912 factor B is added to a culture of the microorganism and the culture is allowed to incubate with the substrate until the deacylation is subtantially complete. The cyclic nucleus thereby obtained is separated from the fermentation broth by methods known in the art. Unlike antibiotic A-30912 factor B, the cyclic nucleus (lacking the linoleoyl side chain) is substantially devoid of antifungal activity.
The cyclic nucleus afforded by the aforedescribed enzymatic deacylation of antibiotic A-30912 factor B, is depicted in Formula II. ##STR4##
A-30912B nucleus has an empirical formula of C34 H51 N7 O14 and a molecular weight of 781.81.
Removal of the side chain group affords a free primary α-amino group in the dihydroxyornithine residue of the cyclic peptide. For convenience, the compound having the structure given in Formula II will be referred to herein as "A-30912B nucleus." As will be apparent to those skilled in the art, A-30912B nucleus can be obtained either in the form of the free amine or of the acid addition salt. Although any suitable acid addition salt may be employed, those which are non-toxic and pharmaceutically acceptable are preferred.
The method of preparing A-30912B nucleus from antibiotic A-30912 factor B by means of fermentation using Actinoplanes utahensis NRRL 12052 is described in the co-pending application of Bernard J. Abbott and David S. Fukuda, entitled "A-30912B NUCLEUS", Ser. No. 103,121, was filed Dec. 13, 1979. A continuation-in-part application of this application, with the corresponding Ser. No. 181,437 is being filed herewith this even date, the full disclosure of which is incorporated herein by reference. Example 3 herein, illustrates the preparation of A-30912B nucleus by fermentation using antibiotic A-30912 factor B as the substrate and Actinoplanes utahensis NRRL 12052 as the microorganism.
The enzyme produced by Actinoplanes utahensis NRRL 12052 may be the same enzyme which has been used to deacylate penicillins (see Walter J. Kleinschmidt, Walter E. Wright, Frederick W. Kavanagh, and William M. Stark, U.S. Pat. No. 3,150,059, issued Sept. 22, 1964).
Cultures of representative species of Actinoplanaceae are available to the public from the Northern Regional Research Laboratory under the following accession numbers:
______________________________________Actinoplanes utahensis NRRL 12052Actinoplanes missouriensis NRRL 12053Actinoplanes sp. NRRL 8122Actinoplanes sp. NRRL 12065Streptosporangium roseumvar. hollandensis NRRL 12064______________________________________
The effectiveness of any given strain of microorganism within the family Actinoplanaceae for carrying out the deacylation of this invention is determined by the following procedure. A suitable growth medium is inoculated with the microorganism. The culture is incubated at about 28° C. for two or three days on a rotary shaker. One of the substrate antibiotics is then added to the culture. The pH of the fermentation medium is maintained at about pH 6.5. The culture is monitored for activity using a Candida albicans assay. Loss of antibiotic activity is an indication that the microorganism produces the requisite enzyme for deacylation. This must be verified, however, using one of the following methods: (1) analysis by HPLC for presence of the intact nucleus; or (2) reacylation with an appropriate side chain (e.g. linoleoyl, stearoyl, or palmitoyl) to restore activity.
It is known that another antibiotic substance possesses the same nucleus as that of antibiotic A-30912 factor B. This substance, which differs from antibiotic A-30912 factor B in that a different acyl group is present in place of the linoleoyl group (R) in Formula I, is tetrahydro-A-30912 factor B (tetrahydro-SL 7810/F-II; tetrahydro echinocandin C) described by R. Traber et al., Helv. Chim. Acta, 62 1252 (1979). Tetrahydro-A-30912 factor B is depicted in Formula I when R is stearoyl. Tetrahydro-A-30912 factor B can be prepared from antibiotic A-30912 factor B by catalytic hydrogenation using PtO2 in ethanol under positive pressure. Tetrahydro-A-30912 factor B can be employed as a substrate in place of antibiotic A-30912 factor B for the enzymatic deacylation using the procedures herein described.
The invention sought to be patented comprehends novel compounds derived by acylating A-30912B nucleus (Formula II). The compounds of the present invention have the chemical structure depicted in Formula III: ##STR5## wherein R1 is C6 -C24 alkyl or C6 -C24 alkenyl; provided that when R1 is alkyl, R1 cannot be n-heptadecyl; and, when R1 is alkenyl, R1 cannot be cis, cis--CH3 (CH2)4 CH═CHCH2 --CH═CH(CH2)7)--.
By the term "alkyl" is meant a univalent saturated, straight-chain or branched-chain hydrocarbon radical. By the term "alkenyl" is meant a univalent, unsaturated, straight-chain or branched-chain hydrocarbon radical containing not more than three double bonds. The double bonds of the unsaturated hydrocarbon chain may be either in the cis or trans configuration. By "C6 -C24 " is meant a hydrocarbon (including straight and branched chains) containing from six to 24 carbon atoms.
In subgeneric aspects, the invention contemplates the following preferred embodiments of the compounds of Formula III:
(a) The compounds wherein R1 is alkyl of the formula CH3 (CH2)n --, wherein n is an integer from 5 to 23, provided that n cannot be 16.
(b) The compounds wherein R1 is alkyl of the formula CH3 (CH2)n --, wherein n is 10, 11, 12, 13, 14, 15, 17, 18, 19, or 20.
(c) The compounds wherein R1 is alkyl of the formula ##STR6## wherein n and m are each, independently, an integer from 0 to 21 provided that n+m must be no less than 3 and no greater than 21.
(d) The compounds wherein R1 is alkenyl containing one cis or trans double bond.
(e) The compounds wherein R1 is cis or trans alkenyl of the formula
CH3 (CH2)n CH═CH(CH2)m --
wherein n and m are each, independently, an integer from 0 to 21, provided that n+m must be no less than 3 and no greater than 21.
(f) The compounds wherein R1 is alkenyl containing two cis or trans double bonds.
(g) The compounds wherein R1 is cis or trans alkenyl of the formula
CH3 (CH2)n CH═CH(CH2)m CH═CH(CH2)p --
wherein n and p are each, independently, an integer of from 0 to 18 and m is an integer from 1 to 19, provided that m+n+p must be no less than 1 and no greater than 19 and that R1 cannot be linoleoyl.
(h) The compounds wherein R1 is:
cis--CH3 (CH2)5 CH═CH(CH2)7 --
trans--CH3 (CH2)5 CH═CH(CH2)7 --
cis-CH3 (CH2)10 CH═CH(CH2)4 --
trans--CH3 (CH2)10 CH═CH(CH2)4 --
cis--CH3 (CH2)7 CH═CH(CH2)7 --
trans--CH3 (CH2)7 CH═CH(CH2)7 --
cis--CH3 (CH2)5 CH═CH(CH2)9 --
trans--CH3 (CH2)5 CH═CH(CH2)9 --
cis--CH3 (CH2)7 CH═CH(CH2)9 --
trans--CH3 (CH2)7 CH═CH(CH2)9 --
cis--CH3 (CH2)7 CH═CH(CH2)11 --
trans--CH3 (CH2)7 CH═CH(CH2)11 --
trans,trans--CH3 (CH2)4 CH═CHCH2 CH═CH(CH2)7 --
cis,cis,cis--CH3 CH2 CH═CHCH2 CH═CHCH2 CH═CH--(CH2)7 --.
The compounds of Formula III inhibit the growth of pathogenic fungi, and are useful, therefore, for controlling the growth of fungi on environmental surfaces (as an antiseptic) or in treating infections caused by fungi. In particular, the compounds are active against Candida albicans and are, thus, especially useful for treating candidosis. The activity of the compounds can be assessed in standard microbiological test procedures, such as in vitro in agar plate disc diffusion tests or in agar dilution tests, or in vivo in tests in mice infected with C. albicans. The compounds are also active against Trichophyton mentagrophytes (a dermatophytic organism), Saccharomyces pastorianus, and Neurospora crassa.
The compounds of Formula III are prepared by acylating A-30912B nucleus at the α-amino group of dihydroxyornithine with the appropriate alkanoyl or alkenoyl side chain using methods conventional in the art for forming an amide bond. The acylation is accomplished, in general, by reacting the nucleus with an activated derivative of the alkanoic acid or alkenoic acid (R1 CO2 H) corresponding to the desired acyl side chain group (R1 CO--). By the term "activated derivative" is meant a derivative which renders the carboxyl function of the acylating agent reactive to coupling with the primary amino group to form the amide bond which links the acyl side chain to the nucleus. Suitable activated derivatives, their methods of preparation, and their methods of use as acylating agents for a primary amine will be recognized by those skilled in the art. Preferred activated derivatives are: (a) an acid halide (e.g. acid chloride), (b) an acid anhydride (e.g. a alkoxyformic acid anhydride or aryloxyformic acid anhydride) or (c) an activated ester (e.g. a 2,4,5-trichlorophenyl ester). Other methods for activating the carboxyl function include reaction of the carboxylic acid with a carbonyldiimide (e.g. N,N-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide) to give a reactive intermediate which, because of instability, is not isolated, the reaction with the primary amine being carried out in situ.
A preferred method for preparing the compounds of Formula III is by the active ester method. The use of the 2,4,5-trichlorophenyl ester of the desired alkanoic or alkenoic acid as the acylating agent is most preferred. In this method, an excess amount of the active ester is reacted with the nucleus at room temperature in a non-reactive organic solvent such as dimethyl formamide (DMF). The reaction time is not critical, although a time of about 6 to about 20 hours is preferred. At the conclusion of the reaction, the solvent is removed, and the residue is purified such as by reversed phase HPLC using LP-1/C18 as the stationary/phase and a mixture of H2 O/CH3 OH/CH3 CN as the solvent system.
An alternative acylation method is a modified Schotten-Baumann procedure in which the nucleus is treated with the acid chloride of the desired alkanoic acid or alkenoic acid at an alkaline pH. In this method, an excess of the acid chloride in a non-reactive organic solvent (such as acetone) is added slowly to a solution of the nucleus in [KHPO4 ]-buffer (pH 7.5 to 8.0) and acetone. The crude reaction product is separated from the reaction product by extraction into an immiscible organic solvent (chloroform and ethyl acetate). Final purification is by reversed-phase HPLC, as described above.
