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Synthetic platelet storage media is disclosed for use with blood preparations intended for in vivo use. The synthetic platelet storage media is prepared by providing sodium chloride, sodium citrate, sodium acetate and sodium phosphate, and formulating in water 45-120 mM sodium chloride, 5-15 mM sodium citrate, 20-40 mM sodium acetate and 20-30 mM sodium phosphate.
1. A magnesium free and glucose free synthetic platelet storage media prepared by the process of:
a) providing sodium chloride, sodium citrate, sodium acetate, and sodium phosphate; and
b) formulating in water:
45-120 mM sodium chloride;
5-15 mM sodium citrate;
20-40 mM sodium acetate; and
20-30 mM sodium phosphate.
2. The synthetic platelet storage media of
3. The synthetic platelet storage media of
4. The synthetic platelet storage media of
The present application is a continuation of patent application Ser. No. 09/732,174, filed Dec. 7, 2000, now U.S. Pat. No. 6,566,046, which is a continuation of patent application Ser. No. 09/240,067, filed Jan. 29, 1999, now U.S. Pat. No. 6,251,580, which is a continuation of patent application Ser. No. 08/692,444, filed Aug. 5, 1996, now U.S. Pat. No. 5,908,742, which is a continuation of patent application Ser. No. 08/338,039, filed Nov. 14, 1994, abandoned, which is a continuation-in-part of patent application Ser. No. 08/072,485, filed Jun. 2, 1993, now U.S. Pat. No. 5,459,030, which is a continuation-in-part of patent application Ser. No. 07/926,477, filed Aug. 7, 1992, now abandoned, which is a continuation-in-part of patent application Ser. No. 07/844,790, filed Mar. 2, 1992 now U.S. Pat. No. 5,288,605, issued Feb. 22, 1994.
The invention generally relates to synthetic media for use with blood preparations intended for in vivo use, including synthetic media used in conjunction with the photodecontamination of platelets.
Whole blood collected from volunteer donors for transfusion recipients is typically separated into its components: red blood cells, platelets, and plasma. Each of these fractions are individually stored and used to treat a multiplicity of specific conditions and disease states. For example, the red blood cell component is used to treat anemia, the concentrated platelet component is used to control bleeding, and the plasma component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia.
Ideally, all blood cell preparations should be from freshly drawn blood and then immediately transfused to the recipient. However, the logistics of operating a blood donor center preclude this possibility in the vast majority of cases. Transfusions are needed day and night and it is difficult, if not impossible, to arrange for donor recruiting at unusual hours. Consequently, modern blood donor centers must use stored blood products.
In the United States, blood storage procedures are subject to regulation by the government. The maximum storage periods for the blood components collected in these systems are specifically prescribed. For example, whole blood components collected in an "open" (i.e., non-sterile) system must, under governmental rules, be transfused within twenty-four hours and in most cases within six to eight hours. By contrast, when whole blood components are collected in a "closed" (i.e., sterile) system the red blood cells can be stored up to forty-two days (depending upon the type of anticoagulant and storage medium used) and plasma may be frozen and stored for even longer periods.
While red cells are stored in the cold, Murphy and Gardner, New Eng. J. Med. 280:1094 (1969), demonstrated that platelets stored as platelet-rich plasma (PRP) at 22� C. possessed a better in vivo half-life than those stored at 4� C. Thus, more acceptable platelet concentrates could be transfused after storage at room temperature. Until recently, the rules allowed for platelet concentrate storage at room temperature for up to seven days (depending upon the type of storage container). However, it was recognized that the incidence of bacterial growth and subsequent transfusion reactions in the recipient increased to unacceptable levels with a seven day old platelet concentrate. Platelet concentrates may now be stored for no more than five days.
One might believe that it is a relatively simple matter to keep the blood preparation sterile during the manipulations needed to concentrate the platelets. After all, blood bags used for platelet concentrate preparation are in themselves sterile, as are the connected satellite bags. However, bacteria can be introduced by at least two different means.
First, if the donor is experiencing a mild bacteremia, the blood will be contaminated, regardless of the collection or storage method. Adequate donor histories and physicals will decrease but not eliminate this problem. See B. J. Grossman et al., Transfusion 31:500 (1991).
A second, more pervasive source of contamination is the venepuncture. Even when "sterile" methods of skin preparation are employed, it is extremely difficult to sterilize the crypts around the sweat glands and hair follicles. During venepuncture, this contaminated skin is often cut out in a small "core" by a sharp needle. This core can serve to "seed" the blood bag with bacteria that may grow and become a risk to the recipient.
Indeed, many patients requiring platelet transfusions lack host-defense mechanisms for normal clearing and destruction of bacteria because of either chemotherapy or basic hematologic disease. The growth of even seemingly innocuous organisms in stored platelets can, upon transfusion, result in recipient reaction and death. See e.g., B. A. Myhre, JAMA 244:1333 (1980) and J. M. Heal et al., Transfusion 27:2 (1987).
The reports assessing the extent of contamination in platelets have differed in their methods, sample size, and bacterial detection schemes. D. H. Buchholz et al., Transfusion 13:268 (1973) reported an overall level of platelet contamination of 2.4% when a large (>1000 bags) sample was examined and extensive measures were taken for bacterial culturing. While some units were heavily contaminated after just 24 hours of storage, the incidence as a whole varied according to the age of the concentrate and increased with the widespread practice of pooling individual units; over 30% of pools were contaminated at 3 days. See also D. H. Buccholz et al., New Eng. J. Med. 285:429 (1971). While other clinicians suggest lower numbers, recent studies indicate that septic platelet transfusions are significantly underreported. See e.g., J. F. Morrow et al., JAMA 266:555 (1991).
Pre-culturing platelets is not a solution to the bacterial contamination problem. The culture assay takes 48 hours to detect growth. Holding platelet units for an additional two days to await the results of the assay would create, ironically, a smaller margin of safety. See Table 2 in J. F. Morrow et al., JAMA 266:555 (1991). While heavily contaminated units would be detected at the outset, lightly contaminated units would be allowed to grow for two days. Older and potentially more contaminated units would end up being transfused.
Washing the blood cells (e.g., with saline) or filtering the bacteria are also not practical solutions. These techniques are time consuming and inefficient, as they can reduce the number of viable blood cells available for transfusion. Most importantly, they typically involve an "entry" into the storage system. Once an entry is made in a previously closed system, the system is considered "opened," and transfusion must occur quickly, regardless of the manner in which the blood was collected and processed in the first place.
Finally, antibiotics are not a reasonable solution. Contamination occurs from a wide spectrum of organisms. Antibiotics would be needed to cover this spectrum. Many recipients are allergic to antibiotics. In addition, there is an every increasing array of drug-resistant strains of bacteria that would not be inactivated.
In sum, there is a need for a means of inactivating organisms from blood components prior to storage and transfusion in a way that lends itself to use in a closed system. This approach must be able to handle a variety of organisms without harm to the blood product or the transfusion recipient.
The invention generally relates to synthetic media for use with blood preparations intended for in vivo use, including synthetic media used in conjunction with the photodecontamination of platelets. By the term "synthetic media" the present invention intends to indicate aqueous solutions (e.g., phosphate buffered, aqueous salt solutions) other than those found as natural fluids (e.g., plasma, serum, etc.). However, it is not intended that such synthetic media be used without the benefit of natural fluids. Indeed, in preferred embodiments, mixtures of synthetic salt solutions and natural fluids are contemplated.
