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"Under the stress of forced dissection—or dog lab, or any other harmful use of an animal—education is thwarted. When forced to use animals in ways the student objects to, the student is traumatized and invariably learns less." 1
Each year, an estimated 20 million animals (10 million vertebrates and 10 million invertebrates) equaling around 170 or more different species are used in the U.S. in all areas of education and grade levels.2 Animals are kept as "pets" in classrooms and used in biology and psychology classes, graduate training courses, and science fair projects.
The studying of animals in science education has taken place since the 1960s and the practice of dissection—the act of cutting into and examining a dead animal—continues to serve as a prominent educational tool for teaching anatomy and other "life" sciences (e.g. physiology, behavior, nutrition, and genetics). Students dissect preserved animals throughout all levels of biology education, from middle school to graduate school. College students who participate in biology courses typically encounter dissection labs. Medical schools perform dissection; however, the students often perform the dissections on human cadavers. Veterinary schools' curricula involve animal dissection and the use of live animals through a number of different ways.
Animals commonly used in education include frogs, fetal pigs, cats, dogs, dogfish sharks, perch, pigeons, rats, mice, rabbits, minks, foxes, bats, turtles, grasshoppers, earthworms, crayfish, clams, cockroaches, sea urchins, squid, and starfish—also known as sea stars. The most frequently dissected vertebrate animals are frogs, fetal pigs, and cats. Many animals—like frogs, salamanders, turtles, and dogfish sharks—are taken from the wild, while others come from licensed animal breeders and dealers, pet stores, fur farms, and slaughterhouses. In addition to buying animals purposely bred for use in research and teaching from licensed animal breeders (known as Class A Dealers), schools and universities also purchase animals from biological supply companies (Class B dealers) and/or random source dealers (Class B random source dealers). Random source dealers obtain live and/or dead animals, like cats and dogs, through a variety of means such as animal shelters and pounds (often referred to as "pound seizure"), from "free animal" ads, or as "stray" animals. These dealers then sell the animals to biological supply companies, schools, and colleges and universities. Supply companies may also purchase animals from any of these sources or from other dealers, and many have direct contracts with animal shelters and pounds both inside and outside the U.S. As of October 1, 2014, the National Institutes of Health (NIH) announced that it will no longer fund research that involves dogs procured from random source Class B dealers. NIH adopted a similar policy regarding cats in 2012.
The treatment of animals destined for use in dissection and other educational purposes involves an inordinate amount of suffering, stress, and inhumane treatment. Millions of animals, such as amphibians, reptiles, fish, birds, and most invertebrates, are "harvested" from natural habitats each year for dissection purposes. Dealers often stockpile animals on top of one another and ship them in crowded containers with no temperature regulation, food, or water. Undercover video footage has exposed that some are still alive as they are pumped full of formaldehyde or other preservatives.
Fetal pigs and organs from cows and sheep, often used in dissection labs, come from slaughterhouses and factory farms. Advocacy groups and undercover investigations have well documented the abuse associated with factory farming. Animals are often kept in cramped, unsanitary conditions while enduring painful "routine" procedures (such as debeaking, tail docking, and castration). Investigative reports have revealed animals still living while being dismembered in the mechanical slaughterhouse assembly line.
Undercover investigations have also documented animal abuse at the hands of Class B dealers and biological supply companies. These companies buy, for example, both live and dead cats captured off the streets in Mexico. Animal protection organizations, in response to complaints of disappearing companion animals in Mexico, found that suppliers trapped thousands of cats in residential neighborhoods, cruelly killed them, and shipped the bodies to the U.S. where suppliers sold them to schools for educational purposes. The New England Anti-Vivisection Society (NEAVS) opposes the surrender of animals by animal shelters and animal control agencies to labs, science and research facilities, or pharmaceutical and educational institutions. In 1983, with the help of thousands of animal activists across the state, we spearheaded a campaign to repeal the 1957 pound seizure law in Massachusetts. The successful campaign resulted in the first and one of the strongest anti-pound seizure laws in the U.S. and protects thousands of lost and abandoned dogs and cats each year from being used in research.
The harvesting of millions of frogs from the wild each year for dissection purposes poses a grave threat to endangered wild populations already decimated by habitat loss, chemical pollution, and global climate change. Given the ongoing destruction and fragility of natural habitats, the yearly removal of animals for dissection is wasteful, creates ecological imbalance, and further compromises already besieged natural resources. Breeding frogs in captivity for dissection is not lucrative, so suppliers use wild harvesting as it reaps greater profit yet poses imbalance and possible extinction in the environment.
Preservation of animals for research presents other significant concerns for the environment. Toxic chemicals such as formaldehyde or derivatives that contain formaldehyde in lesser concentrations preserve millions of animals each year. Formaldehyde is a "reasonably anticipated" carcinogen in humans. "Teachers and students who handle preserved specimens represent potential high exposure groups. The EPA has designated formaldehyde as a hazardous air pollutant, water pollutant, and waste constituent."3 Municipalities and industry have detected formaldehyde in their water supplies, rainwater, lake water, and some waterways. Other substitutes for formaldehyde have not been on the market long enough to truly determine safety and still often contain formaldehyde as a key ingredient. Also, these substitutes do not necessarily preserve specimens in an acceptable form that some teachers claim is useful for dissection. The disposal of specimens by thousands of classrooms every year presents a risk not only to the individual teacher and students involved, but also to the environment and subsequently public health. Studies have documented that not all schools follow strict environmental guidelines for disposing the hazardous waste of dissected animals' remains.
Instead of regarding animal life as valuable, through harmful animal classroom use students can learn that life is disposable and the suffering of someone else is secondary to their own needs and interests. In schools and colleges where student choice is not an option, teachers and professors may make the case that the student is being difficult or disrespectful in straying from the curriculum. Situations are numerous where administration penalized or made a student uncomfortable for not wanting to participate in dissection classes. This of course has serious consequences for the student's grades and science career. Hundreds of students who encountered resistance or were denied or penalized for requesting an alternative to dissection have contacted the Ethical Science Education Campaign (ESEC), the educational affiliate of NEAVS. The growing number of court cases where students—with parental support—are filing litigation against school systems and/or individual teachers indicates that many students are not being granted dissection choice, and as importantly for the evolution of science education ethics, these same students are no longer taking no for an answer. Rather, they are standing with conviction by their ethical and compassionate values and their right to a science education. NEAVS' campaign for dissection choice in Massachusetts led to the enactment of a student choice policy by the MA Department of Education, protecting students who choose not to participate in animal dissection and allowing these students to learn through humane alternatives.