The alkanoic and alkenoic acids employed as starting materials for the acylation reaction, and the activated derivatives thereof (in particular, the acid chlorides and the 2,4,6-trichlorophenyl esters), are known compounds or can be prepared from known compounds by known methods. The 2,4,5-trichlorophenyl esters are conveniently made by treating the acid chloride of the alkanoic or alkenoic acid with 2,4,5-trichlorophenol in the presence of pyridine or by treating the free alkanoic or alkenoic acid with 2,4,5-trichlorophenol in the presence of N,N'dicyclohexylcarbodiimide employed as a coupling agent. The 2,4,5-trichlorophenyl ester derivative can be purified by column chromatography over silica gel in toluene.
When employed systemically, the dosage of the compounds of Formula III will vary according to the particular compound being employed, the severity and nature of the infection, and the physical condition of the subject being treated. Therapy should be initiated at low dosages, the dosage being increased until the desired antifungal effect is obtained. The compounds can be administered intravenously or intramuscularly by injection in the form of a sterile aqueous solution or suspension to which may be added, if desired, various conventional pharmaceutically acceptable preserving, buffering, solubilizing, or suspending agents. Other additives, such as saline or glucose may be added to make the solutions isotonic. The proportions and nature of such additives will be apparent to those skilled in the art.
When employed to treat vaginal candida infections, the compounds of Formula III can be administered in combination with pharmaceutically acceptable conventional excipients suitable for intravaginal use. Formulations adapted for intravaginal administration will be known to those skilled in the art.
The methods of making and using the compounds of the present invention are illustrated in the following examples:
The following procedure, which gives the preparation of the compound of Formula III wherein R1 is CH3 (CH2)11 --, illustrates the method of preparation of the compounds of Formula III by the "active ester" method.
A solution of n-tridecanoic acid (Sigma Chemical Co.) (12.5 g.), 2,4,5-trichlorophenol (11.5 g.), and N,N-dicyclohexylcarbodiimide (12.0 g.) in methylene chloride (650 ml.) is stirred at room temperature for 16 hours. The reaction mixture is then filtered and dried in vacuo to give 2,4,5-trichlorophenyl n-tridecanoate (22 g.). The material is purified by column chromatography over silica gel (Woelm) using toluene as the eluent. Fractions are monitored by TLC using a shortwave UV light for detection. Fractions containing the purified product are pooled and concentrated in vacuo to dryness.
A solution of 2,4,5-trichlorophenyl n-tridecanoate (3.3 mmoles) and A-30912B nucleus (1 mmole) in dimethylformamide (DMF) (200 ml.) is stirred at room temperature for 16 hours. Removal of solvent in vacuo affords a residue. The residue is slurried with methylene chloride (300 ml.) for 45 minutes, and the mixture is filtered. The filtrate is discarded. The remaining solids are extracted with methanol (300 ml.) and the methanol extract is filtered and concentrated in vacuo to give a crude product.
The crude product is purified by reversed-phase HPLC as follows:
A sample of the crude product (1 g.), dissolved in methanol (5 ml.), is injected into a 1×32 in. stainless steel column packed with LP-1/C18 resin (see Example 8). The column is eluted with a solvent system comprising 3:3:4 H2 O/CH3 OH/CH3 CN. The elution is performed at a pressure of 1000-1500 psi with a flow rate of 11-12 ml./min using a LDC duplex pump (Milton-Roy). The effuent is monitered by an ultraviolet detector (ISCO-UA-5) at 280 nm. Fractions are collected every two minutes (21-24 ml.). The fractions containing the desired product are pooled and dried in vacuo to afford the title product. The purified product is analyzed by thin layer chromatography (TLC) using reversed-phase C18 plates (Whatman KC18) and a solvent system comprising 1:2:2 (v/v) H2 O/CH3 OH/CH3 CN. After development, the plates are observed under U.V. light to detect the product.
Employing the method of Example 1, but substituting the appropriate alkanoic acid or alkenoic acid in Step A and the appropriate alkanoic or alkenoic acid 2,4,5-trichlorophenyl ester in Step B, there are obtained the derivatives of A-30912B nucleus shown below:
______________________________________Alkanoyl and Alkenoyl Derivatives of A-30912B Nucleus ##STR7##R1______________________________________CH3 (CH2)10CH3 (CH2)12CH3 (CH2)13CH3 (CH2)14CH3 (CH2)15CH3 (CH2)17CH3 (CH2)18CH3 (CH2)19CH3 (CH2)20cis-CH3 (CH2)5 CHCH(CH2)7trans-CH3 (CH2)5CHCH(CH2)7cis-CH3 (CH2)10 CHCH(CH2)4trans-CH3 (CH2)10 CHCH(CH2)4cis-CH3 (CH2)7 CHCH(CH2)7trans-CH3 (CH2)7 CHCH(CH2)7cis-CH3 (CH2)5 CHCH(CH2)9trans-CH3 (CH2)5 CHCH(CH2)9cis-CH3 (CH2)7 CHCH(CH2)9trans-CH3 (CH2)7 CHCH(CH2)9cis-CH3 (CH2)7 CHCH(CH2)11trans-CH3 (CH2)7 CHCH(CH2)11trans,trans-CH3 (CH2)4 CHCHCH2 CHCH(CH2)7cis,cis,cis-CH3 CH2 CHCHCH2 CHCHCH2 CHCH(CH2).sub.7______________________________________
A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained on an agar slant. The medium used to prepare the slant is selected from one of the following:
______________________________________MEDIUM AIngredient Amount______________________________________Baby oatmeal 60.0 gYeast 2.5 gK2 HPO4 1.0 gCzapek's mineral stock* 5.0 mlAgar 25.0 gDeionized water q.s. to 1 literpH before autoclaving is about 5.9; adjust to pH 7.2by addition of NaOH; after autoclaving, pH is about6.7.______________________________________ *Czapek's mineral stock has the following composition:
FeSO4 . 7H2 O (dissolved in2 ml conc HCl) 2 gKCl 100 gMgSO4 . 7H2 O 100 gDeionized water q.s. to 1 liter______________________________________
______________________________________MEDIUM BIngredient Amount______________________________________Potato dextrin 5.0 gYeast extract 0.5 gEnzymatic hydrolysate of casein* 3.0 gBeef extract 0.5 gDextrose 12.5 gCorn starch 5.0 gMeat peptone 5.0 gBlackstrap molasses 2.5 gMgSO4 . 7H2 O 0.25 gCaCO3 1.0 gCzapek's mineral stock 2.0 mlAgar 20.0 gDeionized water q.s. to 1 liter______________________________________ *N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.
The slant is inoculated with Actinoplanes utahensis NRRL 12052, and the inoculated slant is incubated at 30° C. for about 8 to 10 days. About 1/2 of the slant growth is used to inoculate 50 ml of a vegetative medium having the following composition:
______________________________________Ingredient Amount______________________________________Baby oatmeal 20.0 gSucrose 20.0 gYeast 2.5 gDistiller's Dried Grain* 5.0 gK2 HPO4 1.0 gCzapek's mineral stock 5.0 mlDeionized water q.s. to 1 literadjust to pH 7.4 with NaOH; after autoclaving, pH isabout 6.8.______________________________________ *National Distillers Products Co., 99 Park Ave., New York, N.Y.
The inoculated vegetative medium is incubated in a 250-ml wide-mouth Erlenmeyer flask at 30° C. for about 72 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
This incubated vegetative medium may be used directly to inoculate a second-stage vegetative medium. Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution [see W. A. Dailey and C. E. Higgens, "Preservation and Storage of Microorganisms in the Gas Phase of Liquid Nitrogen," Cryobiol 10, 364-367 (1973) for details]. The prepared suspensions are stored in the vapor phase of liquid nitrogen.
A stored suspension (1 ml) thus prepared is used to inoculate 50 ml of a first-stage vegetative medium (having the composition earlier described). The inoculated first-stage vegetative medium is incubated as above-described.
In order to provide a larger volume of inoculum, 10 ml of the incubated first-stage vegetative medium is used to inoculate 400 ml of a second-stage vegetative medium having the same composition as the first-stage vegetative medium. The second-stage medium is incubated in a two-liter wide-mouth Erlenmeyer flask at 30° C. for about 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
Incubated second-stage vegetative medium (800 ml), prepared as above-described, is used to inoculate 100 liters of sterile production medium selected from one of the following:
______________________________________MEDIUM IIngredient Amount (g/L)______________________________________Peanut meal 10.0Soluble meat peptone 5.0Sucrose 20.0KH2 PO4 0.5K2 HPO4 1.2MgSO4 . 7H2 O 0.25Tap water q.s. to 1 liter______________________________________
The pH of the medium is about 6.9 after sterilization by autoclaving at 121° C. for 45 minutes at about 16-18 psi.
______________________________________MEDIUM IIIngredient Amount (g/L)______________________________________Sucrose 30.0Peptone 5.0K2 HPO4 1.0KCl 0.5MgSO4 . 7H2 O 0.5FeSO4 . 7H2 O 0.002Deionized water q.s. to 1 liter______________________________________
Adjust to pH 7.0 with HCl; after autoclaving, pH is about 7.0.
______________________________________MEDIUM IIIIngredient Amount (g/L)______________________________________Glucose 20.0NH4 Cl 3.0Na2 SO4 2.0ZnCl2 0.019MgCl2 . 6H2 O 0.304FeCl3 . 6H2 O 0.062MnCl2 . 4H2 O 0.035CuCl2 . 2H2 O 0.005CaCO3 6.0KH2 PO4 * 0.67Tap water q.s. to 1 liter______________________________________ *Sterilized separately and added aseptically Final pH about 6.6.
The inoculated production medium is allowed to ferment in a 165-liter fermentation tank at a temperature of about 30° C. for about 42 hours. The fermentation medium is stirred with conventional agitators at about 200 RPM and aerated with sterile air to maintain the dissolved oxygen level above 30% of air saturation at atmospheric pressure.
A fermentation of A. utahensis is carried out as described in Sect. A, using production medium I. After the culture is incubated for about 48 hours, A-30912 factor B, dissolved in a small amount of methanol, is added to the fermentation medium.
Deacylation of A-30912 factor B is monitored by paper-disc assay against Candida albicans or Neurospora crassa. The fermentation is allowed to continue until deacylation is complete as indicated by disappearance of activity.
Whole fermentation broth, obtained as described in Sect. B is filtered. The mycelial cake is discarded. The clear filtrate thus obtained is passed through a column containing HP-20 resin (DIAION High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is discarded. The column is then washed with up to eight column volumes of deionized water at pH 6.5-7.5 to remove residual filtered broth. This wash water is discarded. The column is then eluted with a water:methanol (7:3) solution. Elution is monitored using the following procedure: Two aliquots are taken from each eluted fraction. One of the aliquots is concentrated to a small volume and is treated with an acid chloride such as myristoyl chloride. This product and the other (untreated) aliquot are assayed for activity against Candida albicans. If the untreated aliquot does not have activity and the acylated aliquot does have activity, the fraction contains A-30912B nucleus. The eluate containing A-30912B nucleus is concentrated under vacuum to a small volume and lyophilized to give crude nucleus.
