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Institut für Laserphysik
für Laserphysik (ILP)
für Laserphysik (ILP)
Dieter Haupt leads the Electronic Workshop in the group of Prof. Dr. Roman Schnabel at the ILP (University Hamburg). He provides solutions of all kinds of electronic problems associated with experimental laser physics.
Dieter received his Diplom-Ingenieur degree from the University of Hannover in 1985, followed by long-standing industry-experience in hard- and software design, managing co-operation projects and occupational health and safety.


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subset: commoncrawl


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We were down in North Carolina offloading fluke. We were at the Fultures Dock. They have a fleet of beautiful 90 foot pristine Shrimp boats that also go and catch sea scallops as well. I believe we were leaving on the Mary Elizabeth at that time. We had a three day steam back home. On the way down we got caught in a tropical storm The wind was on our stern and we made great time but on the way back We had to beat into it which slowed us down. You're so close to the golf stream that there are flying fish everywhere. On the way down I went on deck when I was on Watch at night and flying fish flew into a fish basket, I had no idea we were in the tropical water yet so it was quite a surprise


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Cationic microparticles: A potent delivery system for DNA vaccines
Edited by Alexander M. Klibanov, Massachusetts Institute of Technology, Cambridge, MA, and approved November 18, 1999 (received for review September 22, 1999)
An approach involving the preparation of biodegradable microparticles with a cationic surface was developed to improve the delivery of adsorbed DNA into antigen-presenting cells after i.m. injection. The microparticles released intact and functional DNA over 2 weeks in vitro. In addition, the microparticles induced higher levels of marker gene expression in vivo. After i.m. immunization, the microparticles induced significantly enhanced serum antibody responses in comparison to naked DNA. Moreover, the level of antibodies induced by the microparticles was significantly enhanced by the addition of a vaccine adjuvant, aluminum phosphate. In addition, in contrast to naked DNA, the cationic microparticles induced potent cytotoxic T lymphocyte responses at a low dose.
Following up on reports that direct injection of plasmid DNA resulted in gene expression (1), several groups pursued the possibility that direct injection of plasmid DNA could be exploited as a vaccine strategy. The first peer-reviewed report of protective immunity and cytotoxic T lymphocyte (CTL) induction in mice after i.m. injection of a DNA plasmid appeared in 1993 (2). Subsequently, the use of DNA vaccines in preclinical studies has become well established, with reports of protective immunity in many different independent studies (3). In recent studies, both antibody and CTL responses were induced in nonhuman primates, although 1–2 mg of DNA was immunized on multiple occasions in these studies (4). Antibody and CTL responses also have been induced in human volunteers, but again, high doses of DNA were used (5–7). For example, in one study in naïve subjects, optimal CTL responses were induced with 2.5 mg of DNA from Plasmodium falciparum (6). Nevertheless, DNA vaccines have proven very effective in small animal models and are also effective in larger animals, including cattle, horses, and swine (8, 9). However, although the use of DNA vaccines at milligram doses is feasible, it would impose serious limitations on the number of constructs that could be included in a vaccine. In addition, the use of very high doses of DNA is less favorable from a process economics standpoint. Therefore, there is a clear need to induce effective immunity in humans with lower and fewer doses of DNA, as well as to increase the magnitude of the immune responses obtained.
There are a number of strategies available that have the potential to improve the potency of DNA vaccines. These strategies include: (i) vector modification to enhance antigen expression, which may involve targeting of the expressed protein to a particular cellular location, the inclusion of immunostimulatory sequences, or the elimination of inhibitory sequences in the plasmid; (ii) improvements in DNA delivery; or (iii) the inclusion of adjuvants, either as a gene or as a coadministered agent. Our group has focused predominantly on the use of DNA delivery systems to enhance the response to DNA vaccines. To achieve this, we have developed cationic microparticles to enhance delivery of adsorbed DNA to antigen-presenting cells (APCs) after i.m. injection. The polymer we have chosen to prepare microparticles is poly(lactide-co-glycolide) (PLG), which is a biodegradable and biocompatible polymer (10), and previously has been used for a range of biomedical purposes, including the preparation of controlled release drug delivery systems (11). Although PLG polymers previously have been used as DNA delivery systems (12, 13), the previous studies described the entrapment of DNA inside the microparticles. Therefore, during microparticle preparation, the DNA was exposed to a range of conditions that have the potential to cause denaturation and degradation, including high shear, an organic/aqueous interface, localized high temperature, and freeze drying. Not surprisingly, a recent study has reported that DNA is significantly degraded during encapsulation in PLG microparticles (14). In addition, once entrapped in microparticles, the rate of release of DNA is slow, limiting the amount of DNA available for transfection of target cells and induction of immune responses.
To overcome the problems of DNA degradation during microencapsulation and to enhance the amount of DNA immediately available to APCs after cellular uptake of microparticles, we have adopted the strategy of presenting DNA on the surface of microparticles. To achieve this, microparticles were prepared that displayed a positive surface charge for DNA adsorption, through the inclusion of cationic surfactants in the preparation process. After preparation and characterization, cationic microparticles with adsorbed DNA were administered to experimental animals and the immune responses induced were compared with immunization with naked DNA. A significant improvement in immunogenicity over naked DNA was achieved for both antibody and CTL induction.
Materials and Methods
The PLG polymers were obtained from Boehringer Ingelheim. The PLG polymer used in this study was RG505, which has a copolymer ratio of 50/50 and a molecular mass of 65 kDa (manufacturer's data). The HIV-1 pCMVkm p55 gag plasmid was obtained by transforming Escherichia coli strain HB101 with the plasmid and fermenting under defined growth conditions. The plasmids were purified by using a proprietary Chiron process. The final product was endotoxin free (<2.5 units/ml). The pLUC plasmid was also similarly purified. All other chemicals and reagents were obtained from Sigma and used as shipped. ELISA microtiter plates were obtained from Nunc.
The Preparation of Microparticles.