The present invention contemplates that the activating means comprises a photoactivation device capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm, and in particular, between 320 nm and 380 nm. It is preferred that the intensity is less than 25 mW/cm2 (e.g. between 10 and 20 mW/cm2) and that the mixture is exposed to this intensity for between one and twenty minutes (e.g. ten minutes).
By the phrase "the maximum solubility of psoralen in water" the present invention intends a concentration derived experimentally in an aqueous solution in the absence of organic solvents (e.g., DMSO, ethanol, etc.) at approximately room temperature. Concentrations exceeding this level are detected by the presence of precipitate, which is undesirable for intravenous infusion.
A saturated solution of 8-methoxypsoralen can be made by simply dissolving the compound (over a number of hours at room temperature) in distilled water until precipitate is apparent. If the solution is simply centrifuged, the supernatant can have a concentration of over 50 ug ml. On the other hand, if the solution is filtered (e.g., glass wool), the concentration of 8-methoxypsoralen has been found to be under 50 ug/ml. If, instead of centrifuging or filtering, the saturated solution is dialyzed against distilled water (over a number of days at room temperature), the compound is found to have a maximum solubility of approximately 39 ug/ml. It has been found that, when placed in a container (not glass) to shield the compound from the light, a 0.9% NaCl solution of 8-methoxypsoralen at a concentration of 30 ug/ml is stable.
When weighing components, some experimental variability is expected. The present invention employs the term "approximately" to reflect this variability. This variability is typically plus or minus 5% and usually less than 10%.
In one embodiment, the present invention contemplates a synthetic platelet storage media, comprising an aqueous solution of: 45-120 mM sodium chloride; 5-15 mM sodium citrate; 20-40 mM sodium acetate; and 20-30 mM sodium phosphate. In a preferred embodiment, the aqueous solution comprises: approximately 86 mM sodium chloride; approximately 10 mM sodium citrate; approximately 30 mM sodium acetate; and approximately 26 mM sodium phosphate. The solution has a pH of approximately pH 7.2 and an osmolarity of approximately 300 mOsm/Kg. By not containing glucose or magnesium, the media is readily autoclaved.
The invention generally relates to synthetic media for use with blood preparations intended for in vivo use, including synthetic media used in conjunction with the photodecontamination of platelets.
As noted previously, whole blood is collected and typically separated into red blood cells, platelets, and plasma. Each of these fractions are individually stored under specific conditions prior to in vivo use. In many cases, the extent of contamination is related to the storage time because of growth. A process that inactivated microorganisms at the time of blood collection would be expected to prevent growth during storage.
In one embodiment, the present invention contemplates inactivating blood products after separation but before storage. In this embodiment, a nucleic acid binding compound is selectively employed to treat contamination by microorganisms.
In one embodiment, the nucleic acid binding compound is selected from the group comprising furocoumarins. In a preferred embodiment, the furocoumarin in a psoralen that is activated by a photoactivation device.
Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts to pyrimidine bases upon absorption of longwave ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985); and Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to a psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a second photon can lead to formation of a diadduct which functions as an interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058 (1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982); J. Tessman et al., Biochem. 24:1669 (198S); and Hearst et al., U.S. Pat. Nos. 4,124,589, 4,169,204, and 4,196,281, hereby incorporated by reference.
Psoralens have been shown to inactivate viruses in some blood products. See H. J. Alter et al., The Lancet (ii:1446) (1988); L. Lin et al., Blood 74:517 (1989); and G. P. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, hereby incorporated by reference, describe the use of a combination of 8-methoxypsoralen (8-MOP) and irradiation. They show that 300 ug/ml of 8-MOP together with one hour or more of irradiation with ultraviolet light can effectively inactivate viruses. However, these treatment conditions cause harm to the blood product because of energy transfer. Their approach is only feasible if the damage to cells is specifically suppressed by limiting the concentration of molecular oxygen, a difficult and expensive process.
The inactivation method of the present invention provides a method of inactivating single cell and multicellular organisms, and in particular, bacteria, fungi, mycoplasma and protozoa. In addition, the present invention contemplates inactivation of viruses (e.g. HIV virus). In contrast to previous approaches, the method of the present invention does not cause harm to the blood product. There is no significant damage to cells and, therefore, no need to limit the concentration of molecular oxygen.
The present invention contemplates using much lower concentrations of nucleic acid binding compounds than previously employed. For example, the present invention contemplates using 8-MOP at concentrations of 30 ug/ml or less. Indeed, a preferred concentration of 8-MOP for bacterial decontamination in platelet concentrates is 3 ug/ml or less, i.e., a one hundred-fold lower concentration than employed by G. P. Wiesehahn et al., supra. Because lower concentrations are employed, solvents like DMSO (used to increase the solubility of 8-MOP) are unnecessary.
The present invention, furthermore, contemplates using much lower doses of irradiation than previously described. This is accomplished with lower intensity irradiation sources, with wavelength cutoff filters (see below), and/or shorter irradiation times. In a preferred embodiment, the time of irradiation is variable and controlled from 1 second to 99 minutes, in one second increments.
In one embodiment, the device of the present invention is mounted on an agitator, giving horizontal unidirectional and sinusoidal motion of variable frequency and amplitude. In another embodiment, heat from the lamps, ballasts and other sources is blocked from the bags.
While it is not intended that the present invention be limited by the theory of inactivation, the use of lower compound concentrations and irradiation doses comes from and understanding that, where the present invention is applied to the decontamination of a single cell or multicellular organism, a lower level of nucleic acid binding will achieve inactivation. In addition, it is recognized that it is not essential that inactivation be complete. That is to say, partial inactivation will be adequate as long as the viable portion is unable, within the storage period, to grow to levels sufficient to cause disease.
To appreciate that, in any given case, an inactivation method may or may not achieve complete inactivation, it is useful to consider a specific example. A bacterial culture is said to be sterilized if an aliquot of the culture, when transferred to a fresh culture plate and permitted to grow, is undetectable after a certain time period. The time period and the growth conditions (e.g., temperature) define an "amplification factor". This amplification factor along with the limitations of the detection method (e.g., visual inspection of the culture plate for the appearance of a bacterial colony) define the sensitivity of the inactivation method. A minimal number of viable bacteria must be applied to the plate for a signal to be detectable. With the optimum detection method, this minimal number is 1 bacterial cell. With a suboptimal detection method, the minimal number of bacterial cells applied so that a signal is observed may be much greater than 1. The detection method determines a "threshold" below which the method appears to be completely effective (and above which the method is, in fact, only partially effective).
This interplay between the amplification factor of an assay and the threshold that the detection method defines, can be illustrated. For example, bacterial cells can be applied to a plate; the detection method is arbitrarily chosen to be visual inspection. Assume the growth conditions and time are such that an overall amplification of 104 has occurred. The detectable signal will be proportional to the number of bacterial cells actually present after amplification. For calculation purposes, the detection threshold is taken to be 106 cells; if fewer than 106 cells are present after amplification, no cell colonies are visually detectable and the inactivation method will appear effective. Given the amplification factor of 104 and a detection threshold of 106, the sensitivity limit would be 100 bacterial cells; if less than 100 viable bacterial cells were present in the original aliquot of the bacterial culture after the sterilization method is performed, the culture would still appear to be sterilized.