Dr. Theodora Capaldo has published several papers including one on research regarding the psychological effect on students of the harmful use of animals at all educational levels.4 If forced to participate, these students suffer severe psychological trauma as the result of seeing themselves or others engaged in behavior that they find ethically objectionable yet sanctioned by authorities. Their cognitive abilities may become impaired, resulting in less learning. They may withdraw and lose interest in science. Students—even those who believe they are willing participants—become desensitized and may develop a utilitarian view of animals, thereby diminishing their capacity for compassion and ethical decision-making. Qualified, compassionate people—especially women—often decide to end their career in science rather than compromise their values. This loss contributes to the gender gap in science and to individuals feeling disappointed and derailed in their career aspirations.
Despite the availability of non-animal alternatives, some teachers still feel that dissection is a productive tool for teaching anatomy. Others feel that students must undergo a "rite of passage" to become prepared for medical or veterinary careers. They do not, however, factor in that the vast majority of their students will not enter these fields, and that even those who do will now stand a good chance of attending a college, university, or professional school that will not require the harmful use of animals in their science curriculum.
Comparative studies have shown time and again that alternatives to dissection—from computer programs to models that are more realistic than preserved "specimens"—are as educationally effective, and in most cases more so, than animal dissection. In April 2008, the National Science Teachers Association (NSTA) revised its dissection position to acknowledge the educational value of non-animal learning methods as replacements for animal dissections, and to establish the principle of dissection choice for all classrooms. The NSTA's acknowledgement of the educational efficacy of these alternatives is further evidence of their viability as learning tools and the future of science education.
Many science teachers and school systems today are sensitive to their students' requests and are knowledgeable about dissection choice and the use of non-animal alternatives. Dissection choice allows students who have ethical concerns to use alternatives to animal specimens and ensures a future generation of compassionate scientists by guaranteeing that students at all levels of science education have the right to learn without harming or killing animals. From dissection in high school and college to animal labs in veterinary and medical school, science education now deters those who refuse to harm animals from entering the field of science. Fortunately times are changing and a choice for science education without animal cruelty in, for example, veterinary and medical training, is becoming the norm rather than the exception to the rule. Guaranteeing the choice of a cruelty-free science education is the only way to allow, support, and encourage compassionate students to enter the field of science and ultimately change the way science does science.
To date, 18 states plus the District of Columbia now have dissection choice laws and/or policies in place to protect students—campaigns to which NEAVS/ESEC lent support. In addition to our ongoing work to pass dissection choice laws across the nation and to change medical and veterinary school training, NEAVS successfully ended the use of animals in "terminal labs" at the first veterinary college in the U.S. In 2000, Tufts University Cummings School of Veterinary Medicine became the first veterinary school in the country to no longer require terminal labs, in which students trained by practicing on animals before killing them. This victory—the result of years of collaboration between NEAVS and Tufts—led the way for other veterinary schools to follow suit. Today in the U.S., 50 percent of veterinary schools no longer require terminal labs and 96 percent of U.S. medical schools no longer use live animals in their medical student training.
NEAVS/ESEC works to guarantee students at all levels of science education the right to learn science without having to harm or kill animals. Through ESEC, we offer dissection alternative programs as a humane way for students to learn about science, anatomy, and physiology. ESEC supports students and programs at the high school, college, and professional school levels.
[2] Hart, L. A., Wood, M. W., & Hart, B.L. (2008). Why Dissection? Westport, CT: Greenwood Press.
[3] National Toxicology Program. (1998). Report on Carcinogens, Eighth Edition. Research Triangle Park, NC: U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program.
[4] Capaldo, T. (2004). The Psychological Effect on Students of Using Animals in Ways that They See as Ethically, Morally and Religiously Wrong. ATLA, 32, 525-531.


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Good Friday 2017 is a day that our Jesus Christ who died on the cross for our sin's as we know he is our Savior, who saves us from our sin's. He born from mother Mary's womb from there he went to John to get baptism, and he started fasting prayers in hill, on good Friday 2017 he carried his cross on his shoulders from Jerusalem town to Calvary hill while carrying his cross he lot of punishments from soldiers .after reaching Calvary hill he was surrounded by soldiers and he brought on to cross his palm's on to cross with big nails, his legs also attached with big nails to cross on this Good Friday Jesus thought a lesson to all his followers that we should carry our sins as he carry cross, because of our sins he was dead on cross on his good Friday 2017 .actually on this good Friday 2017 two more things also hung on cross, one of them asked Jesus is your a really a Savior save us and save yourself. Another person asked I believe that you're a son of God, please give me some space in your world. Jesus gives a reply to the second person as he wants on this Good Friday 2017 after 3 pm one of the Jerusalem temple's Curtains was separated into two parts and the day became night. Jesus asks his father, "Father please bring me to your place I can't hold this pain anymore "he told this and died.
This is happening on this Good Friday 2017. That's why it is a very important day to every Catholic and Christian. On this God Friday 2017, every Catholic will participate in the procession with the holy cross, and they will do fasting prayers.
The day which is death day of Jesus is become's Good Friday 2017.
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subset: commoncrawl


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Phil Jevon, PGCE, BSc, RN, is resuscitation officer/clinical skills lead, Manor Hospital, Walsall
Applying cricoid pressure helps to prevent the passive regurgitation and aspiration of gastric contents during bag-mask ventilation and attempted tracheal intubation (Nolan et al, 2005). Cricoid pressure is also routinely used in other situations, for example during emergency induction of general anaesthesia and before anaesthesia for Caesarean section.
The cricoid cartilage, the first tracheal cartilage, is situated below the thyroid cartilage from which it is separated by the cricothyroid membrane. It is the only complete ring of cartilage in the respiratory system (Koziol et al, 2000).
The other tracheal cartilages are c-shaped (incomplete rings of cartilage), which are liable to collapse if excess pressure is applied to them (Feinstein and Owens, 1992).
Increased Risk of Regurgitation and Pulmonary Aspiration
During resuscitation, gastric inflation, together with an incompetent oesophageal sphincter, increase the risk of regurgitation and pulmonary aspiration (Nolan et al, 2005).
The risk of regurgitation is higher during pregnancy, in obese patients and in patients with a 'full stomach'.
There is even a risk for those patients who have been fasted before surgery if there is delayed gastric emptying, for example bowel obstruction, opiate administration and trauma (Koziol et al, 2000).
Impaired laryngeal function leading to diminished laryngeal sensation and protective airway reflexes will increase the risk of pulmonary aspiration.
There are several situations when it may be necessary to apply cricoid pressure and these include:
- During cardiopulmonary resuscitation (CPR) until the airway is secured by a cuffed tracheal tube;
- Immediately prior to induction of general anaesthesia in the non-fasted patient;
- Delayed gastric emptying;
- Incompetent lower oesophageal sphincter, for example in late pregnancy;
- Difficult intubation - can help with the visualisation of the vocal cords.