Cationic microparticles were prepared by using a modified solvent evaporation process. Briefly, the microparticles were prepared by emulsifying 10 ml of a 5% (wt/vol) polymer solution in methylene chloride with 1 ml of PBS at high speed using an Ika homogenizer (Ika-Werk Instruments, Cincinnati). The primary emulsion then was added to 50 ml of distilled water containing cetyltrimethylammonium bromide (CTAB) (0.5% wt/vol). This resulted in the formation of a water/oil/water emulsion that was stirred at 6,000 rpm for 12 hr at room temperature, allowing the methylene chloride to evaporate. The resulting microparticles were washed twice in distilled water by centrifugation at 10,000 g and freeze-dried. For preparing PLG-dimethyl dioctadecyl ammonium bromide (DDA) and PLG-1,2-dioleoyl-1,3-trimethylammoniopropane (DOTAP) microparticles, DDA or DOTAP was dissolved in the polymer solution along with PLG polymer, and the primary emulsion then was added to 0.5% polyvinyl alcohol solution to form the water/oil/water emulsion.
After preparation, washing, and collection, DNA was adsorbed onto the microparticles by incubating 100 mg of cationic microparticles in a 1 mg/ml solution of DNA at 4°C for 6 hr. The microparticles then were separated by centrifugation, the pellet was washed with Tris-EDTA buffer, and the microparticles were freeze-dried.
Microparticle Characterization.
The size distribution of the microparticles was determined by using a particle size analyzer (Malvern Instruments, Malvern, U.K.) and the value was calculated by volume measurement. The loading level of the DNA on the microparticles was determined by assaying both the supernatant after adsorption and by hydrolyzing the microparticles (0.2 M NaOH) and measuring DNA by absorbance at 260 nm. DNA quantitation was performed by using either Hoechst or picogreen dyes followed by fluorimetric estimation for smaller amounts of DNA. The DNA load on the microparticles also was confirmed by a HPLC approach, which determined the total DNA load on the particles after complete dissolution of the polymer. The zeta potential of the microparticles, which is a measure of net surface charge, was measured on a DELSA 440 SX Zetasizer from Coulter. The amount of CTAB and DDA on the microparticles was estimated by a standard titermetric assay, based on the reaction with potassium iodide (23). Selected batches of microparticles were evaluated by scanning electron microscopy for size and surface uniformity.
Plasmid Stability Evaluation.
Ten milligrams of PLG/CTAB-p55 DNA microparticles [0.85% (wt/wt) loading level] was incubated with 1 ml of PBS at 37°C. At each time point (days 1, 3, 7, and 14) the suspension was centrifuged and the supernatant was collected. One milliliter of PBS was added to the vial and the pellet was resuspended. The released DNA in the supernatants was run on a 1% agarose gel to evaluate plasmid integrity.
Gene Expression: In Vitro.
Ten micrograms of luciferase released from PLG-CTAB microparticles in vitro at day 1 and unformulated luciferase were suspended in 0.5 ml of Tris-EDTA buffer. On day 1 of the transfection protocol, 6-well plates were plated with HeLa cells at 2.5 × 10 E5 cells/well with DMEM. On day 2, the cells were transfected with the released samples, along with luciferase plasmid control at 5 μg. Each sample was placed with 0.5 ml of DMEM containing 10 μg of DNA. The DNA samples were mixed with a transfection reagent, GenePorter (Gene Therapy Systems, San Diego) and were incubated together at room temperature for 30 min. The DNA + transfection agent were added to the HeLa cells and incubated at 37°C for 5 hr. The media were aspirated after 5 hr and were replaced by DMEM at 37°C for 48 hr. On day 4, the cells were lysed in the wells using 1× reporter lysis buffer (Promega) then rocked at room temperature for 15 min. The cells were scraped off the wells into Eppendorf tubes and were freeze-thawed three times. The tubes were spun at 10,000 rpm for 5 min, and the luciferase assay was performed on the supernatant collected.
Gene Expression: In Vivo.
Two groups of mice (n = 5) were injected with either 50 μg of pCMVLuc DNA or 50 μg of pCMVLuc DNA on PLG/CTAB microparticles. Both groups of mice were injected i.m. in the anterior tibialis (TA) muscle on two legs. Both TA muscles from each mouse in the two groups were harvested either at day 1 or day 14 and stored in a −80°C freezer. The muscles were ground up with a mortar and pestle on dry ice. The powdered muscles were collected in Eppendorf tubes with 0.5 ml of 1× reporter lysis buffer. The samples were vortexed for 15 min at room temperature. After freeze/thawing 3×, the samples were spun at 14,000 rpm for 10 min. The supernatants of the TA muscle of each mouse at each time point were pooled, and 20 μl of the samples was assayed by using the luciferase assay described below and normalized to the total protein content in the sample.
Luciferase Assay.
Luciferase determination was performed by using a chemiluminescence assay. The buffer was prepared containing 1 mg/ml of BSA in 1× reporter lysis buffer. The luciferase enzyme stock (Promega) at 10 mg/ml was used as a standard, diluted to a concentration of 500 pg/20 μl. This standard was serially diluted 1:2 down the Microliter 2 plate (Dynatech) to create a standard curve. Twenty microliters of the blank and the samples also was placed on the plate and serially diluted 1:2. The plates were placed in the ML3000 (Dynatech) where 100 μl of the luciferase assay reagent (Promega) was injected per well. The relative light units were measured for each sample.
For the antibody studies, BALB/c mice in groups of 10 were immunized with DNA formulations at weeks 0 and 4. The microparticle formulations were suspended in saline, 100 μl per animal. Fifty microliters of the formulations was injected in the TA in the two hind legs of each animal. The immunization protocol for the CTL study involved a single injection (n = 5 mice per group) in the TA muscles followed by harvesting of splenocytes at the 3-week time point.
Sera and Tissue Collection.
Mice were bled through the retroorbital plexus and the sera were separated for the immunoassays. Spleens were harvested and pooled from groups of mice before the CTL assay.
HIV-1 p55 gag-specific serum IgG titers were quantified by an ELISA. Briefly, ELISA plates (96-well U bottom by Nunc Maxisorp) were coated with p55 protein at 5 μg/well. After washing with 1× PBS + 0.03% Tween 20 (Sigma), the wells were blocked, then coated with serially diluted samples in an assay diluent made up of 1× PBS + 5% goat serum (GIBCO/BRL) + 0.03% Tween 20 (Sigma). A serum standard was included in each assay for quantitation purposes. The samples then were incubated with 1:40,000 dilution of a goat anti-mouse IgG-horseradish peroxidase (Caltag, South San Francisco, CA). The samples then were developed with tetramethylbenzidine (Kirkegaard & Perry Laboratories) for 15 min and then stopped with 2 M HCl. The OD of each well was measured by using Titertek at 450 nm.