Such a situation is common for bacterial growth assays. The sensitivity of the assay is such that viable bacterial cells are present but the assay is unable to detect them. This may explain, at least in part, the variability in results obtained by researchers attempting to determine the extent of bacterial contamination of blood products. See D. H. Buchholz, et al., Transfusion 13:268 (1973), wherein such variability is discussed.
It should be noted that, in many countries, contamination of blood products by cellular organisms is more pervasive and, therefore, more serious than viral contamination. For example, in South America, the most important blood-borne organism is T. cruzi, which is the etiologic agent of Chagas disease. Approximately 16-18 million people are infected in the Americas (including 11% of the population of Chile). It is contemplated that the decontamination method of the present invention is well-suited for inactivation of this protozoa.
The present invention contemplates specific synthetic media formulations for use with blood preparations. The formulations of the present invention are particularly useful for platelet storage. These formulations are also useful when employed in conjunction with the photodecontamination of platelets.
While synthetic media have not previously been employed in conjunction with photodecontamination, synthetic media formulations have been described for platelet storage. For example, U.S. Pat. No. 4,828,976 to Murphy stresses the importance of having a synthetic blood platelet storage medium that is essentially free of glucose and calcium. The specification notes that glucose consumption and lactate production continue as long as glucose is present at the concentration above 1.5 mM (Col. 12, line 13). Lactate production is thought to be the cause of loss of pH stability and consequent impairment of platelet function. See also U.S. Pat. Nos. 4,925,665 and 4,992,363.
U.S. Pat. No. 4,447,415 to Rock describes a synthetic medium consisting essentially of: a balanced, physiologically compatible, saline solution; an anticoagulant; and one or more additives to enhance stability of the platelets selected from: (a) nutrients to improve the storage life of the platelets; (b) reversible inhibitors for platelet activation; (c) substances to raise cyclic adenosine monophosphate levels which have reversible effects on platelets; and (d) buffering agents which are physiologically compatible. The specification stresses the need to remove plasma. The specification indicates that this is achieved either by centrifugation and washing, or by "extraction" (see Examples 1 and 2 of the '415 patent). See also U.S. Pat. No. (Reissue) 32,874.
U.S. Pat. No. 4,704,352 to Miripol discloses a medium which contains either magnesium L-ascorbate-2-phosphate or calcium L-ascorbate-2-phosphate. U.S. Pat. No. 4,695,460 to Holme describe media that contain sodium bicarbonate. U.S. Pat. No. 4,390,619 to Harmening-Pittiglio discloses a media capable of supporting platelet metabolism, containing a water-insoluble polymer containing releasable phosphate or bicarbonate ions.
In contrast to these previous attempts, the formulations of the present invention: i) utilize residual plasma; and ii) avoid bicarbonate. With respect to plasma, the present invention contemplates that a standard plasma expression method (e.g., such as currently used in most blood banks) will be used, such that platelets are suspended in a residual plasma concentration between 8 and 25% by volume, and more commonly 12 to 20%. Bicarbonate has been found to be extremely difficult to control in a system employing gas permeable blood bags. The present invention therefore contemplates a phosphate buffer to control the pH of the blood preparation.
The present invention contemplates devices and methods for photoactivation and specifically, for activation of photoreactive nucleic acid-binding compounds. The present invention contemplates devices having an inexpensive source of electromagnetic radiation that is integrated into a unit. In general, the present invention contemplates a photoactivation device for treating photoreactive compounds, comprising: a) means for providing appropriate wavelengths of electromagnetic radiation to cause activation of at least one photoreactive compound; b) means for supporting a plurality of blood products in a fixed relationship with the radiation providing means during activation; and c) means for maintaining the temperature of the blood products within a desired temperature range during activation. The present invention also contemplates methods, comprising: a) supporting a plurality of blood product containers, containing one or more photoreactive compounds, in a fixed relationship with a fluorescent source of electromagnetic radiation; b) irradiating the plurality of blood products simultaneously with said electromagnetic radiation to cause activation of at least one photoreactive compound; and c) maintaining the temperature of the blood products within a desired temperature range during activation.
The major features of one embodiment of the device of the present invention involve: A) an inexpensive source of ultraviolet radiation in a fixed relationship with the means for supporting the sample vessels; B) rapid photoactivation; C) large sample processing; D) temperature control of the irradiated samples; and E) inherent safety.
A. Electromagnetic Radiation Source
A preferred photoactivation device of the present invention has an inexpensive source of ultraviolet radiation in a fixed relationship with the means for supporting the sample vessels. Ultraviolet radiation is a form of energy that occupies a portion of the electromagnetic radiation spectrum (the electromagnetic radiation spectrum ranges from cosmic rays to radio waves). Ultraviolet radiation can come from many natural and artificial sources. Depending on the source of ultraviolet radiation, it may be accompanied by other (non-ultraviolet) types of electromagnetic radiation (e.g., visible light).
Particular types of ultraviolet radiation are herein described in terms of wavelength. Wavelength is herein described in terms of nanometers ("nm"; 10−9 meters). For purposes herein, ultraviolet radiation extends from approximately 180 nm to 400 nm. When a radiation source, by virtue of filters or other means, does not allow radiation below a particular wavelength (e.g., 320 nm), it is said to have a low end "cutoff" at that wavelength (e.g., "a wavelength cutoff at 300 nanometers"). Similarly, when a radiation source allows only radiation below a particular wavelength (e.g., 360 nm), it is said to have a high end "cutoff" at that wavelength (e.g., "a wavelength cutoff at 360 nanometers").
For any photochemical reaction it is desired to eliminate or least minimize any deleterious side reactions. Some of these side reactions can be caused by the excitation of endogenous chromophores that may be present during the photochemical activation procedure. In a system where only nucleic acid and psoralen are present, the endogenous chromophores are the nucleic acid bases themselves. Restricting the activation process to wavelengths greater than 320 nm minimizes direct nucleic acid damage since there is very little absorption by nucleic acids above 313 nm.
In blood products, the nucleic acid is typically present together with additional biological chromophores. If the biological fluid is just protein, the 320 nm cutoff will be adequate for minimizing side reactions (aromatic amino acids do not absorb above 320 nm). If the biological fluid includes cells and/or cellular constituents, there will be many other chromophores, including hemes and flavins.
Hemes are abundant in blood products where they arise from the lysis of red cells. Flavins, like hemes, are required for metabolic respiration. Both of these endogenous chromophores will cause damage to cells if excited by photoirradiation.
Hemes have three principle absorption bands: two are in the red region of the visible spectrum; the other is centered about 400 nm. Flavins have two principle absorption peaks: one at 450 nm and the other at 370 nm.
In view of the presence of these endogenous chromophores in blood products, it is intended that the device of the present invention be designed to allow for irradiation within a small range of specific and desirable wavelengths, and thus avoid damage to cells caused by energy transfer. The preferred range of desirable wavelengths is between 320 and 350 nm.
Some selectivity can be achieved by choice of commercial irradiation sources. For example, while typical fluorescent tubes emit wavelengths ranging from 300 nm to above 400 nm (with a broad peak centered around 360 nm), BLB type fluorescent lamps are designed to remove wavelengths above 400 nm. This, however, only provides an upper end cutoff.