Crude A-30912B nucleus, obtained as described in Section C, is dissolved in water:acetonitrile:acetic acid:pyridine (96:2:1:1). This solution is chromatographed on a column filled with Lichroprep RP-18, particle size 25-40 microns (MC/B Manufacturing Chemists, Inc. E/M, Cincinnati, Ohio). The column is part of a Chromatospac Prep 100 unit (Jobin Yvon, 16-18 Rue du Canal 91160 Longjumeau, France). The column is operated at a pressure of 90-100 psi, giving a flow rate of about 60 ml/minute, using the same solvent. Separation is monitored at 280 nm using a UV monitor (ISCO Absorption Monitor Model UA-5, Instrumentation Specialties Co., 4700 Superior Ave., Lincoln, Nebr. 68504) with an optical unit (ISCO Type 6).
On the basis of absorption at 280 nm, fractions containing A-30912B nucleus are combined, evaporated under vacuum and lyophilized to give purified A-30912B nucleus.
A-30912B nucleus is prepared and purified by the method of Example 3 except that tetrahydro-A-30912B is used as the substrate.
A. Shake-Flask Fermentation
A culture of Aspergillus nidulans var. roseus NRRL 11440 is prepared and maintained on an agar slant prepared with medium having the following composition:
______________________________________Ingredient Amount______________________________________Glucose 5 gYeast extract 2 gCaCO3 3 gVegetable juice* 200 mlAgar** 20 gDeionized water q.s. to 1 liter(initial pH 6.1)______________________________________ *V-8 Juice, Campbell Soup Co., Camden, N.J. **Meer Corp.
The slant is inoculated with Aspergillus nidulans var. roseus NRRL 11440, and the inoculated slant is incubated at 25° C. for about seven days. The mature slant culture is covered with water and scraped with a sterile loop to loosen the spores. The resulting suspension is further suspended in 10 ml of sterile deionized water.
One ml of the suspended slant growth is used to inoculate 55 ml of vegetative medium in a 250-ml flask. The vegetative medium has the following composition:
______________________________________Ingredient Amount______________________________________Sucrose 25 gBlackstrap molasses 36 gCorn-steep liquor 6 gMalt extract 10 gK2 HPO4 2 gEnzymatic hydrolysateof casein* 10 gTap water 1100 ml(initial pH 6.5-6.7)______________________________________ *N-Z-Case, Humko Sheffield Chemical, Lyndhurst, N.J.
The inoculated vegetative medium is incubated at 25° C. for 48 hours at 250 rpm on a rotary-type shaker. After 24 hours, the medium is homogenized for one minute at low speed in a blender (Waring type) and then returned to incubation for the remaining 24 hours. Alternatively, the inoculated vegetative medium can be incubated for 48 hours and then homogenized for 15 seconds at low speed.
This incubated vegetative medium may be used to inoculate shake-flask fermentation culture medium or to inoculate a second-stage vegetative medium. Alternatively, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: The vegetative cultures are mixed volume/volume with a suspending solution having the following composition:
______________________________________Ingredient Amount______________________________________Glycerol 20 mlLactose 10 gDeionized water q.s. to 100 ml______________________________________
The prepared suspensions are distributed in small sterile screw-cap tubes (4 ml per tube). These tubes are stored in the vapor phase of liquid nitrogen.
A stored suspension thus prepared can be used to inoculate either agar slants or liquid seed media. Slants are incubated at 25° C. in the light for 7 days.
B. Tank Fermentation
In order to provide a larger volume of inoculum, 10 ml of incubated first-stage vegetative culture is used to inoculate 400 ml of a second-stage vegetative growth medium having the same composition as that of the vegetative medium. The second-stage medium is incubated in a two-liter wide-mouth Erlenmeyer flask at 25° C. for 24 hours on a shaker rotating through an arc two inches in diameter at 250 rpm.
Incubated second-stage medium (800 ml), prepared as above described, is used to inoculate 100 liters of sterile production medium selected from one of the following:
______________________________________MEDIUM IVIngredient Amount______________________________________ZnSO4 . 7H2 O 0.00455 g/LSoluble meat peptone* 30.5 g/LSoybean meal 15.5 g/LTapioca dextrin** 2.0 g/LBlackstrap molasses 10.5 g/LEnzymatic hydrolysateof casein*** 8.5 g/LNa2 HPO4 4.5 g/LMgSO4 . 7H2 O 5.5 g/LFeSO4 . 7H2 O 0.1 g/LCottonseed oil 40.0 ml(Antifoam)**** 1.0 mlTap water 1000.0 ml(initial pH 6.8-7.0)______________________________________ *O.M. Peptone, Amber Laboratories, Juneau, Wisc. **Stadex 11, A.E. Staley Co., Decatur, Ill. ***N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J. ****P2000, Dow Corning, Midland, Michigan
______________________________________MEDIUM VIngredient Amount______________________________________Glucose 2.5%Starch 1.0%Soluble meat peptone* 1.0%Blackstrap molasses 1.0%CaCO3 0.2%MgSO4 . 7H2 O 0.05%Enzymatic hydrolysate ofcasein** 0.4%(Antifoam)*** 0.02%Tap water q.s. to volume______________________________________ *O.M. Peptone **N-Z-Amine A ***Antifoam "A", Dow Corning
The inoculated production medium is allowed to ferment in a 165-liter fermentation tank at a temperature of 25° C. for about 7 days. The fermentation medium is aerated with sterile air, maintaining the dissolved oxygen level above approximately 50 percent of air saturation.
C. Third-Stage Vegetative Medium
Whenever the fermentation is carried out in tanks larger than those used for 100-liter fermentation, it is recommended that a third-stage vegetative culture be used to seed the larger tank. A preferred third-stage vegetative medium has the following composition:
______________________________________Ingredient Amount______________________________________Sucrose 25 gBlackstrap molasses 25 gCorn-steep liquor 6 gEnzymatic hydrolysateof casein* 10 gMalt extract 10 gK2 HPO4 2 gTap water 1000 ml(initial pH 6.1)______________________________________ *N-Z-Case
Whole fermentation broth (4127 liters), obtained by the method described in Example 5 using production medium V, is stirred thoroughly with methanol (4280 liters) for one hour and then is filtered, using a filter aid (Hyflo Super-cel, a diatomaceous earth, Johns-Manville Products Corp.). The pH of the filtrate is adjusted to pH 4.0 by the addition of 5 N HCl. The acidified filtrate is extracted twice with equal volumes of chloroform. The chloroform extracts are combined and concentrated under vacuum to a volume of about 20 liters. This concentrate is added to about 200 liters of diethyl ether to precipitate the A-42355 complex. The precipitate is separated by filtration to give 2775 g of the A-42355 complex as a gray-white powder.
A-42355 complex is separated as described in Example 6 except that the concentrated chloroform extracts (285 L) are chromatographed over a silica-gel column (150 L of Grace silica-gel, grade 62) at a flow rate of 2 L/min. The column is washed with chloroform (200 L), eluted with acetonitrile (500 L), and then continuously eluted with acetonitrile:water (98:2) at a flow rate of 1 L/min. Fractions having a volume of approximately 200 L are collected and analyzed individually for biological activity. The bioassay is performed by a paper-disc assay on agar plates seeded with Candida albicans. Fractions 77 through 103 (1365 L) are combined and concentrated under vacuum. The concentrated solution (4.5 L) contains a precipitate which is removed by filtration to give 119 g of factor B-enriched A-42355 complex. The filtrate is concentrated to dryness; the residue obtained is redissolved in an appropriate volume of methanol. The methanol solution is added to diethyl ether (10 volumes) to precipitate the factor-B-containing antibiotic complex. This precipitate is also separated by filtration and dried to give an additional 24 g of factor-B-enriched A-42355 complex as a gray powder.
Factor-B-enriched A-42355 complex thus obtained (1.0 g) is dissolved in 8 ml of methanol:water:acetonitrile (7:2:1). This solution is filtered and introduced onto a silica-gel column (3.7-cm I.D.×33-cm Michel-Miller Column) through a loop with the aid of a valve system. The column is packed with LP-1/C18 silica-gel reversed-phase resin (10-20 microns), prepared as described in Example 8, in methanol:water:acetonitrile (7:2:1) through a loop with the aid of a valve system. The slurry packing procedure described in Example 9 is used. The solvent is moved through the column at a flow rate of 10 ml/min at ca. 100 psi, using an F.M.I. pump with valveless piston design. One fraction is collected every minute. Elution of the antibiotic is monitored using a UV monitor at 280 nm as in Example 3. Fractions 102-110 are combined and concentrated under vacuum to give an oil. The oil is dissolved in a small volume of tert-butanol and lyophilized to give 22 mg of A-30912 factor B.
The individual A-30912 factors can be identified by the use of thin-layer chromatography (TLC). The Rf values of A-30912 factors A-G, using silica gel (Merck, Darmstadt) TLC, a benzene:methanol (7:3) solvent system, and Candida albicans bioautography are given in Table I.
TABLE I______________________________________A-30912 Factor Rf Value______________________________________A 0.35B 0.45C 0.54D 0.59E 0.27F 0.18G 0.13______________________________________
The approximate Rf values of A-30912 factors A, B, C, D, and H in different solvent systems, using silica gel TLC (Merck-Darmstadt silica gel #60 plates, 20×20 cm) and Candida albicans bioautography, are given in Table II.
TABLE II______________________________________ Rf Values - Solvent SystemsA-30912 Factor a b c d______________________________________Factor A 0.28 0.14 0.28 0.43Factor B 0.39 0.21 0.42 0.47Factor C 0.46 0.31 0.51 0.58Factor D 0.50 0.38 0.57 0.61Factor H 0.42 0.27 0.36 0.53______________________________________ Solvent Systems a: ethyl acetate:methanol (3:2) b: ethyl acetate:methanol (7:3) c: acetonitrile:water (95:5) d: ethyl acetate:ethanol:acetic acid (40:60:0.25)
A-30912 factors A, B, D and H can also be indentified by analytical HPLPLC using the following conditions:
______________________________________Column: glass, 0.8 × 15.0 cmPacking: NucleosilŽ 10-C18 (Machery- Nagel and Company); packed using slurry-packing pro- cedure of Example 8Solvent: methanol:water:aceto- nitrile (7:2:1)Sample Volume: 8 mclSample Size: 8 mcgColumn Temperature: ambientFlow Rate: 1.8 ml/minPressure: ca. 200 psiDetector: UV at 222 nm (ISCO Model 1800 Variable Wavelength UV-Visible Absorbance Monitor)Pump: LDC Duplex MinipumpInjection: loop injection______________________________________
The approximate retention times for A-30912 factors A, B, D, and H under these conditions are summarized in Table III.
TABLE III______________________________________ Retention TimeA-30912 Factor (seconds)______________________________________A 792B 870H 990D 1,140______________________________________
Step 1: Hydrolysis
LP-1 silica gel (1000 g from Quantum Corp., now Whatman) is added to a mixture of concentrated sulfuric acid (1650 ml) and concentrated nitric acid (1650 ml) in a 5-L round-bottom flask and shaken for proper suspension. The mixture is heated on a steam bath overnight (16 hours) with a water-jacketed condenser attached to the flask.
The mixture is cooled in an ice bath and carefully filtered using a sintered-glass funnel. The silica gel is washed with deionized water until the pH is neutral. The silica gel is then washed with acetone (4 L) and dried under vacuum at 100° C. for 2 days.
Step 2: First Silylation
The dry silica gel from Step 1 is transferred to a round-bottom flask and suspended in toluene (3.5 L). The flask is heated on a steam bath for 2 hours to azeotrope off some residual water. Octadecyltrichlorosilane (321 ml, Aldrich Chemical Company) is added, and the reaction mixture is refluxed overnight (16 hours) with slow mechanical stirring at about 60° C. Care is taken so that the stirrer does not reach near the bottom of the flask. This is to prevent grinding the silica gel particles.
The mixture is allowed to cool. The silanized silica gel is collected, washed with toluene (3 L) and acetone (3 L), and then air-dried overnight (16-20 hours). The dried silica gel is suspended in 3.5 L of acetonitrile:water (1:1) in a 5-L flask, stirred carefully at room temperature for 2 hours, filtered, washed with acetone (3 L) and air-dried overnight.
Step 3: Second Silylation
The procedure from the first silylation is repeated using 200 ml of octadecyltrichlorosilane. The suspension is refluxed at 60° C. for 2 hours while stirring carefully. The final product is recovered by filtration, washed with toluene (3 L) and methanol (6 L), and then dried under vacuum at 50° C. overnight (16-20 hours).
This procedure is employed for packing a silica gel C18 reversed phase resin such as that prepared by the method of Example 8.
Generally, a pressure of less than 200 psi and flow rates between 5-40 ml/minute are required for this slurry packing technique; this is dependent on column volume and size. Packing pressure should exceed the pressure used during actual separation by 30-50 psi; this will assure no further compression of the adsorbent during separation runs.
A sudden decrease in pressure may cause cracks or channels to form in the packing material, which would greatly reduce column efficiency. Therefore, it is important to let the pressure drop slowly to zero whenever the pump is turned off.
The approximate volume of columns (Ace Glass Cat. No., unpacked) are No. 5795-04, 12 ml; No. 5795-10, 110 ml; No. 5795-16, 300 ml; No. 5795-24, 635 ml; and No. 5796-34, 34 ml.
The time required to pack a glass column will vary from minutes to several hours depending on column size and the experience of the scientist.
1. Connect glass column to a reservoir column via coupling (volume of reservoir column should be twice that of the column). Place both columns in vertical positions (reservoir column above).
2. Weigh out packing material (ca. 100 g for 200 ml column).
3. Add ca. five volumes of solvent to packing material; use a mixture of 70-80% methanol and 20-30% water.
4. Shake well until all particles are wetted, let stand overnight or longer to assure complete soaking of particles by solvent. Decant supernatant liquid.
5. Slurry the resin with sufficient solvent to fill reservoir column. Pour swiftly into reservoir. The column must be pre-filled with the same solvent and the reservoir column should be partly filled with solvent before slurry is poured. The use of larger slurry volumes may also provide good results; however, this will require (a) larger reservoir or (b) multiple reservoir fillings during the packing procedure.
6. Close reservoir with the Teflon plug beneath the column (see FIG. 1 of U.S. Pat. No. 4,131,547, plug No. 3); connect to pump; and immediately start pumping solvent through system at maximum flow rate if Ace Cat. No. 13265-25 Pump or similar solvent-delivery system is used (ca. 20 ml/minute).
7. Continue until column is completely filled with adsorbent. Pressure should not exceed maximum tolerance of column during this operation (ca. 200 psi for large columns and 300 psi for analytical columns). In most cases, pressures less than 200 psi will be sufficient.
8. Should pressure exceed maximum values, reduce flowrate; pressure will drop.
9. After column has been filled with adsorbent, turn off pump; let pressure drop to zero; disconnect reservoir; replace reservoir with a pre-column; fill pre-column with solvent and small amount of adsorbent; and pump at maximum pressure until column is completely packed. For additional information, see general procedure.
Always allow pressure to decrease slowly after turning off pump--this will prevent formation of any cracks or channels in the packing material.
10. Relieve pressure and disconnect pre-column carefully. With small spatula remove a few mm (2-4) of packing from top of column; place 1 or 2 filter(s) in top of column; gently depress to top of packing material, and place Teflon plug on top of column until seal is confirmed. Connect column to pump, put pressure on (usually less than 200 psi) and observe through glass wall on top of column if resin is packing any further. If packing material should continue to settle (this may be the case with larger columns), some dead space or channelling will appear and step 9 should be repeated.
A-30912 factor B is dissolved in ethanol. PtO2 in absolute ethanol is reduced to form Pt, which in turn is used to reduce the A-30912 factor B catalytically, using hydrogenation under positive pressure until the reaction is complete (about 2-3 hours). The reaction mixture is filtered and concentrated under vacuum. The residue is dissolved in a small amount of tert-butanol and lyophilized to give tetrahydro-A-30912B.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3150059 *||Dec 26, 1962||Sep 22, 1964||Lilly Co Eli||Penicillin deacylation via actinoplanaceae fermentation|
|US3978210 *||Mar 6, 1975||Aug 31, 1976||Toyo Jozo Kabushiki Kaisha||Antibiotic aculeacins|
|US4024245 *||Aug 25, 1976||May 17, 1977||Eli Lilly And Company||Antibiotic A-30912 and process for production thereof|
|US4024246 *||Aug 25, 1976||May 17, 1977||Eli Lilly And Company||Antibiotic A-22082 and process for production thereof|
|US4050989 *||Apr 5, 1976||Sep 27, 1977||Eisai Co., Ltd.||Process of preparing desacyl-pepsidine with bacillus pumilus|
|US4173629 *||Jun 28, 1978||Nov 6, 1979||Sandoz Ltd.||Antibiotic S 31794/F-1|
|BE834289A1 *||Title not available|
|BE851310A1 *||Title not available|
|BE859067A1 *||Title not available|
|BE866095A1 *||Title not available|
|CH568386A5 *||Title not available|
|JPS385867B1 *||Title not available|
|1||*||C. Keller-Juslen et al., Tetrahedron Letters 4147-4150, 1976, vol. 46.|
|2||*||F. Benz et al., Helv. Chim. Acta 57, 2459, (1974).|
|3||*||J. M. Weber et al., J. Antibiotics 31(4), 373-374, (1978).|
|4||*||J. Shoji et al., Ibid (12), 1268-1280, (1976).|
|5||*||J. Shoji et al., Ibid 29(4), 380-389, (1976).|
|6||*||J. Shoji et al., J. Antibiotics 28, 764-769, (1975).|
|7||*||R. Traber et al., Helv. Chim. Acta 62, 1252 (1979).|
|8||*||S. Chihara et al., Agr. Biol. Chem. 37(11), 2455-2463, (1973).|
|9||*||S. Chihara et al., Ibid. 37(12), 2709-2717, (1973).|
|10||*||S. Chihara et al., Ibid. 38(10), 1767-1777, (1974).|
|11||*||S. Chihara et al., Ibid. 38(3), 521-529, (1974).|
|12||*||T. Kato et al., J. Antibiotics 29(12), 1339-1340, (1976).|
|13||*||T. Suzuki et al., J. Biochem. 56 (4), 335-343, (1964).|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US4537717 *||Sep 21, 1984||Aug 27, 1985||Eli Lilly And Company||Derivatives of A-21978C cyclic peptides|
|US4931352 *||Jun 7, 1989||Jun 5, 1990||Merck & Co., Inc.||Antifungal fermentation product|
|US4968608 *||Oct 7, 1987||Nov 6, 1990||Merck & Co., Inc.||Process for antifungal fermentation product|
|US5021341 *||Mar 12, 1990||Jun 4, 1991||Merck & Co., Inc.||Antibiotic agent produced by the cultivation of Zalerion microorganism in the presence of mannitol|
|US5021403 *||Mar 19, 1990||Jun 4, 1991||Merck & Co., Inc.||Antibiotic agents|
|US5028590 *||Apr 11, 1988||Jul 2, 1991||Eli Lilly And Company||Derivatives of A54145 cyclic peptides|
|US5049546 *||Mar 19, 1990||Sep 17, 1991||Merck & Co., Inc.||Antibiotic agents|
|US5166135 *||Aug 9, 1991||Nov 24, 1992||Merck & Company, Inc.||Method for the control of pneumocystis carinii|
|US5202309 *||Aug 19, 1991||Apr 13, 1993||Merck & Co., Inc.||Antibiotic cyclopeptide fermentation product|
|US5310726 *||Mar 19, 1990||May 10, 1994||Merck & Co., Inc.||Lipopeptide compounds|
|US5330973 *||Mar 19, 1990||Jul 19, 1994||Merck & Co., Inc.||Lipopeptide derivatives|
|US5385884 *||Aug 27, 1991||Jan 31, 1995||Merck & Co. Inc.||Lipopeptide derivatives|
|US5386009 *||Mar 19, 1990||Jan 31, 1995||Merck & Co. Inc.||Lipopeptide derivatives|
|US5386010 *||Jun 15, 1992||Jan 31, 1995||Merck & Co., Inc.||Lipopeptide compounds|
|US5428009 *||Jul 16, 1990||Jun 27, 1995||Merck & Co., Inc.||Lipopeptide derivatives|
|US5502033 *||Mar 28, 1994||Mar 26, 1996||Fujisawa Pharmaceutical Co., Ltd.||Antimicrobial polypeptide compound a pharmaceutical composition comprising the same, and a method for treating infectious diseases|
|USH1638 *||Sep 26, 1994||Mar 4, 1997||Furuta; Takahisa||Use of the polypeptide compound|
|WO2010064219A1||Dec 4, 2009||Jun 10, 2010||Ranbaxy Laboratories Limited||Process for the preparation of a novel intermediate for caspofungin|
|U.S. Classification||530/317, 435/71.1, 930/270, 435/68.1, 930/190|
|International Classification||A61K38/00, C07K7/56|
|Cooperative Classification||C12R1/045, Y10S930/19, Y10S930/27, C12R1/62, A61K38/00, C07K7/56|
|European Classification||C12R1/62, C07K7/56, C12R1/045|
|Nov 27, 1981||AS||Assignment|
Owner name: ELI LILLY AND COMPANY; 307 EAST MCCARTY ST., INDIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:ABBOTT, BERNARD J.;FUKUDA, DAVID S.;REEL/FRAME:003929/0720
Effective date: 19800821