Calculating the appropriate cricoid pressure
According to Mehrotra and Paust (1979), the appropriate amount of pressure to be applied to the cricoid is that which would cause the patient pain if it were to be applied to the bridge of the nose.
Cheek and Gutsche (1993) suggest that an inability to swallow equates to appropriate cricoid pressure.
A more scientific approach is that cricoid pressure generates a force that can be expressed in newtons (N) (Koziol et al, 2000) and 9.81N equates to the force of gravity on a mass of 1kg (Parbrook et al, 1990).
For convenience 10N approximately equates to 1kg (Vanner and Asai, 1999). Therefore, 30N (3kg) of force needs to be applied to the cricoid in order to achieve oesophageal occlusion (Vanner and Pryle, 1992) (less when applied prior to induction).
Mode of action
When applied with the correct pressure, cricoid pressure compresses the proximal oesophageal lumen situated between the cricoid cartilage and the cervical vertebrae (Fig 1).
This occludes the lumen of the oesophagus, helping to prevent regurgitation and aspiration of gastric contents. This will also minimise gastric inflation, often associated with bag-mask ventilation during CPR.
- Place a pillow under the patient's head and shoulders;
- Locate the cricoid cartilage - the first complete ring of cartilage below the thyroid cartilage (Adam's apple) (Fig 2);
- Using the dominant hand, place the index finger and thumb on either side of the cricoid cartilage (Fig 3);
- Apply cricoid pressure. Following pre-oxygenation, but prior to intravenous induction, apply a force of 10N (1kg) and following loss of consciousness increase the force to 30N (3kg) (this force should also be applied during CPR) (Fig 4).
- Apply counter pressure to the back of the patient's neck if there is a suspected cervical spine injury (Fig 5). This will reduce movement of the cervical spine.
- Release cricoid pressure once a cuffed tracheal tube protects the airway, if the patient actively vomits or on the anaesthetist's request.
- If lung inflation is not possible, either reduce the pressure that is being applied or release the pressure completely (Nolan et al, 2005).
Complications of cricoid pressure include the following:
- Retching and death from aspiration and ruptured oesophagus if excess force is applied in an awake patient;
- Difficult tracheal intubation;
- Difficult ventilation;
- Aggravation of an existing cervical spine injury.
This article has been double-blind peer-reviewed
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Good Morning!
I wanted to get this link out to you so you can help me tweak a program I am working on. If you are interested, leave me your information and I will be in touch. Oh, by the way, the beta program is completely free for you! Click the title link above to sign up. Thanks again and enjoy your weekend!
Jeremy LaRochester


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Details: London to Paris. Standard. 2h15m.
Seats: Eurostar's standard seats are among the best in European rail — spacious, with decent-sized trays and tables and a reasonable amount of room for luggage. My partner and I were traveling with our two-year-old, who fit snugly between us on a double seat. And when my daughter needed to sleep, I was grateful of the extra seats in the communal areas between carriages.
Entertainment: The new Eurostar trains due in service by 2015 promise wi-fi-connectivity and entertainment channels, but until then passengers must content themselves with newspapers and the entertaining Metropolitan magazine.
Food & drink: The snacks provided by Waitrose were generally OK. But if the bacon sandwiches I had on the outward journey were average, the pizza I ordered on the return was very good. However, the general buffet area is a bit dated and in need of a refresh.
Service: All the staff members we dealt with were friendly, although the till behind the buffet counter had broken down, which meant ordering food was a slightly chaotic experience.
Contact: eurostar.com
Last word: The spacious seating is ideal for families but the incoming entertainment upgrades can't come soon enough and the buffet carriage needs an update
Words By: Glen Mutel
Published in the Winter Spring 2014 issue of ASTAnetwork


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The new owners, Sun TV Network, officially renamed Deccan Chargers as Sunrisers on Thursday and also unveiled the new logo of the team, which is depicting an eagle soaring high and looking at the rising sun. "I am happy and excited about being the mentor and probably the strategic analyst for the team. We have people like Tom Moody, VVS Laxman, Dale Steyn and Sangakkara. With the grace of sun god, we hope to shine brighter. We are confident of making a big impact," Srikkanth said.
Sun Group took over the cash-strapped Hyderabad franchise earlier this year for a whopping Rs 425.2 crore after the Board of Control for Cricket in India (BCCI) terminated the contract of the Deccan Chargers.
Srikkanth said majority members of the previous team have been retained and some more would be picked up when the auction takes place next month. "We are keen on having players like Ambati Rayudu, Smith, who have been released by Pune, and the Pakistani players if they are available," he said.
Sri Lanka's Kumar Sangakkara would continue to play a role as captain of the new franchise. Moody, who did not have much success during his coaching stint with Kings XI Punjab team, is confident of a good show in the next edition of the tournament.
"We are confident that the team will be successful in the forthcoming season of IPL 2013. We are keenly looking forward to it and our game will stand testimony to the competition we will give to other teams," he said.
The team would operate from both Chennai, where Sun Group is headquartered, and also Hyderabad, the Sunrisers CEO K Shanmugam said. (PTI)


text:
Sony CD Boombox with Digital AM/FM Tuner (Red)
From Sony Boombox
Product Description
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Product Details
Consumer Electronics
Color: Red
Brand: Sony Boombox
Model: ZSE5RED
Dimensions: 4.00" h x 10.50" w x 10.50" l, 4.41 pounds
Compact body for easy portability
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Digital AM/FM Tuner
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By:Sony Boombox


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Disabling Links
I have a client who wants me to replicate a site that I created, but as an introduction to the actual site. The items that will be available for sale on the previous site, he wants to have about 95% of it as un-clickable links that still appear on the page.
He wants folks to be able to access the rest of the content and even download it. This, of course, is so that he can give a sample of what they (hopefully) will go on to purchase as a whole.
I was thinking of just adding an Active column to the database and make the entire site's links inactive, but visible, and then he could select a handful to activate through the admin as he sees fit.
Are there any tools within Data Bridge that anyone could recommend to get me started on this or do I need to write some JavaScript that will make the inactive links not work.
I plan on using CSS to make those links greyed and with a line-through text-decoration, but how can I use MySQL and JavaScript (or just one or the other) Actually make the links appear and not be clickable or appear and be clickable based on the Active checkbox I am going to provide my client?
I may have confused or over-explained this, but I think if I could get a point in the right direction I could figure it out.


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Journal of Biomedicine and Biotechnology
Volume 2009, Article ID 837608, 9 pages
Functional Expression of a DNA-Topoisomerase IB from Cryptosporidium parvum
Departamento de Ciencias Biomédicas (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain
Received 20 February 2009; Revised 20 May 2009; Accepted 28 May 2009
Academic Editor: Ali Ouaissi
Copyright © 2009 César Ordóñez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only when CpTopIB ORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38) was assessed.