In a preferred embodiment, the device of the present invention comprises an additional filtering means. In one embodiment, the filtering means comprises a glass cut-off filter, such as a piece of Cobalt glass. In another embodiment, the filtering means comprises a liquid filter solution that transmit only a specific region of the electromagnetic spectrum, such as an aqueous solution of Co(No3)2. This salt solution yields a transmission window of 320-400 nm. In a preferred embodiment, the aqueous solution of Co(No3)2 is used in combination with NiSO4 to remove the 365 nm component of the emission spectrum of the fluorescent or arc source employed. The Co—Ni solution preserves its initial transmission remarkably well even after tens of hours of exposure to the direct light of high energy sources.
It is not intended that the present invention be limited by the particular filter employed. Several inorganic salts and glasses satisfy the necessary requirements. For example, cupric sulfate is a most useful general filter for removing the infra-red, when only the ultraviolet is to be isolated. Its stability in intense sources is quite good. Other salts are known to one skilled in the art. Aperture or reflector lamps may also be used to achieve specific wavelengths and intensities.
When ultraviolet radiation is herein described in terms of irradiance, it is expressed in terms of intensity flux (milliwatts per square centimeter or "mW cm−2"). "Output" is herein defined to encompass both the emission of radiation (yes or no; on or off) as well as the level of irradiance. In a preferred embodiment, intensity is monitored at 4 locations: 2 for each side of the plane of irradiation.
A preferred source of ultraviolet radiation is a fluorescent source. Fluorescence is a special case of luminescence. Luminescence involves the absorption of electromagnetic radiation by a substance and the conversion of the energy into radiation of a different wavelength. With fluorescence, the substance that is excited by the electromagnetic radiation returns to its ground state by emitting a quantum of electromagnetic radiation. While fluorescent sources have heretofore been thought to be of too low intensity to be useful for photoactivation, in one embodiment the present invention employs fluorescent sources to achieve results thus far achievable on only expensive equipment.
As used here, fixed relationship is defined as comprising a fixed distance and geometry between the sample and the light source during the sample irradiation. Distance relates to the distance between the source and the sample as it is supported. It is known that light intensity from a point source is inversely related to the square of the distance from the point source. Thus, small changes in the distance from the source can have a drastic impact on intensity. Since changes in intensity can impact photoactivation results, changes in distance are avoided in the devices of the present invention. This provides reproducibility and repeatability.
Geometry relates to the positioning of the light source. For example, it can be imagined that light sources could be placed around the sample holder in many ways (on the sides, on the bottom, in a circle, etc.). The geometry used in a preferred embodiment of the present invention allows for uniform light exposure of appropriate intensity for rapid photoactivation. The geometry of a preferred device of the present invention involves multiple sources of linear lamps as opposed to single point sources. In addition, there are several reflective surfaces and several absorptive surfaces. Because of this complicated geometry, changes in the location or number of the lamps relative to the position of the samples to be irradiated are to be avoided in that such changes will result in intensity changes.
B. Rapid Photoactivation
The light source of the preferred embodiment of the present invention allows for rapid photoactivation. The intensity characteristics of the irradiation device have been selected to be convenient with the anticipation that many sets of multiple samples may need to be processed. With this anticipation, a fifteen minute exposure time or less is a practical goal.
In designing the devices of the present invention, relative position of the elements of the preferred device have been optimized to allow for fifteen minutes of irradiation time, so that, when measured for the wavelengths between 320 and 350 nanometers, an intensity flux greater than approximately 1 mW cm−2 is provided to the sample vessels. In a preferred embodiment, the device irradiates both sides of the bag.
C. Processing of Large Numbers of Samples
As noted, another important feature of the photoactivation devices of the present invention is that they provide for the processing of large numbers of samples. In this regard, one element of the devices of the present invention is a means for supporting a plurality of blood products, and in particular, blood bags. In the preferred embodiment of the present invention the supporting means comprises glass plates between two banks of lights with a capacity of six 50 ml bags (equivalent to Dupont Stericell bag) plus connectors and tubing, at one time. By accepting commonly used commercially available blood bags, the device of the present invention allows for convenient processing of large numbers of samples.
D. Temperature Control
As noted, one of the important features of the photoactivation devices of the present invention is temperature control. Temperature control is important because the temperature of the sample in the sample at the time of exposure to light can dramatically impact the results. For example, conditions that promote secondary structure in nucleic acids also enhance the affinity constants of many psoralen derivatives for nucleic acids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). These conditions are a mix of both solvent composition and temperature. With single stranded 5S ribosomal RNA, irradiation at low temperatures enhances the covalent addition of HMT to 5S rRNA by two fold at 4� C. compared to 20� C. Thompson et al., J. Mol. Biol. 147:417 (1981). Even further temperature induced enhancements of psoralen binding have been reported with synthetic polynucleotides. Thompson et al., Biochemistry 21:1363 (1982).
With respect to bacteria, it should be noted that repair of crosslinks occurs during irradiation. However, where a lower temperature is employed during irradiation, the bacterial repair process is suppressed. Thus, a 15� C. irradiation has a significant effect on the level of inactivation that is observed.
E. Inherent Safety
Ultraviolet radiation can cause severe burns. Depending on the nature of the exposure, it may also be carcinogenic. The light source of a preferred embodiment of the present invention is shielded from the user. This is in contrast to the commercial hand-held ultraviolet sources as well as the large, high intensity sources. In a preferred embodiment, the irradiation source is contained within a housing made of material that obstructs the transmission of radiant energy (i.e., an opaque housing). No irradiation is allowed to pass to the user. This allows for inherent safety for the user.
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); � C. (degrees Centigrade); HPLC (High Pressure Liquid Chromatography).
In some of the examples below, phosphate buffered synthetic media is formulated for platelet storage and treatment. One embodiment is made as follows:
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
As noted above, the present invention contemplates devices and methods for the activation of photoreactive nucleic acid binding compounds. In this example, a photoactivation device is described for decontaminating blood products according to the method of the present invention. This device comprises: a) means for providing appropriate wavelengths of electromagnetic radiation to cause activation of at least one photoreactive compound; b) means for supporting a plurality of blood products in a fixed relationship with the radiation providing means during activation; and c) means for maintaining the temperature of the blood products within a desired temperature range during activation.
The bulbs (101), which are connectable to a power source (not shown), serve as a source of electromagnetic radiation. While not limited to the particular bulb type, the embodiment is configured to accept an industry standard, dual bipin lamp.
The housing (101) can be opened via a latch (105) so that the blood product can be placed appropriately. As shown in
The housing (100) also serves as a mount for several electronic components on a control board (107), including, by way of example, a main power switch, a count down timer, and an hour meter. For convenience, the power switch can be wired to the count down timer which in turn is wired in parallel to an hour meter and to the source of the electromagnetic radiation. The count down timer permits a user to preset the irradiation time to a desired level of exposure. The hour meter maintains a record of the total number of radiation hours that are provided by the source of electromagnetic radiation. This feature permits the bulbs (101) to be monitored and changed before their output diminishes below a minimum level necessary for rapid photoactivation.
Detectors (110A, 110B, 110C, 110D) may be conveniently placed between the plates (103A, 103B, 104A, 104B) of the plate assemblies (103, 104). They can be wired to a printed circuit board (111) which in turn is wired to the control board (107).
In one capture approach, a bag comprised of a psoralen-binding polymer is employed to capture the compound. The Cutter CLX bag has been found to have this property.