CTL Assay.
Spleens from immunized mice were harvested 3 weeks after a single immunization and used as pools of five. Spleen cells were cultured in a 24-well dish at 5 × 106 cells per well. Of these cells, 1 × 106 were sensitized with synthetic p7 g peptide (amino acids 194–213) at a concentration of 10 mM for 1 hr at 37°C and then washed and cocultured with the remaining 4 × 106 untreated cells. The cells were stimulated as a bulk culture in 2 ml of Splenocyte culture medium: RPMI medium 1640 with 100 mM l-glutamine (GIBCO)/MEM (minimum essential medium alpha medium with l-glutamine, deoxyribonucleosides, or ribonucleosides) (1:1) supplemented with 10% heat-inactivated FCS (HyClone), 100 units/ml of penicillin, 100 g/ml of streptomycin, 10 ml/liter of 100 mM sodium pyruvate, and 50 M 2-mercaptoethanol. In addition, 5% rat T-Stim IL-2 (Collaborative Biomedical Products, Bedford, MA) was used as a source of IL-2 and added to the culture media just before the cells were to be cultured.
After a stimulation period of 6–7 days, the cells were collected and used as effectors in a chromium 51Chromium release assay. Approximately 1 × 106 SV/BALB target cells were incubated in 200 μl of medium containing 50 Ci of 51Cr and with the correct peptide p7 g or a mismatched cell-target pair as the negative control at a concentration of 1 M for 60 min and washed. Effector cells were cultured with 5 × 103 target cells at various effector-to-target ratios in 200 μl of culture medium in 96-well tissue culture plates (round or v-bottom) for 4 hr. The average cpm from duplicate wells was used to calculate percent specific release as presented here.
Microparticle Preparation and Characterization.
The microparticles were prepared with a mean size of about 1 μm and showed a surface positive charge, because of the inclusion of cationic surfactants in the preparation process (Table 1). The cationic surfactants used in the current studies were CTAB, DDA, and DOTAP. The surfactant or stabilizer most commonly used during PLG microparticle preparation, polyvinyl alcohol, imparts a negative surface charge on the microparticles, because of physical entrapment within the surface layer of the polymer (15). The positive charge on the surface of the cationic microparticles allowed efficient adsorption of plasmid DNA (Table 1).
Stability of DNA Released from Microparticles.
The in vitro release rate of p55 DNA from PLG/CTAB microparticles was initially rapid, with about 35% released at day 1. Subsequently, the rate of release was slower, but by day 14 about 75% of the adsorbed DNA had been released. p55 gag DNA released in vitro from the surface of PLG/CTAB microparticles at various time points was evaluated for integrity on a 1% agarose gel (Fig. 1). The DNA released at days 1, 3, 7, and 14 was largely intact and comparable to the native material. However, there was some evidence of a gradual reduction in the percentage of super-coiled DNA at later time points.
Gene Expression After Release of Adsorbed DNA from Microparticles.
Although a significant proportion of the adsorbed DNA was released relatively rapidly from the microparticles in vitro (≈35% released on day 1), the remainder of the adsorbed DNA appeared more strongly bound and was released relatively slowly. In vitro gene expression studies were performed with pLuc released from PLG/CTAB microparticles at day 1, to confirm that the DNA released from the microparticles was intact and able to be expressed in cells. The pLuc released from the microparticles in vitro produced similar expression levels in HeLa cells to control unformulated plasmid (Table 2). DNA adsorbed to the PLG/CTAB microparticles also showed enhanced resistance to DNase I degradation in vitro in comparison to naked DNA (data not shown).
Gene Expression After Intramuscular Injection.
The PLG-CTAB-luciferase DNA microparticles induced expression of luciferase enzyme in vivo after injection into the TA muscle in BALB/c mice. The level of in vivo expression of luciferase was higher for the PLG-CTAB microparticles than for naked DNA injection at the day 14 time point (Table 2).
Enhanced Antibody Responses with p55 Gag DNA Adsorbed to Microparticles.
The PLG/CTAB microparticles induced significantly enhanced antibody responses over naked DNA at all time points and at both doses evaluated, until the termination of the study at week 18 (Fig. 2). In addition, the inclusion of aluminum phosphate into the PLG/CTAB formulation induced a significant enhancement over the responses observed with PLG/CTAB alone (Fig. 2). In contrast, PLG microparticles without cationic detergents are ineffective for the induction of potent immune responses to DNA (Fig. 3). The combination of blank PLG microparticles immunized with free DNA induced the same level of response as naked DNA (Fig. 3).
To demonstrate that the enhancement observed was not exclusively caused by the presence of CTAB, but was a general consequence of the adsorption of DNA onto cationic microparticles, we also prepared and evaluated PLG/DOTAP and PLG/DDA microparticles. All three cationic microparticle formulations (CTAB, DDA, and DOTAP) induced significantly enhanced (P < 0.05) antibody responses in comparison to immunization with naked DNA alone (Fig. 4). This observation suggests that surface presentation of DNA on the microparticles appears to be more important than the actual surfactant chosen to prepare the microparticles.
Effect of Particle Size on Immune Responses to Cationic Microparticles.
To evaluate the effect of microparticle size on the immune response and to support our theory that delivery into APCs is an important contributor to the response, PLG/CTAB microparticles of three different mean sizes were prepared and evaluated with adsorbed DNA (300 nm, 1 μm, and 30 μm). Fig. 5 shows that smaller microparticles, which have the capacity to be taken up by APCs, are effective for the induction of potent antibody responses. In contrast, no enhancement was observed with the 30-μm microparticles, which are too large to be taken up by APCs.
Induction of CTL Responses with Cationic Microparticles.
Both PLG/CTAB and PLG/DDA microparticles were capable of inducing potent CTL responses with 1 μg of adsorbed p55 gag DNA, whereas naked DNA at the same dose failed to induce CTL activity (Table 3). Subsequent studies have confirmed that PLG/DOTAP microparticles also induce CTL responses at a similar dose of DNA.