1. Introduction
Cryptosporidium parvum is one of the most important causal agents of human diarrheas during childhood [1]. Infections occur by ingestion from poor quality supply water contaminated with oocysts (resistance form of this microorganism) [2]. In humans the infection is self-limiting in immunocompetent individuals but may be fatal in HIV-immunocompromised patients [3]. In ruminants the infection is particularly severe in neonatal lambs, which suffer from apathy, anorexia, abdominal cramps, diarrhoea, and a large elimination of oocysts in faeces [4].
The high prevalence of this disease may be a consequence of the resistance of oocysts to chemical water treatments, to the poor efficacy of the current drugs used or the lack of an effective vaccine. Therefore, it is an essential integrated approach in the management of this condition that involves the use of disinfectants, host immune-reinforcing agents, and new drugs [5, 6].
During last decade more than hundred antibiotics and antiparasitic drugs have been tested against C. parvum infections with little or no effect [7]. Paromomycin, a poorly absorbed aminoglycoside antibiotic, and more recently nitazoxanide were pointed as promising agents to treat cryptosporidial infections in acquired immunodeficiency syndrome (AIDS) patients [8]. Both compounds showed efficacy in animal models [9, 10] and paromomycin was prophylactic in neonatal calves, lambs, and goats. On the other hand, nitazoxanide—a nitazoxazole benzamide compound—has a wide range of antimicrobial activity against helminthic parasites. More striking is the drastic reduction in the prevalence of opportunistic Cryptosporidium spp. infections associated to AIDS patients treated with highly active antiretroviral therapy (HAART) [11]. Therefore, there is an urgent need for novel compounds that eradicate these infections with poor or no side effects on the host [12].
Enzymes responsible for DNA manipulation have been studied for long as potential pharmacological targets in proliferative processes such as microbial and parasitic infections and tumor processes. DNA topoisomerases (Top) are enzymatic machines catalyzing changes in the topological state of duplex DNA during replication, transcription, recombination, and DNA repair processes. There are two families and several subfamilies of these enzymes: Type I DNA topoisomerases (TopI) are ATP-independent enzymes with relaxation activity for both positively and negatively supercoiled DNA [13, 14]. Type II DNA topoisomerases (TopII) are multimeric proteins that hydrolyze ATP to generate temporary DNA double-strand breaks followed by passage and rejoining. TopII enzymes not only relax positively supercoiled DNA but also knot/unknot and catenate/decatenate close circular DNA [15].
Many compounds have been screened against Top enzymes, and some of them are being used nowadays. Fluoroquinolones were successfully tested as inhibitors of the DNA gyrases in bacteria and some of them are potent antibiotics of choice [16]. On the other hand TopIB is being targeted in the treatment of several tumor processes (Pommier, 2006). Water-soluble derivatives of camptothecin (CPT)—topotecan and the prodrug irinotecan—are part of the current pharmacopoeia against certain types of cancers, but the studies are much scarcer in relation to topoisomerases in protozoan parasites [17, 18]. The amazing finding of a bi-subunit TopIB in Kinetoplastids [19] has opened a new field in the chemotherapy of these parasites, but little is known about Top in apicomplexan. Only Plasmodium falciparum TopIB (PfTopIB) has been cloned, functionally expressed and targeted with CPT derivatives [20, 21].
An early report describing the cloning of a type II DNA topoisomerase from C. parvum was published years ago [22] but no further information about these enzymes has been reported at present. In this paper we describe the functional expression of CpTopIB gene from the apicomplexan protozoa C. parvum, as well as the heterologous expression, kinetic analysis of the recombinant enzyme and the inhibition by CPT and analogues.
2. Materials and Methods
2.1. Reagents
DNA modification and restriction enzymes were procured from Roche (Basel, Switzerland) and Amersham Biosciences. Pyrococcus furiosus (Pfu) polymerase was from Stratagene (La Jolla, Calif, USA). Cell culture media, chemicals and reagents were purchased from Sigma (St. Louis, Mo, USA). Primers for PCR amplification were from Sigma Genosys (UK). CPT and SN-38 were from Tocris (Tocris Bioscience, Ellisville, Mo, USA), topotecan from R&S Pharmachem (Co. Ltd. China).
2.2. Yeast and Cryptosporidial Strains
C. parvum ooquistes were kindly provided by Dr. Teresa Gómez Muñoz; Dpt. Atención Sanitaria, Salud Pública y Sanidad Animal; Universidad Cardenal Herrera (CEU) Valencia (Spain). For protein expression Saccharomyces cerevisiae strains (a gift from Dr. MA Bjornsti, St. Jude Children's Research Hospital, Memphis, TN) EKY3 [MAT α ura3-52 his3 200 leu2 1 trp1 63 top1 ::TRP1] and MBY3 [MAT α ura3-52 his3 200 leu2 1 trp1 63, top1 ::TRP1 rad52 ::LEU2], both deficient in DNA topoisomerase I activity, were used [23].
2.3. Cloning of Cryptosporidial DNA Topoisomerase I
Cloning of C. parvum DNA-topoisomerase I (CpTopIB) was performed by PCR amplification using whole genomic cryptosporidial DNA as template [24] and the primers showed in Table 1. CpTopIB (GenBank accession number XM-628497) gene, was amplified using Pfu-polymerase and nucleotides, and the amplified band was loaded in a low-melting gel agarose, electrophoresed and taken from the gel for cloning. The 1923 nt amplified band was digested with SmaI and HindIII and ligated to YCpGAL (Leu-) vector and sequenced before expression in a TopIB-deficient yeast strain.
2.4. Yeast Expression System
S. cerevisiae strain, EKY3 was transformed with different constructs viz; wild type (CpTopIB); R318A; K358A; R416A; H489A and Y600F, as the different point mutations, by electroporation [25]. The GAL promoted expression vector carries the Leu- selectable marker and is maintained by selection in synthetic complete SC leu- medium. At least five independent clones were selected from each transformation. After 6-hour induction with 2% galactose in SC leu- raffinose medium, the cells were harvested by centrifugation, washed, and resuspended in TEEG buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 10% glycerol) supplemented with 0.2 M KCl, and protease inhibitors cocktail [1 NaF, 1 sodium bisulphite, 2 Complete Mini (Roche Molecular Biochemicals)]. Cell extracts were prepared by disruption with acid-washed glass beads using a bead-beater chamber (5 cycles of 30 seconds on, 30 seconds off) according to a previously described procedure [26]. Briefly, cells were subjected to one freeze/thaw cycle at –80, lysed by vortexing with 425–600 m glass beads and the extracts were cleared by centrifugation at 15000 xg for 30 minutes at 4.