In this example, the decontamination methods of the present invention are applied to inactivate Klebsiella pneumoniae, which is known to be among the organisms associated with bacteremia. See generally, Infect. Control 5:343 (1984). The particular isolate in this case was obtained following a platelet transfusion where the recipient immediately went into shock and later died. The platelet bag was obtained and cultured, and the organism was identified and serotyped.
For the experiment, the strain was kept at ambient temperature and inoculated onto either heart infusion agar (HIA) or heart infusion agar containing 5% (v/v) sheep blood (BAP) by swabbing each plate for confluency via a sterile applicator swab. Cultures were then incubated under static conditions for 16-18 h at 35� C. At the end of the incubation period, cultures were removed and suspended in phosphate buffered saline (PBS;pH 7.2-7.4) and spectrophotometrically standardized to 1.0 at an OD610 using a Spectronic 501 or 601 spectrophotometer (Bausch and Lomb). After standardization, suspensions were diluted 1:10 in PBS to achieve an ca. 108 CFU/ml concentration. This standardized suspension is then split to use an aliquot for the inactivation study, while another portion was plated in duplicate 10-fold serial dilutions onto HIA (or BAP) to ensure appropriate concentrations of the organism.
To assess inactivation of the organism, two ABO compatible freshly outdated human platelet concentrate units were obtained from the Blood Bank of Alameda—Contra Costa Medical Association. They were pooled and redivided into two bags. One bag was infused with the bacteria preparation. The platelets in the second unit were pelleted at 4000�g for 6 minutes and then resuspended in a medium containing 85% saline and 15% plasma. Bacteria was added after platelets were well resuspended.
3 ml aliquots of bacteria containing platelet concentrate were transferred to a Teflon™ minibag (American Fluoroseal Corporation, Silver Spring, Md.) and received specified amounts of 8-MOP and UVA irradiation, except for the controls, which were irradiated without psoralen, or received no treatment. Temperature was maintained at 25� C. during irradiation in the irradiation device described above which is equipped with an air cooling mechanism.
F24T 12-BL-HO fluorescent lamps were used. These are high output "black light" tubes (engineered to emit specific wavelengths by means of an internal phosphor coating) 24 inches in length. Total intensity is less than 25 mw/cm2 and typically between 15 and 20 mw/cm2.
Following irradiation, bacteria were quantified by plating 0.1 ml of serial 10-fold dilutions in broth onto 100 mm petri dishes containing BHI agar. After 24 hr incubation at 35� C., colonies were counted and bacterial concentration was calculated on a per ml basis. The results (
While not limited to any theory as to the mechanism by which this improvement in-decontamination efficiency was achieved, it would appear that these results indicate that either that the optical properties of the synthetic medium are better, or the lower protein concentration allows for a higher concentration of free 8-MOP. In the latter situation, the 8-MOP would be more available for decontamination.
Artuc and co-workers examined the solubility of 8-MOP in human and bovine serum proteins. They showed that, when working with 8-MOP concentrations ranging from 100 to 1000 ng/ml in serum, 75% to 80% of the 8-MOP was bound to albumin. M. Artuc et al., Brit. J. Derm. 101:669 (1979).
In this example, the binding of 8-MOP to Calf Thymus DNA is compared using plasma and a protein free media in order to validate the efficiency of psoralen-nucleic interactions under the decontamination methods of the present invention. Although this measurement used eukaryotic nucleic acid rather than bacterial nucleic acid, it is a useful indicator of the degree of adduct formation for bacteria.
3H-8-MOP was prepared to a concentration of 115 ug/ml in ethanol at a specific activity of 4.7�106 CPM/microgram (hereinafter "8-MOP stock"). Thereafter 130.5 or 22 ul of 8-MOP stock (2 each) for samples containing DNA ("+DNA") and 52.2 or 8.7 ul for samples not containing DNA ("−DNA") were dried down. To +DNA samples, 40 ul of DNA stock (7.7 mg/ml) was added as well as either 460 ul plasma (day old frozen) or 450 ul Tris-EDTA ("TE") buffer. To the latter was also added 10 ul 5M NaCl. For −DNA samples (i.e., the controls), 184 ul plasma and 16 ul water was added.
The samples were mildly vortexed for approximately one hour and the counts were checked to confirm that the 8-MOP dissolved.
Each sample (100 ul) was irradiated on an HRI-100 (HRI Research Inc., Concord, Calif.) at 25� C. for 0, 2, 4, 8, and 16 minutes. Samples were kept at 4� C. overnight after irradiation. Thereafter, the samples were extracted. First, a phenol solution was prepared at pH 8 by equilibrating with 0.1 M Tris pH 8. Each sample was then extracted with 100 ul phenol. Each sample was centrifuged for 5 minutes to remove the aqueous phase to a new tube. A second extraction was performed with 100 ul 1:1 phenol:chloroform. A final extraction was performed with 100 ul chloroform.
The final aqueous phase was precipitated by adding 50 ul NaCl adjusted to give a final concentration of NaCl of 0.2 M and then adding 250 ul ethanol. The samples were again centrifuged (10 minutes). The supernatant was removed and the pellets were dried. The pellets were resuspended in 100 ul TE and reprecipitated. This was repeated for a total of 3 precipitations. The final pellets were brought up in 600 ul water and 100 ul was counted. Each sample was assayed for DNA by measuring absorbance (260 nm). 8-MOP levels were plotted as adducts per 1000 base pairs ("8-MOP:kBP").
The results (
The frequency of 8-MOP-DNA adduct formation in protein free media predicts a high multiplicity of modification of the bacterial genome. Furthermore, this type of biochemical measurement has the potential to provide a means to monitor the efficiency of the photochemical inactivation method.
Photoactivation of psoralens and isopsoralens may result in a variety of photoproducts. "Photoproduct" is best understood by considering the possible reactions of photoreactive compound when exposed to activating wavelengths of electromagnetic radiation. While not limited to any precise mechanism, it is believed that the reaction of photoreactive compound in its ground state ("C") with activating wavelengths of electromagnetic radiation creates a short-lived excited species ("C*"):
With this reaction described, one can now consider the situation where nucleic acid is not available for binding at the time the compound is exposed to activating wavelengths of electromagnetic radiation. Since the excited species is short-lived and has no nucleic acid to react with, the excited species may simply return to its ground state:
The excited species is not limited to reacting with itself. It may react with its environment, such as elements of the solvent ("E") (e.g., ions, gases, etc.) to produce other products:
The present invention and the understanding of "photoproduct" does not depend on which one (if any) of these reactions actually occurs. "Photoproduct"—whatever its nature—is deemed to exist if, following the reaction of a compound and activating wavelengths of electromagnetic radiation, there is a resultant product formed that can interact with other components of the reaction environment.
With psoralens such as 4′-hydroxymethyl-4,5′,8-tri-methylpsoralen (HMT), there are a number of resultant products produced when the HMT is exposed to activating wavelengths of electromagnetic radiation. The major resultant products of HMT are two cyclobutyl photodimers. In one of the dimers, the two pyrone rings are linked in a cis-syn configuration, while in the other dimer, the linkage occurs between the furan end of one molecule and the pyrone end of the other, again with cis-syn configuration. A third resultant product of HMT is a monomeric HMT photoisomer. In this isomer, the central ring oxygens assume a 1, 4 instead of the normal 1, 3 orientation. While the two photodimers would not be expected to have an intercalating activity due to geometrical considerations, the photoisomer remains planer, and accordingly, it is contemplated that it has a positive intercalative association with double stranded nucleic acid and, thus, could be a mutagen.