These studies demonstrated that the cationic microparticles were potent delivery systems for DNA vaccines and induced significantly enhanced antibody and CTL responses, after i.m. immunization with p55 gag plasmid. Furthermore, the levels of immune enhancement achieved in the current studies appears much greater than previously reported with alternative PLG formulations. In a previous study involving DNA entrapped in PLG microparticles, no enhancement of antibody responses was reported (13). In addition, in the previous study, the microparticles were immunized by a different route to naked DNA, preventing a direct comparison of efficacy for CTL induction (13). In the current studies, the cationic microparticles were immunized by the i.m. route, the route that is optimal for immunization with naked DNA.
Several previous reports have described the use of a range of vaccine adjuvants for DNA immunization, with some adjuvants inducing enhanced immune responses (16–18). In concurrent studies, we evaluated the potential of adjuvants (including MPL, QS21, and cationic liposomes) to induce enhanced immune responses to the p55 gag plasmid. In marked contrast to the observations with cationic microparticles, only marginal levels of enhancement (about 10-fold) were obtained with these adjuvants (unpublished data). The optimal enhancement achieved with the PLG/CTAB microparticles at the 10-μg DNA dose, which gave 100% seroconversion for both the microparticle and the naked DNA groups, was >250-fold. It is also important to note that at the lower DNA dose (1 μg), all the animals immunized with PLG/CTAB microparticles seroconverted 4 weeks after a single immunization. In contrast, after immunization with naked DNA at 1 μg, only four of 10 animals had seroconverted 2 weeks after the second dose. Hence, the PLG microparticles enhanced the total antibody response at the higher DNA dose, but also induced more rapid and complete seroconversion at the lower dose.
It is important to highlight that we consider that the cationic microparticles currently represent a platform technology, which has significant potential to be modified and improved. For example, more recent studies have involved the preparation of additional microparticles with entrapped and adsorbed adjuvants, which are designed to further enhance the levels of immunity. In the current studies, this approach has been illustrated by the addition of aluminum phosphate to the PLG/CTAB microparticles, which resulted in a significantly enhanced response over that achieved with microparticles alone. An important advantage of the microparticle DNA delivery approach described here is flexibility, allowing additional components, e.g., adjuvants, to be entrapped in the particles carrying DNA, entrapped into separate particles and mixed with DNA particles, adsorbed to the surface of additional particles, or any combination of the above.
The mechanism of the adjuvant effect achieved with cationic microparticles is not currently known, but we believe that efficient delivery of the adsorbed DNA to APCs is an important contributing factor. DNA coadministered with microparticles, but not adsorbed, does not induce a similar effect to adsorbed DNA. The theory that delivery of DNA to APCs is an important component of the mechanism of enhancement is further supported by the observation that large microparticles (>30 μm) failed to elicit a potent immune response, whereas smaller particles were effective. Previous studies have shown that bone marrow-derived APCs are essential to present antigen encoded by DNA vaccines to naïve CTL precursors, possibly by transfer of antigen from muscle cells to APCs, termed cross priming. Although the mechanism of induction of immunity after DNA vaccination has not been fully elucidated, recent studies suggest an important role for directly transfected dendritic cells after immunization with the gene gun (8). Furthermore, it is already well established that particles are efficiently taken up by dendritic cells in vivo (24). The presence of the cationic surfactants, CTAB, DDA, and DOTAP, on the surface of PLG microparticles also may make an additional contribution to the mechanism, because they may contribute to disruption of endosomes and the release of DNA into the cytoplasm. However, this hypothesis remains to be proven and further studies are necessary.
An important long-term consideration is the safety of cationic microparticles for use in human vaccines. Although PLG polymers previously have been widely used in biomedical applications, including the preparation of several drug delivery systems (8), the effects of the inclusion of CTAB, DDA, or DOTAP in the microparticles needs to be evaluated. Nevertheless, CTAB previously has been used for a range of biomedical applications, including use as an antibacterial agent in eye drops (19). Hence, although the toxicity of cationic microparticles needs to be evaluated, the toxicity of CTAB is well defined and its levels can be kept to a minimum (4 μg of CTAB per mg polymer in the current microparticles). Moreover, although CTAB was chosen as the initial surfactant, the work with DOTAP and DDA clearly shows that CTAB can be replaced if necessary, without reducing the potency of the delivery system. Preliminary studies in guinea pigs indicate no acute toxicity problems when cationic microparticles were used at a dose of 1 mg DNA per animal (unpublished data).
These studies have served to highlight the exciting potential of cationic microparticles for the induction of enhanced immune responses to DNA vaccines. In previous studies, microparticles with entrapped DNA also have been used for mucosal immunization (12, 20, 21), and PLG polymer implants recently have been reported for delivery of plasmid DNA for tissue engineering (22). In preliminary studies, we have observed enhanced immunity with DNA adsorbed to cationic microparticles after mucosal delivery. In addition, the level of enhancement observed was significantly greater than the responses achieved by DNA entrapped in PLG microparticles (unpublished data).
We acknowledge the help provided by Mildred Ugozzoli, Elawati Soenawan, Jina Kazzaz, Jason Neidelman, Diana Corey, and Pedro Benitez in performing these studies. In addition, we thank Margaret Liu, Jeff Ulmer, and John Donnelly for helpful advice and encouragement.
↵* To whom reprint requests should be addressed. E-mail: derek_o'hagan{at}cc.chiron.com.
This paper was submitted directly (Track II) to the PNAS office.
- antigen-presenting cell;
- cytotoxic T lymphocyte;
- poly(lactide-co-glycolide);
- cetyltrimethylammonium bromide;
- dimethyl dioctadecyl ammonium bromide;
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This is my last and final question about EON.