2.5. Protein Purification
Purification of CpTopIB was done according to [27]. Briefly, overexpressing yeast extracts were loaded onto a phosphocellulose (P-11) column previously equilibrated as manufacturer indications. The recombinant proteins (wild-type CpTopIB and mutated forms) were eluted at 4 with a discontinuous gradient of KCl (0.2, 0.4, 0.6, 0.8 and 1 M) in TEEG buffer, supplemented with 0.1 mg/mL sodium bisulphite, 0.8 mg/mL NaF and the protease inhibitors cocktail. The active fractions were mixed with equal volume of 2 M NH2SO4 and loaded onto a phenyl-sepharose column preequilibrated with TEEG containing 1 M NH2SO4 (Sigma-Aldrich, St. Louis, USA). The column was eluted with a discontinuous inverse gradient of NH2SO4 (1, 0.8, 0.6, 0.4 and 0.2 M). In order to reach an appropriate concentration for the different in vitro assays, the eluate from the phenyl-sepharose column was concentrated by Microcon YM-30 (Millipore, Co, Ma, USA). Purity of the samples was analyzed by SDS-PAGE. To store, 40% glycerol was added to preserve the activity and keep in a freezer. Protein concentration was determined colorimetrically [28].
2.6. DNA Relaxation Assays
CpTopIB activity was assayed by the relaxation of a negatively supercoiled plasmid DNA. The reaction mixture in a total volume of 20- contained 0.5 g of supercoiled RF I (ΦX-174) DNA, 10 mM Tris-HCl buffer pH 7.5, 5 mM MgCl2, 0.1 mM EDTA, 15 g/mL bovine serum albumin, 50 mM KCl, and 0.2 g purified protein. The reaction mixtures were incubated for 30 minutes at 37. The enzyme reactions were digested with the addition of 2 g of proteinase K, incubating for 1 hour and adding up to 1% SDS. The extent of plasmid DNA relaxation was assessed by electrophoresis in a 1% agarose gel in 0.1 M Tris borate EDTA buffer pH 8.0 at 2 V/cm for 14 hours. The gels were visualized under UV illumination after being stained with ethidium bromide (0.5 mg/mL) and a posterior electrophoresis in the presence of 0.1 mg/mL ethidium bromide, in order to separate the nicked DNA from the relaxed topoisomers.
2.7. In Vitro Cleavage Assay
To 0.5 g supercoiled RF I DNA, in a total volume of 20 cleavage buffer (35 mM Tris HCl, pH 7.5, 50 mM KCl, 3 mM β-mercaptoethanol, 0.1 mM EDTA, and 50 mg/mL BSA), was added a solution of the appropriate drug dissolved in DMSO or water (final concentration of DMSO in the incubation mixture must not exceed 1%). The DNA-cleavage reaction was initiated by the addition of 2 units of CpTopIB purified as above. After incubation for 30 minutes at 37, the cleavable complex was trapped by the addition of 2 10% SDS, followed by the digestion with proteinase K for a further 30 minutes period at 45. The mixture was purified with phenol/chloroform and electrophoresed in a 0.9% agarose gel containing ethidium bromide (0.5 g/mL). The increase of nicked DNA was estimated as drug-induced CpTopIB-mediated DNA cleavage.
2.8. Site-Directed Mutagenesis
The procedure to obtain the mutated genes was developed according to the QuickChange method, following the instructions procedure; the ORF encoding CpTopIB gene was mutated and subcloned in the YCpGAL(Leu-) vector. Figure 1 describes the distinct mutations performed on CpTopIB.
The sequence of the primers used and their codon changes are listed in Table 1. The PCR reaction contained 20 ng of plasmid YCp-CpTopIB as template depending on the different mutations, 1 M of each oligonucleotide, 10 M dNTPs, 5 of 10 Pfu-buffer, and 2.5 units of Pfu-polymerase for a total volume of 50 L. Reactions were carried out in a Mastercycler gradient thermocycler (Eppendorf) as follows: a 5-minute first step at 94, followed by an amplification cycle at 95 for 1 minute, 60 for 1 min and 72 for 1 minute; repeated for 16 times. Mutants were sequenced (Sistemas Genomicos, Spain) in order to confirm the absence of undesirable additional mutations.
2.9. In Vivo Sensitivity to CPT
Sensitivity of the MBY3 yeast cell lines transformed with va s CpTopIB constructs to CPT was determined, as described earlier. The individual transformed yeast cell lines grown in SC leu- medium plus dextrose were serially diluted 10-fold and 5- of each culture were spotted onto selective SC leu-agar plates supplemented with 25 mM HEPES, pH 7.2, 2% dextrose or galactose, supplemented with 30 M CPT in a final 0.25% DMSO. The controls plates without the drug contained 0.25% DMSO. Viability (growth) of yeast cells was scored, as per the size of the culture spot, following incubation at 30°C for 24 hours.
3. Results
3.1. Cryptosporidium parvum Topoisomerase IB
The active form of CpTopIB (GenBankTM accession number XM-628497) is the expression product of a single 1923 bp ORF. The expressed polypeptide corresponding to the putative CpTopIB is constituted by 641 amino acids with a predicted molecular mass of 74.7 kDa and a pI of 8.7, showing a 60% identity to human DNA topoisomerase IB (hTopIB) (GenBankTM accession number J03250), 75% identity to baker yeast's (yTopIB) (GenBankTM accession number K03007), and 99.8% of identity with C. homini. The protein is much shorter than human's (765 amino acids) and baker yeast's (769 amino acids), but contains all the recognizable domains of a canonical TopIB protein. A recombinant active enzyme was obtained by cloning the PCR-amplified band into the yeast expression vector YCp-GAL(Leu-) driven by GAL1 promoter and expressed in a TopIB-lacking yeast system (EKY3).
Figure 1(a) shows a sequence alignment of CpTopIB with its homologous from human, P. falciparum, and the budding yeast Saccharomyces cerevisiae showing that, as expected, CpTopIB contains a putative nonconserved N-terminal domain (start-Met-Gly 42), a conserved core domain (Ile-43-Val-492), and the C-terminal domain, (Thr-590-Phe-641) which shows high homology with other eukaryotic C-terminal ends [29] (Figure 1(b)). This region includes a phylogenetically conserved "SKINY'' signature in which Tyr-600 (Tyr-723 in the human enzyme) plays role in DNA cleavage. The C-terminal domain is connected to the core by a poorly conserved coiled-coil putative linker (Val-493-Val-589). The putative cryptosporidial core domain has high homology with the one from the human enzyme (Gln 33-Ser 456) and other species, and it includes all the amino acid composing the so-called catalytic tetrad involved in the relaxing activity; Arg-318 (Arg-488 in the human enzyme), Lys-358 (Lys-532 in the human enzyme); Arg-416 (Arg-590 in the human enzyme) and His-489 (His 632 in the human protein).