In this example, the photochemical breakdown of 8-MOP is compared with AMT. The samples were analyzed by reverse phase HPLC using a Rainen Dynamax 300A column. Gradient elution was performed with 0.1 M ammonium acetate/acetonitrile (0-70% acetonitrile over 42 minutes). AMT elutes as a single peak at approximately 24 minutes under these conditions. Detection was by absorption at either 260 or 330 nm. The latter wavelength was used for the plasma containing samples.
Standard solutions of each compound were prepared at various concentrations. These solutions were then diluted 1:10 into water, then 300 ul injected for analysis. All samples were monitored at 300 nm. Peaks were analyzed by measuring either peak height or peak area, then converted to a gh/ml value using the standard plot. Peak area was determining by photocopying the trace, cutting out the copy of the peak, then weighing the resultant trace. The two methods gave essentially the same result.
The results are shown in FIG. 7. Clearly, AMT degrades more quickly than 8-MOP. It would, therefore, be expected to generate more photoproducts—which eventually would end up in the transfusion recipient. By contrast, it is not expected that 8-MOP generates a significant amount of photoproducts. This is important when one considers that the weight of authority has concluded that unactivated 8-MOP is nonmutagenic.
One of the bags (301) of the two bag set (300) contains a synthetic media formulation of the present invention (indicated as "STERILYTE"). In the second step shown in
Following phototreatment, the platelets are transferred from the synthetic media bag (301) into the storage bag (302) of the two bag set (300). The storage bag can be a commercially available storage bag (e.g., CLX bag from Cutter).
From the above, it should be evident that the present invention provides synthetic media formulations for use with blood preparations intended for storage and in vivo use. The formulations include synthetic media formulations to be employed in conjunction with the photodecontamination of platelets.
Patent Citations
Non-Patent Citations
Referenced by
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text:
Primal Scream-Inducing
By dankempster 4 Comments
It's not very often that I get the urge to give up on a game. Sure, I'm prone to putting titles down when I tire of them. I think we all are, to some extent. But I'll always endeavour to pick them back up at a later date and plough on through to the end. For me to irrevocably say "fuck you" to a game is rare. I think the last time I experienced any such feeling was way back in 2005, when the eagerly-awaited Unlimited SaGa turned out to be nothing like what I was expecting. Five years later, it seems to be happening again.
Unfortunately, that couldn't be further from the truth. The promise of fleshed-out worlds is countered with environments that look and feel sparse and empty, leaving me with no motivation to explore them. The environmental puzzles are few and far between, meaning that most of the game seems to be spent running from one end of the environment to the other in order to trigger the next story-advancing cut-scene. As great as the game's graphical presentation is, its interface is clunky and hideous. Worst of all, though, is the combat, which is mapped to the shoulder buttons in such an unwieldy fashion that I seem to be spending most of my time fleeing from enemies just so I don't have to suffer the abysmal controls. Bear in mind, this is coming from somebody who's spent parts of this year with tank controls, characters who plant their feet when they shoot, and camera control mapped to the face buttons. Questionable control schemes are something I usually have no trouble overcoming, but Primal has pushed my tolerance to the absolute limit.
As things stand, I think Primal is going to be the first game in five years to get the brush-off from me. Nobody is more disappointed by this more than me, I can assure you. Ever since I ordered the game last year, I've been excited about the prospect of dipping into its world - a world that is clearly a labour of love, and constructed from themes that really appeal to me. To discover that my ticket into that world has been invalidated by the title's core gameplay is disheartening to say the least. Maybe I'll give Primal one last chance and go back to it over Christmas, or perhaps even next Summer, when I might be more willing to forgive its flaws. Right now, though, I'm not prepared to spend any more time with it, and back on the Pile of Shame it shall go.
With University back in full swing, my time available to play games has once again dwindled away. Most of my game-playing outside of Primal has been spent on titles that are fun to play, but don't demand much attention or commitment in terms of story. My go-to title in this regard has been FIFA 11. It's been four years since Pro Evolution Soccer 6 marked the last time I got excited about a sports game release. After two years of disappointment having spent full-price on Konami's flagship soccer series, I decided to sit out last year, and ended up returning to the field this year after switching teams. I bought FIFA 11 purely on the strength of its demo, and I don't regret a single penny of that £35 purchase. After some abysmal online performances I've confined myself to the game's Career mode, where I'm having a blast playing for Watford. While Pro Evos 2008 and 2009 failed to hold my attention for any longer than a couple of months, I already get the feeling that FIFA 11 could keep me busy not just over the next twelve months, but well into the year beyond that as well. I guess only time will tell.
I've also been dabbling with a couple of Xbox LIVE Arcade titles over the last couple of weeks. The first of these is Sonic the Hedgehog 4: Episode One. As somebody who effectively passed through the gateway to video games via Sonic the Hedgehog 2, I felt somewhat obliged to pick this up back when it released last week. I'm not going to go into any great detail about the game here, so if you'd like to know more about what I thought of it, be sure to check out my review. The other downloadable gem that I've been spending the last couple of days with is Super Meat Boy. I'm not usually one to gravitate towards titles bearing the masocore label, but something about Super Meat Boy seemed irresistible to me (actually, it was probably more the fact that I still had 800 Points kicking around after buying Sonic 4, and I seem totally incapable of leaving any residual Points unspent). I've only played through the first "world", but there's something oddly compelling about its simple gameplay and demand for pixel-perfect jumps. Chances are I'll never finish it, but whatever time I do end up committing to Super Meat Boy I know will be time well-spent and enjoyed.
A final note to those of you who are sitting, waiting patiently and twiddling your thumbs in anticipation of Episode Twelve of Enduring Final Fantasy VII. I must be sounding like a broken record at this point, but I promise I haven't forgotten about the series and I have no intention of stopping. I'm currently in a position where I have the next three episodes planned and ready to write up, but I just don't have the will or the desire to sit down and pump whole afternoons into writing them. I've hit something of a stride in my creative writing recently, too, to the point where I'd much rather be doing that than blogging here on Giant Bomb (hell, the only reason I'm writing this blog is to let you all know that I didn't die during Freshers' Week). When I have more time and more motivation, I will pick the series back up. For now, though, consider it on hiatus. I'm sorry, but I'd rather write those blogs at a time when I want to write them, than at a time when I feel like I have to write them. They'll wind up better that way.
As always, thanks very much for reading. I'll see you around.
Currently playing - Sonic the Hedgehog 4: Episode One (XBLA)

subset: commoncrawl


text:
Husein Alicajic
Husein Alicajic is an Australian filmmaker who writes and directs for film and television,[1] known for award-winning commercials for Foxtel. [2][3]
Born in Sydney to a Bosnian father and a Scottish mother, Alicajic attained degrees in Economics (Hons), Philosophy and Communications before deciding to become a filmmaker.