domain: chemistry


Large UAV performance in an ultra-mobile package
NEMESiS UAV is a VTOL (Vertical Take-off and Landing) system developed to help UAV operators focus on their mission instead of handling the UAV. NEMESiS UAV versatility and ease of use along with its high performance characteristics makes it a valuable tool that is in a VTOL class by itself. The whole system (UAV and GCS) is the most compact UAS currently in use. It fits in a single case that can be carried by one man and it is ready to be deployed on the field and operate in less than 5 minutes.
Vertical Take-off and Landing (VTOL) Engines configuration.
Configurations of 4 or 8 engines are used depending on the desired mission endurance (flight time) and payload capacity.
The options are:
4 motors: 30 minute flight time
8 motors Rev1: 45 minute flight time
8 motors Rev2: 60+ minute flight time
At least 1000 gr of payload in all configurations. An embedded electromagnet onboard the UAV offers the option to release payloads up to 1 Kg.
Both UAV and GCS are battery operated. GCS can be powered additionally by an external power supply of 110-250V or vehicle power supply of 12V/3A
NEMESiS UAV guarantees a rock-solid video and telemetry data link over a range of 7Km. A GCS embedded gyro-stabilized antenna and the unique technology of beamforming array antenna onboard the UAV is the most powerful and interference-free UAV data link system available today.
Optical Sensors and Video Streaming
The NEMESiS UAV employs up to three optical sensors and DUAL video streaming of two sensors simultaneously. The UAV is equipped with a front-side-looking First Person View (FPV) camera plus two optical sensors onboard the gyro-stabilized gimbal (day and thermal – LWIR). The user selects from the GCS which two video sources wants to stream simultaneously to the GCS.
A detachable or retractable landing gear option is offered.
The NEMESiS data link is a high-performance wireless digital data link, providing long-range, high band-width data communication for both real time video and telemetry streaming. It is encrypted and provides frequency diversity in the 5-5.8 GHz license-free spectrum.


text:
Authenticity is what you will find in Seguin. Dated back from 1838 Seguin is full of history, hospitality, and rich food. You will find something for everyone’s palate!
Seguin has a variety of restaurants for everyone’s taste buds – from BBQ joints and homestyle cooking to Asian cuisines and authentic Tex-Mex. You will also find breweries and a winery nestled within Seguin. So feast your eyes on Seguin.
On its third generation, the family owned and operated Davila’s BBQ offers a variety of smoked meat. Their unique bar-b-que recipes have been featured on the Food Network and are worth the trip to Seguin. You can also enjoy their new food truck at numerous community events.
Seguin’s Hispanic culture shines through its food offerings with over a dozen Mexican food restaurants offering traditional Mexican dishes full of spice and flavor. Each restaurant has their own spin and you will want to try them all.
Need your caffeine kick? The right place for you is in the downtown historic district, Court Street Coffee Shop. The owners are proud to serve exceptional coffee roasted by Austin’s Texas Coffee Traders, gourmet ice cream, cakes from The Ultimate Cheesecake Bakery, fresh-baked goods, an impressive salad bar, and soups and sandwiches crafted in the coffee shop kitchen.
If you’re looking for one of the best burgers you’ve ever feasted on, try Kirby’s Korner. Whether you’re hankering for one of their hand pressed burgers or a specialty salad, we’re confident that the price, service, and great taste will have you coming back for more.
Fine dining can be enjoyed at the Chop House located within the Park Plaza Hotel on the downtown square. The 100 year-old hotel re-opened in December 2015 after an extensive rehab and offers a variety of delicious entrees.
Come on in, have a bite to eat, and stay for a while. For more authentic eats, visit www.visitseguin.com

domain: food


Challenges have been hurled from Tehran: There is no place for the Jewish State in the future…The Zionist regime will meet with destruction through unity in the Islamic world.
Iran’s Supreme Leader Ali Khamenei, and its President Mahmoud Ahmadinejad are carrying out a deliberate strategy and plan to mobilize tens of millions of Muslims in the Middle East against Israel. The two have made the most demonic attacks against the Jewish people since the days of Adolf Hitler.
To the best of my knowledge I am the only Jew and Zionist to have met face-to-face with these leaders. A flood of questions about the encounter has reached me from Israeli leaders, U.S. leaders, radio talk show hosts, including Glenn Beck, and other God-fearing men and women. There is no way to understand the events that are unfolding and how they fit into God’s prophetic plan for our age without understanding what is happening and why.
What is going on in the Middle East is part of a spirit war. It is no different than the war fought in ancient Iran (Persia) in the days of the prophet Daniel. Challenges have been hurled from Tehran: “There is no place for the Jewish State in the future…The Zionist regime will meet with destruction through unity in the Islamic world…
Anyone who loves freedom and justice must strive for the annihilation of the Zionist regime…The Zionist regime will be wiped off the map and thrown into the trash pit of history forever…The Israeli machine is a tool in the hands of Zionists who control the Middle East and the world…The Zionist regime is a cancerous tumor; its very existence is an insult to humankind…The Zionists control the world media and financial system.”
These demon-possessed rages which spew forth from the mouths of Iran’s leaders are the same as those which came from the mouth of Nebuchadnezzar in Babylon, and later Hitler. The only difference is that modern-day Iran is a country which is 1.6 million square kilometers with an annual purchasing power of one trillion dollars. It also controls 10 percent of the world’s oil reserves. It is, as a top Israeli atomic expert told me, “A screwdriver turn away from having the capacity to build atomic bombs.”
Simply put, if Iran is allowed to go nuclear, it will precipitate a one trillion dollar atomic arms race in all Gulf Sunni Arab states. Their vast oil wealth gives them the means and the hatred of Shi’a Muslims for Sunnis will give them all the motivation they need. Iran is more than 90 percent Shi’a Muslim with a 200-mile Persian Gulf border. Many experts believe an Armageddon-type conflict could occur within the next decade. If you’ve not read my latest novel, Seven Days, based on an Israeli attack against Iran, you need to do so. It was released July 4, 2012. The book documents two periods of seven days—the first the attack on Iran, the second period on Iran’s planned attack on the U.S. through Hezbollah terrorists. (And three Hezbollah terrorists were arrested in Mexico early in September!)
Israel’s attack on Iran’s reactors could begin with a massive cyber-attack against Iran’s infrastructure in an attempt to shut down the country’s radar and missile launch capabilities. If the electro-magnetic attack (EMP) is successful, then Israel would not need to carry out further strikes with ballistic missiles. The likelihood is great that 1,100 targets would have to be hit in order to prevent Iran from retaliating against the countries that allowed Israel flight clearance. It would also prevent shutting down the Strait of Hormuz and strikes against U.S. bases and allies in the region. Should Iran retain the capacity to retaliate, it would experience a barrage of missiles trained at its nuclear facilities. The initial attack would last approximately seven days, but the effects of it would continue for more than thirty days.
Approximately 50,000 missiles could be launched at Israel from Hezbollah in Lebanon and Hamas in Gaza (both Iranian proxies). Jews would become targets for assassination and Jewish installations besieged by Iranian terrorists worldwide. Recently, I met with my dear friend, Prime Minister Netanyahu. He said, “What is driving this is a belief in the Mahdi who disappeared about 1,000 years ago. It is believed he will return during a great apocalyptic war that will claim the lives of millions.” This radical Islamic belief, a demonic counterfeit of the Christian doctrine of the Second Coming of the Lord, almost mandates this kind of conflagration. No one wants the only country in the world with a 90 percent Shi’a population—the most extreme of the Muslim sects—to acquire the horrific weapons of atomic bombs and missiles to fulfill their twisted ideology.
On September 24, 2010, in New York City, I was granted an exclusive with Ahmadinejad. I invited the Fox News Network to join me and interview the president. I noticed that before the meeting, Iran’s Supreme Leader Khamenei and three additional Islamic clerics were seated together.
The remaining Iranian diplomats exited the room. The veiled woman translator was not permitted in the room. She had to translate from another location. Before the cameras were turned on, they all prayed to Allah to invoke the power of the Mahdi. Ahmadinejad did the same before he opened his mouth to say a word. Be sure and read the excerpts from that interview which follow this article. There is no doubt in my mind that this is a spirit war—a battle between darkness and light.
There are two factors that very few are considering with an Iranian response: First, the concept of risk versus reward. The last time Iran attacked the U.S. by kidnapping U.S. hostages and holding them for 444 days, President Jimmy Carter rewarded Iran by sending $7.9 billion through the Federal Reserve to the Bank of England to buy back the hostages. He also signed a top-secret agreement called the “Algerian Accord,” which guaranteed that the U.S. would not attack Iran.
Obviously, since one Liberal Left president did that in the past, Iran’s leaders will expect a greater reward for their bad behavior this time.
Second, the new wars of the 21st century are fought first as cyber wars. Phase two is economic war; phase three is media war, and then proxy wars. Countries generally no longer attack each other; they hire proxies as subcontractors to do their bidding instead. For example, Iran has the Mahdi Army in Iraq, Hezbollah in Lebanon, and Hamas in Gaza at its disposal. Only in the last and final stage of war do we see “boots on the ground.” By the time that point is reached, the war has already largely either been won or lost. The Holy Grail of understanding the times and God’s prophetic plan for God-fearing Believers concerning these events is that the only war that can be fought effectively is the spirit war.
Daniel waged a spirit war through fasting and prayer in Babylon against the demonic powers behind the throne of Persia. From that battle, the head of the nation was struck down and deliverance came for my people, the House of Israel. Today it is our turn to fight and win that vital spiritual battle so that Israel can again be delivered from destruction.