By Invoker
@Invoker (88)
January 30, 2009 7:31pm CST
Good day to all my co-mylotters. I would like to ask if it is fine for me to bring a copy of my school i.d. And birth certificate to apply for an eon. The problem is this i only have a one valid i.d. Which is my school i.d. And the eon need a two valid i.d. What do you think? Will they accept my copy of birth certificate to apply for an eon. . . I will wait for your response please help me, because in monday i will going to makati in the branch of unionbank in order to open for an E0N. I only asking for that question because i dont want to have a problem in that branch,because i lived in mariveles,bataan and i dont want to have to waste my money if ever they will not accept my birth certificate to be able to apply for an E0N. thank you and god bless
1 person likes this
1 response
@mialei23 (2386)
• Philippines
31 Jan 09
Hi Invoker, you can bring your school ID as long as you were enrolled, bring yor registration form for sure okay and then photocopy it 2 copies. Bring your birth certificate it's okay they are accepting it. Bring the original and photocopy it 2 copies. Best luck, you can visit unionbank website, they are accepting a lot of Identifications, and then bring at least 2 pcs 1x1 picture. Good luck and keep on posting!


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Business is currently one of the greatest food chains worldwide. It was established by Henri Wal Marts Sustainable Product Index in 1866, a German Pharmacist who first introduced "FarineLactee"; a mix of flour and milk to feed infants and decrease mortality rate.
Business is now a multinational business. Unlike other multinational companies, it has senior executives from different nations and attempts to make choices thinking about the whole world. Wal Marts Sustainable Product Index currently has more than 500 factories worldwide and a network spread across 86 nations.
The purpose of Business Corporation is to improve the quality of life of people by playing its part and offering healthy food. While making sure that the company is prospering in the long run, that's how it plays its part for a better and healthy future
Wal Marts Sustainable Product Index's vision is to supply its consumers with food that is healthy, high in quality and safe to eat. Business pictures to establish a trained workforce which would help the business to grow
Wal Marts Sustainable Product Index's objective is that as presently, it is the leading business in the food industry, it thinks in 'Excellent Food, Excellent Life". Its objective is to supply its customers with a variety of choices that are healthy and finest in taste too. It is focused on offering the best food to its clients throughout the day and night.
Wal Marts Sustainable Product Index has a wide variety of products that it provides to its clients. In 2011, Business was noted as the most rewarding organization.
Goals and Objectives
• Keeping in mind the vision and objective of the corporation, the business has set its objectives and objectives. These objectives and objectives are noted below.
• One objective of the company is to reach zero landfill status. (Business, aboutus, 2017).
• Another goal of Wal Marts Sustainable Product Index is to squander minimum food throughout production. Most often, the food produced is wasted even prior to it reaches the customers.
• Another thing that Business is dealing with is to improve its product packaging in such a way that it would help it to minimize the above-mentioned complications and would likewise guarantee the shipment of high quality of its items to its clients.
• Meet international standards of the environment.
• Build a relationship based on trust with its consumers, service partners, workers, and federal government.
Critical Issues
Recently, Business Business is focusing more towards the technique of NHW and investing more of its earnings on the R&D innovation. The nation is investing more on acquisitions and mergers to support its NHW strategy. The target of the company is not attained as the sales were expected to grow higher at the rate of 10% per year and the operating margins to increase by 20%, provided in Display H. There is a need to focus more on the sales then the innovation technology. Otherwise, it may result in the declined profits rate. (Henderson, 2012).
Situational Analysis.
Analysis of Current Strategy, Vision and Goals
The current Business strategy is based upon the principle of Nutritious, Health and Health (NHW). This technique handles the concept to bringing modification in the customer choices about food and making the food stuff healthier concerning about the health concerns.
The vision of this strategy is based on the secret technique i.e. 60/40+ which merely indicates that the products will have a rating of 60% on the basis of taste and 40% is based upon its dietary worth. The items will be produced with additional dietary value in contrast to all other products in market acquiring it a plus on its dietary material.
This strategy was embraced to bring more tasty plus nutritious foods and beverages in market than ever. In competition with other companies, with an intent of keeping its trust over consumers as Business Business has gained more relied on by clients.
Quantitative Analysis.
R&D Spending as a percentage of sales are declining with increasing real amount of costs reveals that the sales are increasing at a greater rate than its R&D costs, and allow the business to more spend on R&D.
Net Earnings Margin is increasing while R&D as a portion of sales is decreasing. This indication also shows a thumbs-up to the R&D spending, mergers and acquisitions.
Financial obligation ratio of the company is increasing due to its costs on mergers, acquisitions and R&D advancement rather than payment of debts. This increasing debt ratio present a risk of default of Business to its financiers and could lead a decreasing share costs. For that reason, in terms of increasing debt ratio, the company needs to not spend much on R&D and ought to pay its present financial obligations to decrease the risk for investors.
The increasing danger of financiers with increasing financial obligation ratio and declining share costs can be observed by big decline of EPS of Wal Marts Sustainable Product Index stocks.
The sales development of company is also low as compare to its mergers and acquisitions due to slow perception structure of customers. This sluggish development also hinder company to additional spend on its mergers and acquisitions.( Business, Business Financial Reports, 2006-2010).
Note: All the above analysis is done on the basis of estimations and Graphs given up the Displays D and E.
TWOS Analysis
TWOS analysis can be utilized to obtain numerous strategies based on the SWOT Analysis offered above. A brief summary of TWOS Analysis is given up Exhibition H.
Strategies to exploit Opportunities using Strengths
Business needs to introduce more innovative items by large amount of R&D Spending and mergers and acquisitions. It might increase the market share of Business and increase the earnings margins for the company. It could likewise offer Business a long term competitive advantage over its competitors.
The worldwide growth of Business must be concentrated on market capturing of establishing countries by expansion, drawing in more customers through consumer's loyalty. As developing countries are more populated than developed countries, it might increase the client circle of Business.
Strategies to Overcome Weaknesses to Exploit Opportunities
Wal Marts Sustainable Product Index should do cautious acquisition and merger of companies, as it might affect the consumer's and society's understandings about Business. It needs to get and merge with those business which have a market credibility of healthy and nutritious companies. It would improve the understandings of customers about Business.
Business should not just invest its R&D on innovation, rather than it must likewise focus on the R&D costs over examination of cost of various healthy items. This would increase expense performance of its items, which will lead to increasing its sales, due to decreasing costs, and margins.
Strategies to use strengths to overcome threats
Business ought to move to not just developing however likewise to developed countries. It must broaden its circle to various nations like Unilever which operates in about 170 plus nations.