When CpTopIB gene was expressed in the yTopIB-defective EKY3 yeast strain, using the YCp-GAL(Leu-) vector, significant relaxation activity was detected. Yeast extracts were pooled and purified as described above (Figure 2(a)). Purified CpTopIB was used for kinetic characterization. Relaxation activity of the supercoiled Rf I close circular DNA from the virus ΦX-174, was measured as a function of protein concentration (Figure 2(b)) and time (Figure 2(c)). Using 2 units of purified CpTopIB and 0.3 g of supercoiled DNA substrate, a distributive relaxation activity with many relaxed topological isomers was obtained as early as 5 seconds, completing the process in 1 minute. Two-fold serially diluted CpTopIB-transformed EKY3 yeast extracts were assayed for relaxation activity (Figure 2(b)) starting from 0.2 g protein (left lane). Significant activity was found after six 2-fold dilutions (0.03 g protein) but no visible topoisomers were detectable beyond.
We further studied the relative in vitro DNA-cleavage potentials of CPT, topotecan, and SN-38—the active metabolite of irinotecan hydrolysis by cellular esterases [30]—at 10 and 100 M final concentrations. The capability of these compounds to stabilize DNA-cleavable-complexes was determined by agarose gel electrophoresis, followed by SDS and proteinase K treatments that allow denaturation and proteolysis of the binary complex, producing an increase in nicked DNA (Figure 3). There is a dose-dependent nicked-DNA increase for CPT and topotecan, but it could not be found for the irinotecan metabolite SN-38.
3.2. Site-Directed Mutagenesis Studies
The role of the amino acids of the catalytic tetrad was analyzed by site-directed mutagenesis, studying the relaxation activity of the mutated proteins. Based on these analogies, we replaced the residues sited at the core domain by the neutral amino acid alanine, which is not protonable at physiological pH, preventing the electrostatic contributions in substrate catalysis and hydrogen bonding (mutations R318A, K358A, R416A and H489A). On the other hand Tyr-600, placed at the C-terminal end, which aligns with a homologous Tyr at position 723 in the human enzyme, was replaced by Phe, which is sterically homologous but lacks the ability to cleave DNA (mutation Y600F). All these mutations were created in the pSK vector, carried to the yeast YCp-GAL(Leu-) plasmid, and heterologously expressed in the TopIB-deficient yeast system (EKY3). Figure 2(b) compares the residual relaxation activity of these mutants with the one found in the recombinant wild-type enzyme. As expected, none of the mutants of the active site created, displayed any significant ability to relax supercoiled DNA under standard assay conditions, using undiluted extracts (0.2 g protein).
3.3. Topoisomerase-Deficient Yeast Sensitization to CPT
CpTopIB is conserved in terms of reaction mechanism and drug sensitivity. The heterologous expression of the wild type dimeric enzyme into a topoisomerase-deficient yeast strain (top1 ) and defective in double-strand breaks repair (rad52 ); MBY3 was able to confer sensitivity to 30 M CPT when cultures where induced with 2% galactose (Figure 4).
The site-directed mutants prepared previously were transfected to the MBY3· yeast-deficient strain plated and induced with 2% galactose in absence and presence of 30 M CPT. Pictures of Figure 4 show that none of the catalytic amino acids (R318A, K358A, R416A, and H489A), nor the active site mutant (Y600F), conferred susceptibility to camptotecin to the transformed yeasts. In addition CpTopIB MBY3—transformed yeast induced a significant drop in the number of viable cells when treated with CPT both in time course (Figure 5(a)) and dose-response (Figure 5(b)) experiments, thus showing the requirement of an active Top enzyme to be inhibited by the drug.
4. Discussion
There is no effective pharmacological treatment to eradicate cryptosporidiosis. It is well established that this disease can be fatal only when the immune system is challenged. Several antibiotics and antiprotozoal agents have been clinically or experimentally assessed, with very limited or no success. However, some drugs may be overviewed along with some new approaches attempted for new drug design and development against cryptosporidiosis. There is an increasing body of evidence that Cryptosporidium oocysts exposed to UV light are not able to regain the infectivity previous to exposure. Rochelle and coworkers explained that the mechanism of nucleotide excision repair (NER) is not sufficient to restore the damage caused to DNA due to mutations or the absence of repair genes [31]. Many Top inhibitors are DNA-damaging agents, causing stable single (SSB) and double strand breaks (DSB) [32]. This particular feature and the fact that these compounds are being used effectively as antineoplasic agents make Top enzymes promising targets for chemotherapy intervention against cryptosporidial infections.
We have cloned and functionally expressed the predicted CpTopIB gene (GenBank XM-628497), with C. parvum genomic DNA as a template. Like trypanosomatids, attempts to express this ORF in a routine bacterial expression system were not successful and a top-deficient yeast strain was required for this purpose [33]. Heterologous expression of CpTopIB-transformed S. cerevisiae strain under galactose pressure produced a single prominent band of 76 kDa, which is consistent with the estimated molecular mass of the putative ORF. Purified yeast extracts by a conventional low-pressure liquid chromatography procedure derived in a highly enriched solution of CpTopIB that was used to relax supercoiled DNA.
CpTopIB contains all the structural and enzymatic features described for all TopIB enzymes in a single polypeptide that relaxes supercoiled DNA distributively. The enzyme was targeted by CPT and the water-soluble derivative topotecan, which established irreversible bonds with DNA, but not by the irinotecan metabolite SN38 that surprisingly was not able to cleave DNA at the concentrations used. Site-directed mutagenesis of the amino acids composing the catalytic tetrad in the putative core domain yielded in no enzymatic activity at all, as well as the point substitution of the cleaving Tyr-600 by Phe. All these results supported the existence of a conserved active centre that matched well with that reported in the human enzyme [29, 34].
Only one apicomplexan TopIB has been studied at present. Riou and coworkers, purified and characterized PfTopIB from infected erythrocytes (Riou et al., [35]). Like C. parvum, plasmodial enzyme is a monomeric protein of 104 kDa corresponding to a peptide of 839 amino acids which gene is located at chromosome 5 of the plasmodial genome, and it is developmentally regulated during the different stages of Plasmodium life cycle [21]. In addition, the enzyme is effectively targeted by CPT that induces cleavable complexes with DNA in vitro [17].