text:
The chemical the many aspects of Clinical Bud, categorised as cannabinoids, take up a strong section inside arena of medical science termed CAM (Complementary and also Vary Medicine). Also, normalization and is purified belonging to the removes need to be upgraded and security of business items through rack house ought to be evaluated (e.g. To prevent CBD destruction simply by prescribing free of sour on ingredients etc.). As a final point, the building care are required to be increased to minimize contamination. CBD cannabis gasoline is certainly non-psychoactive even more beneficial. For anyone hoping to forestall in some cases know supplied THC, Health reform Ganja, Inc. Total variety CBD denotes everything that develops while in the hangman’s halter sow close to that CBD, similar to locate examples of THC together with different cannabinoids such as CBG (cannabigerol) and also antioxidant compounds.
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Hemp engine oil will be an gel done by means of chilly pressing your seed of the hemp sow (Cannabis Sativa). Within animal investigation, THC provides inadequate in general success through minimizing seizures has got recently been been proven becoming a less effective anticonvulsant when compared to CBD THC, as being a psychoactive drug, boasts several unwanted side effects, just as the well-determined euphoric “high” hyperlinked to non-elite benefit from – that would be undoubtedly an essential disincentive for the drug firm to cultivate medicines made up of it. His or her motor oils remain true considering the fact that they choose a remarkable CO2 removal remedy, which unfortunately advances an item diminished price.

domain: medical


text:
Building healthy friendships is like sowing seeds of love and laughter that bloom into lifelong bonds. As parents and caregivers, we hold the guiding hand in nurturing our kindergarten children’s social and emotional growth. In this blog, we will explore heartwarming strategies to assist your little one in building friendships that will last a lifetime. Get ready to embark on a journey filled with giggles, shared secrets, and unforgettable playdates. We’ll delve into various aspects, including how to build friendships, engaging friendship-building games, captivating friendship stories for kids, and recommended friendship books for young readers.
Encouraging Social Interaction
Encouraging social interaction is vital to help your kindergarten child build friendships. Here are a few strategies to promote healthy connections:
- Facilitate Playdates: Arrange playdates with other children from your child’s kindergarten class. These opportunities for shared activities can help foster friendships.
- Volunteer Together: Engaging in community service activities or volunteering together provides a chance for your child to meet and bond with other children who share similar interests.
- Join Extracurricular Activities: Enrol your child in extracurricular activities like sports, arts, or music, where they can interact with peers outside of the classroom.
Teaching Empathy and Kindness
Empathy and kindness form the foundation of healthy friendships. Understanding the importance of active listening, kindness, and empathy lays the foundation for children to learn how to build friendships that flourish.Help your child develop these qualities with the following strategies:
- Lead by Example: Model empathy and kindness in your own interactions with others. Children learn from observing their parents’ behaviour.
- Practice Perspective-Taking: Encourage your child to consider other people’s feelings and perspectives. Prompt them to think about how their words or actions might affect others.
- Role-Playing Scenarios: Engage in role-playing activities where you act out different social situations. This helps your child understand how their behaviour impacts others.
Engaging Friendship Building Games
Games can be a fun and interactive way to foster friendship building. Friendship building games provide a playful avenue for children to develop social skills, teamwork, and camaraderie.. Here are a few game ideas that encourage social interaction and teamwork:
- “Friendship Bingo”: Create a bingo board with different friendship-related activities, such as sharing a toy or complimenting a friend. Encourage your child to complete the activities to fill up their bingo board.
- “Guess Who”: Write positive qualities of different classmates on index cards and play a guessing game where your child has to guess which classmate matches each description.
- “Cooperative Treasure Hunt”: Organise a treasure hunt where children work together to solve clues and find hidden treasures. This game encourages collaboration and communication.
Captivating Friendship Stories for Kids
Reading friendship stories can spark your child’s imagination and teach them valuable lessons about friendship. Here are some captivating books that explore the theme of friendship:
- “The Rainbow Fish” by Marcus Pfister: This beautifully illustrated story teaches children about sharing and the importance of friendship.
- “The Giving Tree” by Shel Silverstein: A heartfelt tale about selflessness and the enduring bond between a tree and a boy.
- “How to Be a Friend: A Guide to Making Friends and Keeping Them” by Laurie Krasny Brown and Marc Brown: This book offers practical advice on making friends, resolving conflicts, and maintaining healthy relationships.
Recommended Friendship Books for Kids
Apart from specific friendship stories, there are numerous other books that celebrate friendship and encourage positive relationship-building in children. Delve into the enchanting world of friendship books for kids, where captivating stories teach valuable lessons about loyalty, understanding, and the beauty of genuine connections. Here are a few recommendations:
- “A Sick Day for Amos McGee” by Philip C. Stead: A heartwarming story about a zookeeper who receives unexpected visits from his animal friends when he falls ill.
- “The Invisible Boy” by Trudy Ludwig: This book explores themes of inclusion, kindness, and empathy, emphasising the importance of appreciating everyone’s unique qualities.
- “You Are Friendly” by Todd Snow: Through vibrant illustrations and simple text, this book highlights the small acts of friendliness that can brighten someone’s day.
Fostering Inclusive Friendships
Inclusivity is the cornerstone of a thriving community of friends. Teaching your child the importance of embracing diversity and fostering inclusive friendships will broaden their understanding and enrich their social experiences. Here are a few strategies to promote inclusivity:
- Encourage Diversity: Expose your child to diverse cultures, traditions, and perspectives through books, films, and community events. This cultivates an appreciation for differences and helps them embrace diversity in their friendships.
- Teach Acceptance: Discuss the value of acceptance and respect for others’ unique qualities. Encourage your child to celebrate differences and discourage any form of discrimination or prejudice.
- Foster Empathy: Help your child develop empathy by encouraging them to put themselves in others’ shoes. Teach them to recognize and understand the challenges faced by individuals who may be different from them, promoting empathy and compassion in their friendships.
- Create an Inclusive Environment: Organise gatherings or playdates that bring together children from diverse backgrounds. This provides an opportunity for your child to interact and form friendships with peers from different cultures, ethnicities, or abilities.
By fostering inclusive friendships, you equip your child with the tools to build connections that transcend boundaries, fostering a sense of unity and understanding among their peers. Embracing diversity at a young age creates a solid foundation for a future filled with harmonious relationships. Nurturing shared experiences and open communication is vital in building healthy friendships that stand the test of time.
Imagine your child walking hand in hand with a newfound friend, their laughter echoing in the air. By implementing these effective strategies and weaving engaging games, captivating stories, and recommended books into your child’s journey, you become a guiding force in their exploration of friendships. Building healthy relationships takes time and patience, but the joy, support, and memories forged along the way are immeasurable. Let’s embark on this adventure together, nurturing the seeds of friendship that will blossom into a lifetime of love and companionship.
P.S. Looking for an environment that nurtures friendship and holistic development? Consider EuroKids, a leading early childhood education provider committed to fostering social, emotional, and cognitive growth in children. With our child-centric approach and expert educators, we create a conducive atmosphere for building lasting friendships. Learn more about us at www.eurokidsindia.com.

domain: sociology


text:
Synthesis and dopamine receptor binding of dihydrexidine and SKF 38393 catecholamine-based analogues
MetadataShow full item record
Dopamine is an important neurotransmitter that regulates numerous essential functions, including cognition and voluntary movement. As such, it serves as an important scaffold for synthesis of novel analogues as part of drug development effort to obtain drugs for treatment of neurodegenerative diseases, such as Parkinson's disease. To that end, similarity search of the ZINC database based on two known dopamine-1 receptor (D1R) agonists, dihydrexidine (DHX) and SKF 38393, respectively, was used to predict novel chemical entities with potential binding to D1R. Three compounds that showed the highest similarity index were selected for synthesis and bioactivity profiling. All main synthesis products as well as the isolated intermediates, were properly characterized. The physico-chemical analyses were performed using HRESIMS, GC/MS, LC/MS with UV–Vis detection, and FTIR, 1H NMR and 13C NMR spectroscopy. Binding to D1 and D2 receptors and inhibition of dopamine reuptake via dopamine transporter were measured for the synthesized analogues of DHX and SKF 38393.

domain: literature


"Nightlife" is from M.E.D.'s "Push Comes to Shove," the latest from the consistently great Stones Throw Records, home of visionary producer Madlib, who provides the beat for this track. And the beat, revolving around a beautiful acoustic bass sample that pushes and pulls stubbornly against the drums, is the reason to listen, as M.E.D.'s stiff, unremarkable rapping leaves a lot to be desired.
Daily Download: "Nightlife," M.E.D.
Bland rapping, but an amazing Madlib beat that makes up for it.
July 12, 2005 11:01AM (UTC)
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Rochdale MP, Tony Lloyd, has called on the government to look again at their decision to pull back levelling up funds which would have improved the gateway into the town centre from Rochdale railway station.
Tony said, “These funds would have helped towards new sustainable homes and much needed redevelopment of the routes that lead into Rochdale’s town centre from the train station but this decision means that local communities and businesses will lose out.
“It was only last year that Prime Minister Sunak admitted on camera to taking money away from ‘deprived urban areas’ yet he’s managed to ensure that parts of his own constituency receive £19m of levelling up funds. Small wonder he’s being accused of ‘levelling up’ favouritism.
“Labour in government will do things differently by ending this type of contest that pits community against community by transferring power out of Westminster through the Take Back Control Act, so local leaders can harness the skills and assets in areas like Rochdale to drive growth.”
Rochdale Borough Council, supported by Tony, submitted a bid for £20million of funding to the Levelling Up fund last year for two of the Rochdale station gateway projects, specifically over 200 new homes on the Central Retail Park site and creation of a new station square.
The plans included a new public square in front of Rochdale train station and the creation of new homes and business spaces, alongside a 500 space park and ride facility on Miall Street. The project aimed to create an improved gateway into Rochdale town centre – removing traffic to give greater priority to pedestrians and cyclists.
Even with the latest funding announcement nearly all places are still worse off, of 151 local authority areas only eight have not experienced real-terms cuts since 2018 even when new levelling up funds are factored in.
Notes for editors:-
• The £2 billion-plus fund is shared between 111 communities across the UK as part of the second round of levelling-up funding. But Of the 80 successful bids in England, which includes Rishi Sunak’s constituency receiving £19m of funding, only half are in the 100 most deprived areas of the country.
• Analysis suggests that of 151 local authority areas, only eight have not experienced real-terms cuts since 2018 even when levelling up funds, including those just announced, are factored in. The other 143 have still had real-terms cuts to the key services that sustain communities.
•During the Tory leadership contest in the summer, Rishi Sunak Tories in Tunbridge Wells that as Chancellor he tried to reverse Treasury formulas “that shoved all the funding into deprived urban areas” so areas like theirs could benefit.