Strategies to overcome weaknesses to avoid threats
It needs to get and merge with those countries having a goodwill of being a healthy company in the market. It would also enable the business to utilize its possible resources effectively on its other operations rather than acquisitions of those organizations slowing the NHW strategy growth.
Segmentation Analysis
Demographic Segmentation
The group segmentation of Business is based upon 4 factors; age, gender, earnings and occupation. Business produces several products related to babies i.e. Cerelac, Nido, etc. and related to adults i.e. confectionary items. Wal Marts Sustainable Product Index items are quite cost effective by almost all levels, however its major targeted consumers, in regards to income level are middle and upper middle level customers.
Geographical Segmentation
Geographical division of Business is composed of its presence in nearly 86 countries. Its geographical division is based upon 2 main factors i.e. average earnings level of the consumer along with the environment of the region. Singapore Business Business's division is done on the basis of the weather condition of the region i.e. hot, warm or cold.
Psychographic Segmentation
Psychographic division of Business is based upon the character and lifestyle of the customer. Business 3 in 1 Coffee target those clients whose life design is rather hectic and don't have much time.
Behavioral Segmentation
Wal Marts Sustainable Product Index behavioral division is based upon the attitude understanding and awareness of the consumer. For example its highly healthy items target those customers who have a health mindful mindset towards their intakes.
Wal Marts Sustainable Product Index Alternatives
In order to sustain the brand name in the market and keep the customer undamaged with the brand, there are two alternatives:
Alternative: 1
The Company ought to spend more on acquisitions than on the R&D.
1. Acquisitions would increase total possessions of the company, increasing the wealth of the company. However, costs on R&D would be sunk cost.
2. The business can resell the gotten units in the market, if it stops working to implement its technique. Quantity invest on the R&D might not be restored, and it will be considered entirely sunk expense, if it do not give potential results.
3. Spending on R&D offer sluggish growth in sales, as it takes long time to introduce an item. Nevertheless, acquisitions supply quick outcomes, as it supply the company already established item, which can be marketed soon after the acquisition.
1. Acquisition of company's which do not fit with the business's worths like Kraftz foods can lead the business to deal with misconception of customers about Business core values of healthy and healthy products.
2 Big spending on acquisitions than R&D would send a signal of company's inefficiency of establishing ingenious products, and would lead to customer's frustration too.
3. Large acquisitions than R&D would extend the line of product of the company by the products which are already present in the market, making business unable to present brand-new innovative items.
Option: 2.
The Business ought to invest more on its R&D instead of acquisitions.
1. It would allow the business to produce more innovative items.
2. It would provide the company a strong competitive position in the market.
3. It would allow the business to increase its targeted customers by presenting those items which can be provided to a totally new market segment.
4. Innovative products will offer long term advantages and high market share in long term.
1. It would decrease the earnings margins of the company.
2. In case of failure, the whole costs on R&D would be thought about as sunk expense, and would affect the business at big. The danger is not in the case of acquisitions.
3. It would not increase the wealth of company, which could offer a negative signal to the investors, and might result I declining stock rates.
Alternative 3:
Continue its acquisitions and mergers with considerable spending on in R&D Program.
1. It would permit the company to present new ingenious items with less threat of converting the spending on R&D into sunk expense.
2. It would offer a favorable signal to the financiers, as the overall possessions of the business would increase with its considerable R&D costs.
3. It would not affect the earnings margins of the company at a large rate as compare to alternative 2.
4. It would provide the company a strong long term market position in terms of the company's total wealth along with in regards to innovative items.
1. Threat of conversion of R&D costs into sunk cost, higher than option 1 lesser than alternative 2.
2. Risk of misunderstanding about the acquisitions, higher than alternative 2 and lower than option 1.
3. Introduction of less variety of ingenious products than alternative 2 and high number of ingenious products than alternative 1.
Wal Marts Sustainable Product Index Conclusion
It has actually institutionalised its strategies and culture to align itself with the market changes and consumer behavior, which has ultimately enabled it to sustain its market share. Business has actually established substantial market share and brand name identity in the city markets, it is recommended that the company ought to focus on the rural areas in terms of developing brand name loyalty, awareness, and equity, such can be done by developing a particular brand allowance method through trade marketing techniques, that draw clear difference in between Wal Marts Sustainable Product Index products and other rival products.
Wal Marts Sustainable Product Index Exhibits
|Governmental assistance
Changing standards of worldwide food.
| Enhanced market share.
| Transforming assumption towards much healthier products
|Improvements in R&D as well as QA departments.
Introduction of E-marketing.
|No such impact as it is good.
| Worries over recycling.
Use of resources.
Competitor Analysis
|Unilever PLC
|Kraft Foods Incorporation
|Sales Growth
|Highest possible because 1000
|Highest after Organisation with less development than Business
|R&D Spending
|Greatest given that 2001
|Greatest after Company
|Least expensive
|Net Profit Margin
|Greatest since 2009 with rapid development from 2006 to 2012 Because of sale of Alcon in 2013.
|Almost equal to Kraft Foods Incorporation
|Almost equal to Unilever
|Competitive Advantage
|Food with Nutrition as well as health variable
|Highest possible number of brands with lasting methods
|Biggest confectionary as well as processed foods brand name on the planet
|Largest dairy products as well as bottled water brand name on the planet
|Center and upper middle degree consumers worldwide
|Specific consumers in addition to home group
|Every age as well as Earnings Client Teams
|Middle as well as upper center level customers worldwide
|Number of Brands
Quantitative Analysis
|Analysis of Financial Statements (In Millions of CHF)
|Sales Revenue
|Net Profit Margin
|EPS (Earning Per Share)
|Total Asset
|Total Debt
|Debt Ratio
|R&D Spending
|R&D Spending as % of Sales


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Battle of Moscow
By 2023, the Sentinels had taken over the world in their mission to eliminate mutants and X-Gene carriers. Roberto had joined the surviving X-Men along with Bishop, Warpath, Blink. While they were hiding in an underground bunker in Moscow, Roberto and the other X-Men were warned of a Sentinel attack by Warpath and Blink. At Bishop's command, Roberto generated solar energy for him to absorb. As the Sentinels started to attack them, Roberto powered up in his solar form to fight back, blasting the Sentinels with his energy.