With director Alex Proyas, Alicajic created a pre-trailer for the 20th Century Fox film I, Robot.[3] In 2008, while with Aritmetic, Alicajic produced a campaign for Foxtel, winning a gold Promax Award.[4] In 2009 he moved from Aritmetic and joined Photoplay.[2][5]
Alicajic collaborated with UK writer Jeff Noon on his IF award-winning script Divine Shadows,[6] which is currently in pre-production for shooting to begin in 2010.[3] As part of a two-picture deal Alicajic has also signed to direct the AMPAS Nicholl Fellowship winning feature screenplay The Secret Boy.[3]
Partial filmography[edit]
- The Red Room (1997)
- Beginnings (2002)
- Still Life (2005/IV)
- Harry the Hunchback (2005)
- Aquamarine (2006)
Awards and nominations[edit]
- 2002, Won Dendy Award for 'Best Short Film' Beginnings at the Sydney Film Festival
- 2005, Won Inside Film Award for 'Best Unproduced Screenplay' Divine Shadows.[6][7]
- 2008, Won Promax Award for 'Best in Show Gold' for his Foxtel Summer campaign
- 2009, Won Golden Trailer Award for 'Best Unproduced Movie Trailer'[3]
- ^ "Husein Alicajic". Screen Australia. Retrieved 6 March 2010.
- ^ a b "HUSEIN ALICAJIC". Photoplay. Retrieved 6 March 2010.
- ^ a b c d e "Wow filmmaker's forum: Director's Vision Through The Cinematographer's Lens". WOW Film Festival. Retrieved 6 March 2010.
- ^ "Kamen Brothers + Husein Alicajic to Photoplay". Campaign Brief. 17 September 2009. Retrieved 6 March 2010.
- ^ "New to Photoplay". Australian Creative. 18 September 2009. Retrieved 6 March 2010.
- ^ a b "Lantana scores big at Australian IF awards". Screen Daily. 11 November 2001. Retrieved 6 March 2010.
- ^ "2001 Inside Film Awards". Inside Film Awards. Retrieved 6 March 2010.
External links[edit]
|This biographical article related to film in Australia is a stub. You can help Wikipedia by expanding it.


text:
Teen pregnancy prevention starts with a foundation in healthy relationships. How do you talk to your young people about healthy relationships?
Teen pregnancy and teen childbearing affect several critical social issues, including poverty and income disparity, overall child well-being, child welfare, out-of-wedlock births, healthy relationships and marriage, responsible fatherhood, health issues, education, substance use and other risky behaviors, and violence.
The Alabama Campaign to Prevent Teen Pregnancy recognizes the complexities of these social and economic issues and believes teen pregnancy prevention requires the thoughtful attention and deliberate action of parents, youth, community leaders, faith leaders, educators, policy-makers, health professionals, and others who have a desire to ensure Alabama's youth live healthy, fulfilling lives.
Sexual Health Education Survey
The Alabama Alliance for Healthy Youth has developed a survey for all Alabama residents to assess their opinions on sexual health education. Please take 2-3 minutes to fill out this important survey!...Read More...
In late May the Washington Post featured an op-ed piece written by three former Surgeon Generals. The piece highlighted the need for comprehensive sex education for all young people, and was a call to action for policy makers, administrators, teacher...Read More...
One July 21-22, 2015, The FOCUS Program will offer its summer conference in Clanton, AL. This conference is for youth-serving professionals working in schools. Registration is due by July 14, 2015....Read More...
Bedsider, a project of the National Campaign, is an online birth control support network for older adolescents. In 2014 an independent evaluation of the network was launched, and the results are in. Among the significant findings were that women who...Read More...
Our 12th Annual Conference, Insights, Answers, and Opportunities, was a great success, thanks to our conference participants and excellent presenters. Feedback from the conference was positive, and we are happy we can provide this content to the yout...Read More...View All News


text:
Simple Sudoku :) Free
By Lanner
Game CenterView More by This Developer
Open iTunes to buy and download apps.
1.Unlimited sudokus in 4 different difficulty levels.
2.Game Center support for leaderboards and achievements.
3.Support for iOS 7.
4.Support for hint.
5.Support for note.
6.Support for Undo and Redo.
7.Funny animations and sharp graphics.
What's New in Version 2.4.1
Bug fixed.
View In iTunes
This app is designed for both iPhone and iPad
- Free
- Category: Games
- Updated: Jun 29, 2014
- Version: 2.4.1
- Size: 5.7 MB
- Languages: English, German, Japanese, Simplified Chinese, Traditional Chinese
- Seller: Yuangang Sheng
- © com.app510
Compatibility: Requires iOS 6.0 or later. Compatible with iPhone, iPad, and iPod touch. This app is optimized for iPhone 5.


text:
Hello to you here on Audiko.net! We offer you to download a free Finley ringtones for your iPhone or mobile. Elect your favorite trim from your favorite piece of music - Gruppo Randa, Diventerai una Star, Ad Occhi Chiusi, or any other track. Then download the prepared Finley ringtones to your phone, and enjoy Italian music by Finley every time your phone rings. If you enjoy Italian or maybe Italian Rock and punk rock music, then you will probably like ELISA, Vasco Rossi, or Ligabue as well. For all who want a free Finley ringtone, we also recommend listening to Pop Rock, Pop Punk, and Emo. You may obtain your ringtone both in the MP3 and m4a formats, compatible with both mobile phones and iPhones. Just select a track, and choose free Finley ringtones download option.
Download Finley Ringtones
Top free ringtones
|Diventerai una Star
|Il tempo di un minuto
|Sole di Settembre
|Scegli Me
|Gruppo Randa
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- Meek Mill - All Eyes On You (feat. Chris Brown & Nicki Minaj)
- Rihanna - Bitch Better Have My Money
- omi - Cheerleader
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- Taylor Swift - Bad Blood (feat. Kendrick Lamar)
- Trey Songz - Slow Motion
- Young Thug - Check
- Future - Trap Niggas
- Tink - Treat Me Like Somebody
- Tink - Million


text:
La Cage Aux Folles OUT Night
7:30pm Thu, Oct 3, 2013
Venue: North Shore Music Theatre
62 Dunham Road, Beverly, MA 01915Contact:
Karen Nascembeni
0 people saved this
La Cage Aux Folles OUT at the North Shore. An Evening for the Gay and Lesbian Community!
10 #NoSummerRegrets Scenes Showing June at Its Best
Summer is here, and that means plenty of sunshine and breezy temperatures: the perfect conditions...
Search All Listings Here.


text:
I am trying to connect to remote sftp server over ssh with JSch (0.1.44-1) but during "session.connect();" I am getting this exception:
com.jcraft.jsch.JSchException: Algorithm negotiation fail
at com.jcraft.jsch.Session.receive_kexinit(Session.java:529)
at com.jcraft.jsch.Session.connect(Session.java:291)
at com.jcraft.jsch.Session.connect(Session.java:154)
Logs from JSch:
INFO: Connecting to xx.xx.xx.xxport 22
INFO: Connection established
INFO: Remote version string: SSH-2.0-WeOnlyDo 2.0.6
INFO: Local version string: SSH-2.0-JSCH-0.1.44
INFO: CheckCiphers: aes256-ctr,aes192-ctr,aes128-ctr,aes256-cbc,aes192-cbc,aes128-cbc,3des-ctr,arcfour,arcfour128,arcfour256
INFO: aes256-ctr is not available.
INFO: aes192-ctr is not available.
INFO: aes256-cbc is not available.
INFO: aes192-cbc is not available.
INFO: arcfour256 is not available.