text:
Tim Klüter and Matthias Weuster contributed equally to this work.
The authors declare that they have no competing interests.
TK drafted the manuscript, participated in its design and performed the statistical analysis. MW carried out the surgical procedures and participated in its design and coordination. SB carried out the surgical procedures and the biomechanical testing and performed the statistical analysis. LM carried out the surgical procedures and carried out the biomechanical testing. SF conceived of the study and carried out the histologic assessment. NS carried out the histologic staining and participated in its design and coordination. YA carried out the histologic staining. TP participated in the design of the study and performed the statistical analysis. DV participated in the biomechanical testing. AS conceived of the study and participated in its design. SL designed the study, participated in its coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The prescription of the oral anticoagulant rivaroxaban to prevent thromboembolic episodes associated with orthopaedic surgery has dramatically increased since it was introduced. Rivaroxaban is beeing prescribed although recent in-vitro studies revealed that it impaired osteoblast metabolism. In this study we analysed the effect of rivaroxaban on fracture healing in a rat femur fracture model.
Femur fractures were created by a 3-point-bending device in 48 Wistar rats and subsequently stabilized by intramedullary nailing. After the surgical procedure animals were randomised into four groups. Two groups were fed with 3 mg rivaroxaban per kg body weight per day and two control groups were fed with chow only. Animals were euthanized 28 or 49 days after surgical procedure. Femurs underwent undecalcified histologic staining micro CT scanning and biomechanical testing. The statistical significance was evaluated using one-way Anova with Bonferroni correction.
Micro CT-scans revealed significantly increased volume of bone tissue in the fracture zone between day 28 and 49. During the same time callus volume decreased significantly. Comparing the fracture zone of the rivaroxaban group to the control group the treated group revealed a larger callus and a marginal increase of the tissue mineral density. The torsional rigidity was not influenced by the treatment of rivaroxaban.
In the present study we were able to demonstrate that rivaroxaban does not impair fracture healing in a rat femur fracture model. Considering the fact that low molecular weight heparins delay fracture healing significantly, rivaroxaban might be an improved alternative.
Haake DA, Berkman SA. Venous thromboembolic disease after hip surgery: risk factors, prophylaxis, and diagnosis. Clin Orthop Relat Res. 1989;242:212–31. PubMed
Salzman EW, Harris WH. Prevention of venous thromboembolism in orthopaedic patients. J Bone Joint Surg Am. 1976;58(7):903–13. PubMed
Handoll HH, Farrar MJ, McBirnie J, Tytherleigh-Strong G, Milne AA, Gillespie WJ. Heparin, low molecular weight heparin and physical methods for preventing deep vein thrombosis and pulmonary embolism following surgery for hip fractures. Cochrane Database Syst Rev. 2002;4:Cd000305. PubMed
Palmer AJ, Koppenhagen K, Kirchhof B, Weber U, Bergemann R. Efficacy and safety of low molecular weight heparin, unfractionated heparin and warfarin for thrombo-embolism prophylaxis in orthopaedic surgery: a meta-analysis of randomised clinical trials. Haemostasis. 1997;27(2):75–84. PubMed
Stinchfield FE, Sankaran B, Samilson R. The effect of anticoagulant therapy on bone repair. J Bone Joint Surg Am. 1956;38-a(2):270–82. PubMed
Long A, Zhang L, Zhang Y, Jiang B, Mao Z, Li H, et al. Efficacy and safety of rivaroxaban versus low-molecular-weight heparin therapy in patients with lower limb fractures. J Thrombosis Thrombolysis. 2014;38(3):299–305. CrossRef
Tiedeman JJ, Lippiello L, Connolly JF, Strates BS. Quantitative roentgenographic densitometry for assessing fracture healing. Clin Orthop Relat Res. 1990;253:279–86. PubMed
- Rivaroxaban does not impair fracture healing in a rat femur fracture model: an experimental study
Stefanie Fitschen- Oestern
- BioMed Central
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text:
What We Know and What We Need to Know
In the 1960s food aid made up nearly 20 per cent of overall overseas development aid, today that figure is five per cent. Increasingly food aid is provided as emergency relief rather than as a form of long term support and many donors have expressed strong interest in switching from food to cash assistance. Food aid continues to generate heated debate, particularly around what effect it has on food consumption and nutrition, food markets, and labour supply, as well as the extent to which it exacerbates or mitigates conflict. In the UNU-WIDER working paper 'Mapping the Impacts of Food Aid: Current Knowledge and Future Directions', Amy Margolies and John Hoddinot provide an overview of what we currently know about the impact food aid has on these issues and highlight key areas in which further research is needed.
The impact of food aid
The most common way by which food aid is distributed to a beneficiary population is through a 'standard food ration', which typically consists of a basket of dry or canned food items being shipped to recipients by international donors. Food aid is also delivered through school meals, in return for labour and through the distributing food purchased locally. There is a diverse range of literature discussing the effects such aid has on recipient households and agricultural practices. A number of interesting findings can be drawn from these discussions.
Margolies and Hoddinot point out that food aid transfers do seem in general to increase household calorie consumption. The evidence for this comes from two countries where food transfer programmes have been studied in detail, Bangladesh and Ethiopia. In Bangladesh the amount of this increase differed from programme to programme. The highest increase recorded was associated with the transfer of micro-nutrient fortified whole wheat flower (66 kcal per taka), the lowest with a program that offered participants rice in return for participating in a public works projects (23kcal per taka). In Ethiopia studies show that food transfers put into place after the drought in 2002 increased both total consumption and pre-school nutritional status.
One particular concern of many critics of food transfers is that they will disincentives recipients from participating in the labour market and acts as a substitute for local food production. However a number of studies show that this is not the case. For example, an analysis of data from rural Ethiopia revealed no evidence of negative dependency effects, and, in fact, showed a potential positive effect on labour supply. One might fear disincentives to agricultural production if food transfers provide an adequate replacement. However, in a strange case of aid inefficiency having a positive unintended consequence, due to the inconsistent timing of the deliveries associated with food transfer programs food aid does not seem to lead to recipients not participating in the labour market.
Some critics of food aid argue that it leads to lower food prices. The increased supplies are thought push down prices, and thus disincentivize agricultural production in the area. Early studies into the impact of food aid provided evidence consistent with this hypothesis, however Margolies and Hoddinot point out that empirical work done over the last fifteen years has largely failed to replicate these adverse results. In particular a study of the effect of food aid, in the form of yellow maize, in Mozambique found that the program had no noticeable effect on prices and studies of programs of Kenya and Swaziland have replicated these results. While some studies have found that food aid has had this effect they have put this down to programmatic failure rather than problems with food aid itself.
Emergency Food Aid
Food aid is becoming increasingly restricted to emergency settings that arise often as a result of internal strife in recipient countries. There are particular considerations that need to be taken into account by both practitioners and policy makers when thinking about food aid provided to countries experiencing these circumstances. In particular it is important to question whether food aid is likely to exacerbate or mitigate any ongoing conflict.
Margolies and Hoddinot suggest that if the conflict is driven by grievances about the distribution of resources or by considerations of survival then food aid does have the potential to serve as a mitigating force. The disbursal of food aid to regions which feel they have been deprived is a mechanism by which the inequalities at the heart of the conflict can be diminished. In the case of conflicts driven by considerations of survival, if food aid is delivered at a level sufficient to meet the survival thresholds of both sides then it can clearly reduce the incentives for conflict.
However there are a number of cases where it appears that food aid has prolonged conflicts. There is evidence that during the Rwandan genocide the government redistributed food aid to elites, thus further exacerbating tensions and perpetuating inequalities. Similarly in Sudan and Ethiopia there is evidence that food aid was diverted by the governments to supportive constituencies and the military. While there may be a theoretical basis for believing food aid has the potential to mitigate conflict, in practice there are many accounts of its failure to do so and few, if any, documented successes. Indeed, Margolies and Hoddinot point out that one of the few studies focusing specifically on the relationship between food aid and conflict found that increasing food aid has the effect of increasing the incidences of conflict, an effect which is even larger in countries without a civilian government. There is evidence that other forms of aid aimed at community and peace building can in some circumstances help reduce the incidence and duration of conflicts. Given the increasing tendency for food aid to be restricted to emergency settings, more work clearly needs to be done in this area.
Alternative approaches: food, cash or something else?
On the face of it there seems to be a number of reasons why both donors and recipients might prefer cash transfers to food. Cash transfers are in theory more economically efficient because they allow beneficiaries to buy what they need most and thus have a less distorting effect on production and consumption choices. Cash may also stimulate agricultural production by raising demand in the local market. It is also much more expensive to distribute food rather than cash as administrative costs which are 20 to 25 per cent higher. Furthermore a study of the effects of food aid in the aftermath of the Asian Tsunami of 2005 found that recipients of cash, rather than food, transfers were more likely to improve the variety of their diets. Given the supposed benefits of cash transfers it is important to consider whether there is any reason why they should not replace all food aid.
One potential reason is that cash transfers could be ineffective under certain local conditions. If recipients have no physical access to markets then having money to spend will do them little good. Furthermore, if traders lack the capability to bring extra food to the market without significantly raising prices then a large portion of a cash transfer will be eaten up by inflation. Cash transfers to areas with a very uncompetitive market may simply allow traders to raise prices.
It is also the case that many recipients simply prefer food to cash. This preference is obviously dependent on prevailing food prices and tends to increase as prices rise, however recipients also claim local traders, aware of the cash transfers, collude to raise food prices to levels they know their customers can newly afford. Margolies and Hoddinot argue that we should take it as a general principle that recipients should have some say in the form of the assistance they receive, given this, the preference food is clearly an argument in favour of that kind of transfer.
There are a number of factors that should effect the decision about whether cash or food transfers are preferable and many of them are not yet fully understood. Further research in this area is particularly important given the growing gulf between donors (who prefer cash transfers) and recipients (who prefer food).
The authors conclude by pointing out that while programme designers need to be mindful of the potential negative effects of food aid, there is little evidence that it disincentives its recipients to work or that it has adverse effects on food markets. However with regard to the impact different food aid modalities have at a household level and during times to crisis the author argue that more work needs to be done.