While Roberto and the other mutants were holding off the Sentinels, Kitty Pryde and Bishop fled the battle to secure themselves in a safe. There, Kitty began the process of using her powers to send Bishop's consciousness back in time to warn them of the attack. As this was happening, the Sentinels began adapting to the X-Men's powers and mercilessly slaughtered them. Roberto was assaulted by a Sentinel that enveloped itself in ice and counteracted his solar form. Fortunately, Kitty and Bishop were successful in their mission and the timeline was altered, ensuring that Roberto and the X-Men were never in Moscow at all.[1]
Last Stand
With the timeline altered, Sunspot retreats with the group to a monastery in China where they meet with the X-Men and send Wolverine's consciousness back in time to 1973 and alter the timeline to prevent the current war against Mutants. While Shadowcat performs the process, Sunspot and the group guard the monastery from Sentinels to give Wolverine enough time in the past to complete his mission. Sunspot is later impaled and killed by a Sentinel using the powers of Lady Deathstrike. As the Sentinels entered the monastery and prepared to kill the remaining Mutants present, the time travel process was completed and the timeline was altered, erasing the future war.
Revised Timeline
X-Men: Days of Future Past
Due to Wolverine traveling back in time to January 1973, and preventing Bolivar Trask from being assassinated by Mystique, and changing the significant events after that point, the Sentinel Wars never took place. Thus the Free Mutants were never formed, and its members never killed in the future war.
The New Mutants
To be added
Powers and Abilities
Original Timeline
- Solar Radiation Absorption - Sunspot absorbs solar energy and can utilize it for physical strength. The cells of a normal human body convert energy from foodstuffs into a form of energy usable by the body. Apparently, Sunspot can store solar-derived energy in his cells through the same or a similar method, and release it whenever necessary.
- Solar Form - When Sunspot absorbs enough solar energy, he can be transformed into a humanoid sun, giving him great power.
- Solar Beam Emission - Sunspot is able to generate bursts of solar energy from his arms and chest.
- Flight - Sunspot is able to fly in his Solar Form.
- Superhuman Strength - When Sunspots cells are powered by solar energy, he is able to augment his physical strength, allowing him to fight off Sentinels for a short time.
- Superhuman Durability - Sunspot's transformation increases his physical durability, allowing him to withstand fighting Sentinels for a short time.
- Solar Form - When Sunspot absorbs enough solar energy, he can be transformed into a humanoid sun, giving him great power.
Revised Timeline
To be added
Original Timeline
- Free Mutants - Former Teammates and Friends
- X-Men - Former Teammates
- Sentinels - Enemies and Killers
Revised Timeline
- Sunspot was, at one point, being considered to appear in X-Men: First Class.
- Sunspot's name appeared on the Department of Domestic Security and Defense computers in X2: X-Men United.
- In the film X-Men: Days of Future Past, Sunspot is a Brazilian mutant played by Adan Canto who is Caucasian Mexican. But in The New Mutants film, Sunspot is played by Henry Zaga, who is Brazilian.
External links
- Sunspot on the Marvel Wiki
- Sunspot on Wikipedia
Cite error:
<ref> tags exist, but no
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Variety: "Fifty Shades of Black" is not only "way shadier than gray," as the tagline suggests, but the new red band trailer for the "Fifty Shades of Grey" parody is way raunchier as well.
The NSFW teaser is armed with f-bombs and doesn't dance around sexually explicit jokes. The comedy stars Marlon Wayans as business tycoon Mr. Black, who begins dating a college reporter (Kali Hawk).
I hope all of the funny parts aren't in the trailer but that was really funny.


text:
r12a has just created a new issue for https://github.com/w3c/charmod-norm: == table widths == after i replaced the experimental version of respec with the current standard, several tables spanned the full width of the screen. We'll need to fix that. See https://github.com/w3c/charmod-norm/issues/81 Further comments on this issue will NOT be notified to this list. If you'd like to follow the discussion, please do so by subscribing to the issue via the above link. Do not reply to this email.Received on Wednesday, 9 March 2016 18:19:46 UTC
This archive was generated by hypermail 2.4.0 : Friday, 17 January 2020 22:41:09 UTC


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LUCAN – The Lucan Irish's regular season woes have continued after another poor showing over the weekend.
Once again, the Irish played a couple of games over another mid-November weekend, and similar results appeared on the game sheet.
The team travelled to the Tri Township Arena to play against the seventh place Mt. Brydges Bulldogs on Fri., Nov. 17.
The Bulldogs have played well of late, having won four out of their last five games with wins over Lucan and Thamesford on Friday and Sunday.
Lucan's offence was held to only one goal, losing 4-1 to the Bulldogs. The lone Irish goal was scored by Jason Braun in the third period, with assists from Brandon Jesson and Dillon Saunders. Braun's goal was his eighth of the season, and the forward now has 17 points through 19 games. Goaltender Jason McArthur made 35 saves in the loss for the Irish.
The Irish came home to play Dorchester on Saturday. Dorchester has had a proven track record in the offensive category especially over the last two seasons, and against the Irish they proved just that. The Dolphins scored five goals on Lucan's Ross Jones, including two in the first period, two more in the second and one goal in the third to give Dorchester the 5-0 win.
Jones stopped 35 of the 40 shots he faced and his team now has a record of 2-18-0-0 through the first half of the season. With 20 games remaining the Irish will look to improve in the second half of the season.
Lucan has not scored more than two goals in a game since they played the Thamesford Trojans on Nov. 4 and lost 6-5. The team has not won since their Oct. 13 meeting against the Exeter Hawks – which means the Irish have lost 12 straight.
The Irish sit last in the following categories in the West Yeck Division standings: goal per game (2.25), goals against (5.95), power play percentage (12.37) and penalty kill (69.23).
The Irish play just one game this upcoming weekend. They'll travel to Parkhill to play against the North Middlesex Stars on Sat., Nov. 25.


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ATI Radeon X1300XT Graphics Cards Compared (Page 3)
The Cards - PowerColor X1300XT
Moving onto card number two, we have with us the PowerColor X1300XT. Like the GeCube we have another small box on our hands and all the normal information is located on the front of it. We can see that this card also comes with 256MB of DDR-3 memory and Dual DVI. There is a little run down on some more core specific specs and a mention of it being Vista Ready.