In conclusion, there is an increasing resistance of C. parvum to such a broad range of chemotherapeutic agents, some of which are effective against other apicomplexan parasites. This might be related to the intracellular stages of the life cycle that are practically engulfed inside the host cytoplasm. Therefore, there is an urgent need of novel targets for drug screening. The above mentioned results show that C. parvum contains an active TopIB enzyme that is presumable involved in resolving structural problems in DNA during replication, transcription, and repair processes. Unlike other protozoa, the enzyme comprises a unique protein encoded by a single gene, which contains all the structural features defining a fully active TopIB. Understanding of distinct molecular characteristics of the CpTopIB and regulation of expression of the enzyme during parasite life cycle may be useful for development of novel selective inhibitors of CpTopIB as promising anticryptosporidial agents.
|Type IB DNA-topoisomerase
|Type II DNA-topoisomerase
|Gene encoding Cryptosporidium parvum topoisomerase I
|Gene encoding human topoisomerase I
|Gene encoding Plasmodium falciparum topoisomerase I
|Acquired immunodeficiency syndrome
|Highly active antiretroviral therapy
|Nucleotide excision repair
|Single strand breaks
|Double strand breaks
This research was supported by Ministerio de Ciencia e Innovación (Grants AGL2003-06976/GAN and AGL2006-07420/GAN) and GR238 (Junta de Castilla y León) from the Spanish Kingdom.
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Our Liberal Government has promised real change in Canada. The good news is, Canadians are hopeful for the Liberal government's plan for real change and they will get it.
That means investing in middle class families and making sure they have every opportunity to succeed. One of the first course of action taken by the new Liberal government was the introduction and passage of the middle class income tax cut.
On the question of what is needed to better support the middle class: Investing in Jobs and Training is #1 priority. Without question, the economy is the top priority. Now is the time to make significant investments in a strong middle class. It means creating jobs by making the right investments in roads, bridges, transit, housing and green infrastructure. It means asking those who have a lot, to give a little more to those who need it.
Canada's Finance Minister, the Honourable Bill Morneau will present the federal budget on March 22nd. Budget 2016 will present an excellent opportunity for Canadians. We know there's never been a better time than right now to make the right investments. Ones that create jobs, put more money in people's pockets, and support economic growth.
Please stay tuned.


text:
what happens when you aren't a piano prodigy
You have to understand, my dad is a brilliant pianist. He could always play, from the time he was a little kid. For no reason that anyone could explain or understand, he could sit down and make music. He never learned to read music, but if he heard something, he could play it right back, so when he finally took lessons from a local teacher, he fooled her for months. "Can you just play it for me so I can hear how it should sound?" he'd ask when she gave him a new assignment. She'd play the piece through, and he'd instantly memorize it. Later, he'd pretend to be studying the notes on the page. She was furious when she discovered his trick, or so the story goes.
When he grew up, my dad bought a grand piano and then was too poor to fix his car for a whole winter. My parents were barely in their twenties, proud of their tiny square house, running their own tiny business out of the basement, and there was this sleek, giant Yamaha grand filling the whole living room. Almost a decade before I was born, that piano was my dad's baby.
When I was a baby, he sat me on his lap while he played, my hands on his hands.
(I don't have a photo of that, but here I am as a baby, with my hair looking much like a wig, playing)
And of course, when I was old enough, he taught me some pieces, and eventually signed me up for lessons. He still couldn't read music very well, but he wanted me to learn, so I was sent to a piano school that specialized in early classical training. The word "pedagogy" floated around in the halls.
I don't know where my ferocious competitiveness came from. It's been there for as long as I can remember, an engine too big for my body, eternally revving. It wasn't enough to just play—I wanted to be the best.
I dreaded the finger exercises. I was bored by the endless arpeggios. The flick of the thumb under the third finger going up the scale—the movement had to be perfectly smooth, subtle, the arrangement of the hand needed to stay even. I wanted to play Rachmaninoff, with the crashing depths of the low register. Lush, chocolate-y Brahms. I wanted to play cascading Debussy, with the blur of rich sound, and Chopin, of course, with the delicate right hand trilling up at the very top while the left hand ran darkly around the bass. I wanted pounding drama and thunder and thrill. I wanted to close out the annual recital with a show-stopper. I was jealous of the kid who got to do it, while I was still playing in the middle of the pack. I was also terrified of performing.
Kate on February 19th 2014 in being sad, family, fear, life, Little Victories, perfection, uplifting


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Adger W. Cowans
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Who is Adger W. Cowans?
Adger W. Cowans is an actor.
- also known as Adger W. Cowan, Adger Cowan, Adger Cowans
- born in (79 years ago)
- profession: Actor
- read more about Adger W. Cowans (actor)
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Blog Directory Submissions
Posted in Blog Traffic
Top Blog Directories List to Submit Your Blog To
This post is part 3 of the Traffic Action Plan, see part 1 here: –
and part 2 here: –
Submitting Articles For Traffic
This is another "old school" and "un-sexy" method that gets overlooked by most people now for getting traffic to new websites.
One of the most important things to remember when getting new backlinks to a fresh new website to help get it to rank well in the search engine results is to use a VARIETY of backlink building tactics so that the links are not all coming from one source or type of source. Mixing in some blog directory links is an easy way of ensuring this, but you will also discover you actually get visitors from some of these blog directories, as well as the valuable backlinks.
The directories below are specifically fro Blogs, if your site is not a blog then the best way to approach directory submissions is to pick up a simple [and cheap] piece of software that will automate the whole process for you, I've recommend taking a look at the one here: –


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NCSE was featured in Climate Fwd: — The New York Times's e-newsletter "with stories and insights about climate change" — for February 21, 2018: "climate change is presented as a controversial subject in a significant number of American classrooms, according to research from the National Center for Science Education."
Florida's House Bill 827 and Senate Bill 1644 — similar bills that would make it easier for creationists and climate change deniers to smuggle instructional materials they favor into public school classrooms — were front and center in a recent report (February 23, 2018) in the journal Nature on legislation targeting the integrity of science education.
"After three years of resistance at the GOP-dominated Idaho Statehouse, including more pushback from House Republicans this session, the Senate Education Committee voted 6-3 on Thursday to approve revised school science standards as-is — with no parts relating to climate change deleted," reports the Idaho Statesman (February 22, 2018).
Florida's House Bill 827 — which would make it easier for creationists and climate change deniers to smuggle instructional materials they favor into public school classrooms — was approved, with slight modifications, by the House Education Committee on a 19-0 vote on February 15, 2018.
"For the second time in a decade, a state appeals court has told a taxpayer that he can't sue his school district because it teaches the theory of evolution in science class," reported PennLive (February 16, 2018).
National Center for Science Education, Inc.
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Moving Mayhem - Raspberry Cupcake Edition
Call it prophecy or call it coincidence… either way, it's somewhat humorous that I made this post one year ago and titled it Moving Mayhem – Bread Pudding Edition. It's almost as if I was setting myself up for another edition. I just hope there isn't another Moving Mayhem post in 2013. This move should go much more smoothly than last year's, even though we're basically just doing the second half of that move in reverse. If the weather forecasters are right, we at least won't be dodging tornados. So, since this move is only 500 miles and we'll be doing it in basically 2 days, I'm not getting rid of everything. That meant I could make whatever I wanted and didn't have to worry about using up my good stuff. It will be going with me. With that in mind, I decided I wanted raspberry muffins, but since Seeley recently made muffins, I decided to change it to raspberry cupcakes, and the cupcakes vs. muffins discussion began.