If Twitch has banned you, you may wonder how to regain your account. There are several options available, including appeals and shadow bans. You can also use VPNs to unblock Twitch chat. However, these options are only available to some users.
The idea of shadow bans on Twitch has generated a lot of debate and criticism. Some think these bans are a good idea, while others believe they are a waste of time. Some say that they give users a sense of ownership and empower them to give feedback to the site’s moderators. You should think carefully before implementing a shadow ban, regardless of your stance.
One way to avoid a shadow ban is to use a VPN. However, some VPNs block Twitch chat from your country. However, it is possible to connect to a VPN and keep access to the site. The VPN can help you watch content from restricted regions or use features of the Twitch site that aren’t available in your region.
Another way to avoid a shadow ban on Twitch is to avoid submitting links related to yourself. Subreddits ban self-promotion and other topics, so reading the comments carefully before submitting links is essential. Many subreddits list their rules in their “About” or sidebar. You should also avoid harassing other users or abusing moderators.
One recent case involving a shadow ban is Dr. Disrespect’s Twitch ban. It is unclear why he was banned. Many people speculated that it was due to a criminal element, but the reality is unknown. Nevertheless, Dr. Disrespect has defended himself and called out E.A.’s reaction to his ban on Twitch.
Shadowbans on Twitch is a very subtle means to combat spammers. These tactics can prevent the spammers from setting up multiple accounts. The shadowbans are less noticeable than those telling users their history has been banned. However, it’s important to note that the shadowban is more effective than notifying users about it. The shadowban is often a symptom of a more severe problem, like a bot-driven account. In most cases, it can be used to weed out spammers, but the user is never informed of it.
As with any other social network, social media platforms have rules about what’s acceptable. Shadowbans are often the result of violating the terms of service. It’s essential to follow these guidelines to avoid being shadow-banned. In addition to avoiding spamming or posting illegal content, you should also ensure that you don’t post links or other offensive or controversial materials.
Twitch has been grappling with hate-based incidents in the past year on its streaming platform. In a nutshell, this means that Twitch has decided to suspend a popular streamer for using the word ‘cracker’ in a video or attempting to manipulate a game’s odds of winning. While these cases are rare, they do occur and often target Twitch streamers marginalized by the wider community.
In response to this problem, the streaming platform has updated its reporting and appeals processes. The first one involves the new account suspension appeals portal, which will allow users to keep an eye on the status of their appeals. While Twitch is constantly cracking down on “bad actors,” these new changes should ensure a consistent experience for streamers.
Another case involving Twitch and politics is the Trump campaign’s Twitch page, which rebroadcasts a 2016 rally. In it, Trump claimed that Mexico was not sending the best people to the U.S. but instead sending rapists and crime to our shores. The comments violated Twitch’s rules, and Twitch suspended the account of Trump for two weeks.
While the Twitch community has become renowned for its live streaming of video games, users have pushed the platform in new directions. Adding diversity and fresh ideas to the mix is healthy for the platform’s community. Variety inspires creativity and progress. Regardless, keeping the rules in mind is always a good idea.
While this ban is unlikely to be permanent, Greekgodx remains a Twitch partner. However, if Twitch decides to ban someone permanently, he’ll lose his partnership status. Some Reddit users speculate on the reason behind the ban, with nudity cited as a possible cause. Male nudity is illegal under Twitch’s terms of service.
Other causes for temporary suspensions on Twitch include broadcasting offensive content or inappropriate music. While these are minor offenses, the Twitch community has a strict policy that requires streamers to adhere to the rules and guidelines of the community. If they break the rules, Twitch may ban them for good.
Appealing a ban on Twitch
Appealing a ban on Twitch is possible, but you have to be careful because Twitch can’t permanently reinstate banned accounts. The first step in this process is to build a strong case for yourself. This way, you can push your complaint to the Twitch support team. Depending on the severity of your ban, you can recover your account in a short time.
Twitch is working to make the appeals process more transparent and user-friendly. Users can share their perspective on a suspension, see updates on their Twitch ban, and receive email updates. They are also working to make their appeals process faster. The new system will consider the context of the ban, the nature of the offense, and the severity of the ban.
Once you’ve logged into your Twitch account and clicked on ‘appeal,’ you’ll need to provide some information about yourself and your budget. Then, submit your appeal. You’ll have to wait a few days to be approved if unsuccessful. You’ll get a reply from Twitch if your request is accepted. If you’ve received a ban for an action or activity, you can appeal it within 60 days.
You must explain why you’ve been banned and the reasons for it. For example, if your account was banned due to content related to sexuality, you must explain why you did that. A ban can last up to 90 days, so if you feel your actions are wrong, keep going. Using a VPN can help you get back on Twitch without hassle.
Once your account is banned, you can’t use it again until the ban is lifted. A VPN server will help you temporarily change your I.P. address so you can sign in with a different account. You can even choose a new name for your account if you want to be recognized as someone other than you were before. This will help you to hide your I.P. address, which Twitch will be able to trace.
Twitch has strict rules regarding harassment, trolls, and violence. They do this to keep the community safe. The more abusive and violent a user is on the platform, the higher the chance they will be banned.
VPNs that can unblock Twitch chat
If you’re looking to unblock Twitch chat, you’ll need a VPN that offers servers in countries where Twitch is available. Some VPNs only have one server in the U.S., while others have as many as 15. The more servers you have, the better, and the better your connection will be. You can also choose a VPN with additional privacy features.
A good VPN for Twitch will have many servers available worldwide, ensuring you can always find a server that works. Ideally, it will have a world map that shows all available servers. Once you’ve selected a server, you can use it to watch your favorite Twitch streamers. Make sure you have administrator rights on your device to avoid logging out.
Another thing to consider when choosing a VPN for Twitch is how many I.P. addresses it has. VPNs with over 100,000 IPs can circumvent blocking mechanisms and protect users from DDoS attacks. They also offer better speeds and protection against hackers and other threats. Moreover, many of the best VPNs for Twitch are highly affordable and easy to use.
NordVPN has a vast network of servers and reliable connections. Its no-logs policy and kill-switch are also essential factors. Apart from this, it uses the fastest connection protocols. NordVPN is one of the best VPNs for Twitch, and it even has a browser extension for easier access.
Using a VPN can help you get around throttling by ISPs. They can check the amount of internet traffic a user sends to Twitch and decide whether they are hurting their servers. But there is no foolproof method, and there are other security features that you should consider.
A good VPN will also offer a no-logs policy and ensure your privacy. This is especially important if you live in a country with censorship and internet surveillance. Additionally, a good VPN should support multiple protocols, which broadens your options and makes your connection scalable.


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Celent Ranks PRODUCT4 Tops
PRODUCT4 is pleased to announce that independent research and consulting firm Celent has published its ranking of branch solution providers, and PRODUCT4 ranked the highest in both features and technology.
The press release can be read by clicking here.
Branch Automation Solutions: The Convergence of Teller, Platform, and CRM
In a new two-part series Celent discusses the trends in the industry on both the business and technical sides. It examines the technical environment choices that make the most sense for a bank when choosing a vendor. Finally the report analyzes the vendors using Celent's ABCD framework for analysis.
Celent is a research and consulting firm focused on the application of information technology in the global financial services industry.
Founded in 1999 by veteran financial services technology analysts, Celent is an independent, self-financed, and profitable firm dedicated to providing unbiased information and advice to financial services firms.
Celent's analyst team is organized by financial services industry vertical: Banking, Securities & Investments, as well as Insurance. These analysts produce monthly research reports for our members, and are available to members to further explore issues and questions that may arise. This direct access to Celent's analysts means that members can immediately contact established experts on a large variety of issues within financial services technology.
Headquartered in Boston, Celent maintains offices in New York, San Francisco, Tokyo, and Paris. With its team of internationally experienced and multi-lingual analysts, Celent is able to provide its clients with global perspective.

domain: finance


MySQL service is in error again. If it happens many times, you will be scared of wasting time. The error content is as follows.
Can't connect to local MySQL server through socket '/tmp/mysql.sock'
|Ruby On Rails||v220.127.116.11|
[Survey 1] Is MySQL running? I'm using "Sequel Pro", so I'll try connecting. → Connection error
Check the operating status of the process
CMD>ps -ef | grep mysql 501 1287 1 0 11:45AM ?? 0:00.06 /bin/sh /usr/local/opt/[email protected]/bin/mysqld_safe --datadir=/usr/local/var/mysql 501 6400 1287 0 11:55AM ?? 0:00.51 /usr/local/opt/[email protected]/bin/mysqld --basedir=/usr/local/opt/[email protected] --datadir=/usr/local/var/mysql --plugin-dir=/usr/local/opt/[email protected]/lib/plugin --log-error=ichikawadaisukenoMacBook-Air.local.err --pid-file=ichikawadaisukenoMacBook-Air.local.pid 501 6402 804 0 11:55AM ttys000 0:00.01 grep mysql
!! ?? Looks like it's working! ??
[Survey 2] First of all, from the error content. It is said that there is no socket file, so I tried using the following command.
Try creating a socket file.
CMD>cd application folder CMD>touch /tmp/mysql.sock
→ As a result, no improvement.
[Survey 3] Check the log.
Check the MySQL log
CMD>more /usr/local/var/mysql/ichikawadaisukenoMacBook-Air.local.err (Partially omitted) mysqld_safe A mysqld process already exists
It looks like the process is running multiple times. Stop an existing running process.
CMD>kill -9 Target service process
This time it just improved. However, the following error message is still output.
200616 16:04:42 mysqld_safe A mysqld process already exists
I will deal with it at a later date.


text:
Welcome to the new website for The Friary Surgery.
Here you will find up to date information on services offered by our practice & guidance on healthcare. The ‘In Your Community’ section you will find local & national services, support groups & activities for all ages & conditions.
You can keep track of our appointment schedule as this has recently changed since the Covid-19 pandemic and we are still not back to normal yet. Any announcements and news will be posted on our latest news section, so we advise all our patients to visit our website on a regular basis.
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A Level Biology for OCR Year 2 Student Book (BOK)
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Written by curriculum and specification experts in partnership with OCR, this Student Book supports and extends students through the new linear course while delivering the breadth, depth, and skills needed to succeed in the new A Levels and beyond. It develops real subject knowledge as well as essential exam skills. This Student Book covers the second year of content required for the new OCR Biology A specification.

domain: library