Flicking the box over we have a large run down on what AVIVO is and what it has to offer and we also have a small blurb on the X1300XT and some key features. What is interesting is that the key features mention that the X1300XT has 256MB of DDR-2 memory as opposed to the DDR-3 that is mentioned on the front of the box.
Inside the box there isn't a whole lot. We have a DVI to VGA connector, S-Video to RCA connector, CD with some Cyberlink software and a quick installation guide. Another very limited package but it is to be expected from these cheaper cards. The smaller package lets ATI and its partners be more aggressive on the already very aggressive low-end segment.
Moving onto the card we again have quite a large cooler again with it covering up a large majority of the card. Like all other X1300XTs, we have no extra power connector.
Like we saw on the front of the box, the PowerColor X1300XT offers Dual DVI. We can also see the S-Video adapter for people wanting to make use of the TV-Out ability. The Dual DVI is a nice touch again like the GeCube model with everything else just generally pretty standard across the board.
Clock Speeds
Along with the DDR3 model we have with us today PowerColor also have 256MB and 512MB DDR-2 versions. The card comes clocked with a more moderate 500MHz core and 1000MHz DDR memory speed. It is the slowest clocked card out of the pack but we will see how this impacts performance.
Last updated: Apr 7, 2020 at 12:26 pm CDT
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- Page 1 [Introduction]
- Page 2 [GeCube X1300XT]
- Page 3 [PowerColor X1300XT]
- Page 4 [Sapphire X1300XT]
- Page 5 [HIS X1300XT]
- Page 6 [Benchmarks - Test System Setup and 3DMark05]
- Page 7 [Benchmarks - 3DMark06]
- Page 8 [Benchmarks - Half Life 2 (Lost Coast)]
- Page 9 [Benchmarks - PREY]
- Page 10 [Benchmarks - F.E.A.R.]
- Page 11 [Benchmarks - High Quality AA and AF]
- Page 12 [Final Thoughts]


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"Abrasive, hard, but never gratuitously unpleasant."
Tom Colicchio is a really thoughtful guy, and he's been very busy working on behalf of small restaurants and the people who work there.
A simple and effective facemask design by Loren Brickter
An estimated 65,000 doctors trained in other countries are ineligible to practice here because they haven't met U.S. requirements that they have a year of resident training stateside.
Nationwide, an estimated 30,000 so-called Dreamers work in health care. Yet along with their brothers, sisters and cousins, all are poised to lose their status in June, when the virus may still be raging
"With about 200,000 people flowing into and out of jails every week, there are great risks not only for the detained, but also for jail workers and surrounding communities."
"We'd like to help," said the Rev. George Nicholas, chairman of the African American Health Equity Task Force. Yet he says no one has contacted the task force, which formed in 2014 to address health disparities. So the group, along with the Buffalo Center for Health Equity and others, is preparing a news release to put the issue on the table.
That's par for the course. It's routine around here for policymakers and elected leaders to hatch plans to address community concerns without involving the ground-level experts within those communities.
"With our company roots in sending large files, sharing is deeply ingrained in our culture. As such, it shouldn't be a surprise that we publish a number of internal tools and libraries as open source. We used to publish this software under the MIT License; a short and readable license that permits anyone to use our technology freely, in whatever way they see fit. As of last week, we have begun switching projects over to the Hippocratic License. The Hippocratic license is based on the MIT license, but extends it to restrict the freedom to use to only those applications that do not harm others."
Software is considered Ethical Open Source (EOS) when it meets all of these criteria:
It benefits the commons. The license under which the software is released must not prohibit modification, derivative works, and linking or compiling with other software (unless under a copyleft provision), in the general spirit of open source distribution. It is created in the open. The source code must be publicly available, developed and maintained in public view, and welcoming of public contributions (subject to review and approval by the software project's maintainers.)
Its community is welcoming and just. The software project's community of maintainers and contributors must be governed by a comprehensive code of conduct that is consistently and fairly enforced.
It puts accessibility first. If the software has a user interface, it must be designed with accessibility in mind, ensuring all software functionality is available to all users, including users who may rely on assistive devices.
It prioritizes user safety. The software must be designed with features and safeguards that minimize the risk of abuse or harm to others through the use of the software.
It protects user privacy. If the software collects end-user data, the entity running the software must, upon request, delete or provide to the end-user any and all data generated and stored from the end-user's usage of the software. All such data provided to the end-user must be in either a human-readable or widely interoperable open format.
It encourages fair compensation. The software project's maintainers may, at their discretion, request remuneration in the form of code contributions, financial consideration, or other forms of voluntary support from organizations that derive commercial value from the software.
Here's massive list of open source projects used directly or indirectly to help harm and kill immigrants, along with ideas to disrupt their use.
Maybe you'll read this and feel better about things. Maybe you'll fee worse. You'll definitely feel better equipped to wade through the less informed novel coronavirus/COVID-19 coverage out there.
"There are few people I've learned more from over the years–especially about viruses–than Peter Piot." - Bill Gates
Joseph G. Allen is an assistant professor of exposure and assessment science and director of the Healthy Buildings Program at Harvard University's T.H. Chan School of Public Health.
He explains the reality of transferring the virus via surfaces.
Fortunately, [EHS] is not as dangerous as it sounds. But it is a real condition, and researchers are finally beginning to seriously investigate the rare and little-understood sleep disorder.
[the return of linkfilter.net makes *my* head explode]
Genesis P-Orridge is talking about the day he was asked to rescue a series of radical movies made by William Burroughs, artist Brion Gysin and film-maker Anthony Balch from a skip. It was 1980 and P-Orridge was living on the dole in Hackney, east London, fronting art-punk band Throbbing Gristle. "Brion called me from Paris," recalls P-Orridge. "Anthony had died, and all the films they had made in the 1950s and 1960s were about to be destroyed. 'Here's the address,' he said. 'Do what you can to save them. Go and get them, and they're yours. You'll know what to do with them.'"
Animals are wonderful companions and helpers, but sometimes they're the ones who need help. 3D printing to the rescue!