INFO: SSH_MSG_KEXINIT sent
INFO: SSH_MSG_KEXINIT received
INFO: Disconnecting from xx.xx.xx.xx port 22
I am able to log in to remote server with linux sftp command. I was trying to find any kind of clue in the internet but I failed. Any help would be greatly appreciated.
Marek Dominiak
Debug output from sftp console:
OpenSSH_5.5p1-DAM_1.2, OpenSSL 0.9.8r 8 Feb 2011
debug1: Reading configuration data /etc/DAM/ssh/ssh_config
debug1: Applying options for *
debug1: Applying options for *.*
debug1: Connecting to xx.xx.xx.xx port 22.
debug1: Connection established.
debug1: identity file /**/spv_id_rsa.key type -1
debug1: identity file /**/spv_id_rsa.key-cert type -1
debug1: Remote protocol version 2.0, remote software version WeOnlyDo 2.0.6
debug1: no match: WeOnlyDo 2.0.6
debug1: Enabling compatibility mode for protocol 2.0
debug1: Local version string SSH-2.0-OpenSSH_5.5p1-DAM_1.2
debug1: SSH2_MSG_KEXINIT sent
debug1: SSH2_MSG_KEXINIT received
debug1: kex: server->client aes256-cbc hmac-md5 none
debug1: kex: client->server aes256-cbc hmac-md5 none
debug1: sending SSH2_MSG_KEXDH_INIT
debug1: expecting SSH2_MSG_KEXDH_REPLY
debug1: Host 'xx.xx.xx.xx' is known and matches the RSA host key.
debug1: Found key in ~/.ssh/known_hosts:8
debug1: ssh_rsa_verify: signature correct
debug1: SSH2_MSG_NEWKEYS sent
debug1: expecting SSH2_MSG_NEWKEYS
debug1: SSH2_MSG_NEWKEYS received
debug1: Roaming not allowed by server
debug1: SSH2_MSG_SERVICE_REQUEST sent
debug1: SSH2_MSG_SERVICE_ACCEPT received
debug1: Authentications that can continue: publickey
debug1: Next authentication method: publickey
debug1: Trying private key: /**/spv_id_rsa.key
debug1: read PEM private key done: type RSA
debug1: Authentication succeeded (publickey).
debug1: channel 0: new
debug1: Entering interactive session.
debug1: Sending subsystem: sftp
Connected to xx.xx.xx.xx.
Ok the problem is solved, I have found that I the remote server had only these encryption options: .
To enable aes256-cbc I had to apply "Java Cryptography Extension (JCE) Unlimited Strength Jurisdiction Policy Files" as it was written in http://www.jcraft.com/jsch/README.
After this I can connect to remote server without problems.


text:
News from Ali Curung, Alice Springs, Brunette Downs, Jervois, Kulgera, Tennant Creek, Territory Grape Farm, Uluru, Watarrka, Yuendumu and surrounding areas.
Alice Springs Local News
Latest News
Perfect conditions have greeted visitors to the Alice Springs Show, and the animal pavilions are again among the most visited ex...Friday 3 July 2015 4:10pm
The great-grandson of renowned Aboriginal artist Albert Namatjira is sentenced to eight-and-a-half years in prison for the mansl...Thursday 2 July 2015 5:36pm
A pen of 87 hereford-cross-angus steers has set a record price for the Alice Springs Show cattle sale, reaching $2.94 per kilo...Thursday 2 July 2015 5:08pm
Alice Springs painters Marina Strocchi and Wayne Eager launch a joint exhibition at the heritage-listed Residency building.Thursday 2 July 2015 4:04pm
Police praise Territory Day revellers after paramedics enjoy the best cracker night in memory, with relatively few serious incid...Thursday 2 July 2015 9:37am
A man dies in Alice Springs after a woman police believe he knew ran him over.Thursday 2 July 2015 7:12am
The month of June provided some unusual weather events for the Northern Territory.Wednesday 1 July 2015 3:24pm
Thirty years ago Tom Vandeler was a South Australian grain and sheep farmer. These days he trains pigs to fly.Wednesday 1 July 2015 10:56am
A Barkly Tablelands cattleman has expressed safety concerns about the shutdown of the Tennant Creek weather radar in the Norther...Tuesday 30 June 2015 2:41pm
Pastoralists and locals have slammed the decision to the shut down the Tennant Creek weather radar and are amazed by the lack of...Monday 29 June 2015 5:35pm
A man dies after being hit by a light truck near Elliott, halfway between Darwin and Alice Springs on Monday morning, the day af...Monday 29 June 2015 2:56pm
Northern Territory Police are forced to retreat from a town camp near Alice Springs for the second night in a row as rocks are t...Sunday 28 June 2015 10:03am
Two people are dead and two people are seriously injured after three separate weekend car crashes in the Northern Territory.Saturday 27 June 2015 9:05am
Young and old embrace the enduring allure of Toastmasters in Alice Springs.Friday 26 June 2015 11:21am
As part of testing the water for more land releases, the Northern Territory Government has sold two 1000-hectare lots in Ti Tree...Wednesday 24 June 2015 5:36pm
A Red Centre emu thinks it is a horse.Wednesday 24 June 2015 2:48pm
Calls for 'conversation' on banning pet cats in Alice Springs after video of tabbies hunting wildlifeA conservationist is calling for a "conversation" on prohibiting pet cats in Alice Springs after a feral cat video monitoring pr...Wednesday 24 June 2015 1:36pm
Students from 10 Alice Springs schools, including School of the Air, have just finished painting cattle.Tuesday 23 June 2015 2:28pm
A Northern Territory artist begins documenting the peculiar art of decorating termite mounds, a practise which is an outback tra...Tuesday 23 June 2015 2:27pm
A Northern Territory Government program will see $850,000 of public money given to nine mining exploration companies in a bid to...Tuesday 23 June 2015 9:53am
Former Country Liberals president Sue Fraser-Adams becomes latest woman to leave Northern Territory partyFormer Country Liberals president Sue Fraser-Adams becomes the latest high-profile woman to resign her membership of the party.Monday 22 June 2015 1:49pm
A man who went on a New Year's Day rampage in a four-wheel drive in Alice Springs while high on ice is jailed for almost five ye...Monday 22 June 2015 11:51am
After years of buck-passing about bodies rotting at home, coronial investigations being put at risk and cadavers criss-crossing ...Monday 22 June 2015 11:45am
The iconic outback event, the Brunette Downs Races, in its 105th year, were held on the Barkly Tableland in the Northern Territo...Monday 22 June 2015 11:28am
A 52-year-old man dies in what police believe is a domestic incident in Alice Springs.Sunday 21 June 2015 11:30pm
ABC News: NT Edition
News and information, from your state, for your state
Around the clock coverage of news events as they break.Watch Now
Latest videos
Latest audio
- 15-year-old William Weir from Ammaroo Station has cleaned up at the Alice Springs Show
- Geoff Miers is one of the horticulture stewards at the Alice Springs Show.
- Thirty years ago Tom Vandeler was a South Australian grain and sheep farmer. These days he trains pigs to fly.
- Barkly Tablelands cattleman Chris Towne has expressed safety concerns about the shutdown of the Tennant Creek weather radar in the Northern Territory.
- The Bureau of Meteorology's (BOM) acting regional director Ben Suter said he understood frustration about the short notice but said the BOM had been c