Either way, here's what you'll need:
½ cup butter
¾ cup sugar
2 teaspoons vanilla
2 eggs, separated
⅔ cup buttermilk
1 ½ cups flour
½ teaspoon soda
½ teaspoon powder
1 cup raspberries
So, I have to admit to not getting the best looking berries. One of the other brands had much bigger, prettier berries, but they didn't smell at all. These ones, on the other hand, smelled amazing. In fruit, aroma almost always trumps appearance.
If you can't find any that are good, you could probably use frozen ones. Just make sure they have thawed and come to room temperature. In fact, it's important to note that everything in this recipe must be room temperature. So, on that note, throw your softened butter into the bowl of your mixer.
Add the sugar and beat on medium speed for about 2 minutes, or until it's light and fluffy.
Next, separate your eggs and put the whites aside.
Add the yolks and vanilla to your butter mixture and beat until thoroughly incorporated.
In a separate bowl, whisk together your dry ingredients.
Add ⅓ of the dry mixture to the batter and mix to combine.
Follow that with half of the buttermilk.
Then another third of the dry, the other half of the wet, and finally the last of the dry. You should have a nice smooth batter. Obviously, everything needs to be scraped down.
Now back those egg whites you set aside.
Whip them until you have stiff peaks.
Now, rinse your raspberries and add them to the bowl.
You don't want to totally decimate the raspberries, so just mix enough to break them up some. It will be more evenly distributed after you've finished folding in the egg whites.
Which you're going to do now. Start with half of the egg whites. Just drop them on top.
Then fold them in. This will lighten up the batter some, which will makes the second edition easier to incorporate.
Fold that in now.
Um, somewhere in that process you should have preheated your oven to 350°. Scoop the batter, filling the cups almost to the top. I used a ¼ cup measuring cup for this process. Somehow I haven't managed to buy a muffin scoop. At some point after I've moved, I'll probably remedy that situation.
Just before you put the cupcakes into the oven, turn the temperature up to 425°. Place the cupcakes in the oven, leaving the temperature at 425° for 5 minutes, and then turn it back down to 350°. That extra heat helps to keep your cupcakes nicely rounded on top and stops them from sinking in the middle. Bake them for a total of 25-30 minutes, or until a toothpick inserted into the middle comes out clean, or perhaps with a few crumbs on it.
Remove them from the pan as soon as you can and place them on a rack to cool.
Now it's time for what I think makes a cupcake a cupcake… the frosting. I keep reading about this amazing flour/cornstarch based frosting that is the most amazing frosting ever made. I guess it's time I gave it a try. After much research, I took the parts I liked from different sources and put them together to form my own version of said frosting.
Here's what you'll need:
¾ cup milk
¾ cup sugar
2 Tablespoons cornstarch
¼ teaspoon salt
1 ½ sticks (¾ cup) unsalted butter
1 teaspoon vanilla
When making frosting it's important to use unsalted butter. Salty frosting… not such a good thing. Place your sugar, salt, and cornstarch into a medium saucepan.
Whisk them together until there are no more clumps of cornstarch.
Add the milk.
Cook over medium heat, stirring constantly, until the sugar is dissolved and the mixture thickens. You don't want it to reach a rolling boil. You'll know it's ready when you start to see the pan while you're stirring.
When you've reached that stage, remove it from the heat and allow it to cool. It's important that it is room temperature, and not even a little bit warm when you start the next part. Place the softened butter into your mixing bowl. You'll want to use the whisk attachment this time. Whip the butter until it's nice and fluffy.
Pour in the cooled milk mixture and add the vanilla.
Then just start mixing. At first you're going to have a curdled mess.
But just keep mixing. We're talking like high speed for at least 5 minutes. I actually had to walk away from mine for a bit so it could do its thing without me panicking and hovering. Eventually, you'll have this. A smooth, creamy, and quite frankly, perfect frosting.
I think the reviews I've read were right. Not only is the flavor and texture of this frosting seriously amazing, but it pipes beautifully.
Pipe it on or spread it, whichever you prefer, then just top with a fresh raspberry, and you have cupcake perfection.
These cupcakes are moist and tender, and the slightly tart berries pair perfectly with the sweet frosting. Seriously divine.
So what's the difference between a cupcake and a muffin to you?


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Submitted by jaylon01 • February 17, 2014 www.articlesnatch.com
If you want to have some adventure at night in the darkness, you can enjoy yourself at Kaamos. In Finland, the sun doe not rise for two months. Though there is no absolute darkness, but you can enjoy the twilight by going for a ski or any other winter sports.
- Category: Travel


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Eells Ranch Annexation to Garden Valley Fire Protection District, Project No. 2018-03
NOTICE OF PUBLIC HEARING January 23, 2019
Effective January 1, 2019, the California Department of Fish and Wildlife will increase the filing fees for Notices of Determination.
The diverse special districts, cities, county service areas that make up the area our Local Agency Formation Commission works with.
The following information is designed to help you move your project through the various stages of the LAFCO process. State law and El Dorado LAFCO Policies and Guidelines require a series of steps and…


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4. Colby has been battling ear infections for the past couple of weeks. In case you've paid close attention to the blog, you might recall that he got his second set of ear tubes a mere 2.5 months ago, so this has been a really special development.
PSA for the day: kids can still get ear infections while they have ear tubes.
Second PSA for the day: the world's tiniest bottle of ear drops used to treat them costs $38. Lame.


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Advertising and Marketing Graduate Programs (On-Campus)
In marketing graduate programs, students learn how to conduct analysis and apply marketing theory and technique to campaigns across industries and cultures. In master's degree programs, they might develop their skills as managers and leaders, and in doctorate degree programs, their focus may be on becoming educators of the future generation of marketing professionals.
At the graduate level of education, students enhance their basic marketing knowledge and skill set and broaden their knowledge to include more nuanced areas of marketing, such as digital marketing, marketing analytics, or international advertising. They do so in a bricks and mortar classrooms with their classmates and professors in an on-campus setting.
Why Pursue a Marketing and Advertising Degree on Campus?
Marketing and advertising professionals interact with people on a regular basis. In general, this creative and innovative crew is one comprising people-persons and professionals who want to communicate as successfully as possible. Therefore, having the skills to listen, speak clearly, and interact with a variety of types of people is key. In the classroom, interacting with classmates and professors can help you develop these critical skills.
At the graduate level of education, students also commonly develop their skills as leaders and managers. By navigating team dynamics on group projects and applying newly learned skills in the flesh, students can begin to develop who they are as leaders and collaborators.
On-campus marketing and advertising programs offer students access to clubs and other groups geared toward students' professional interests. A student studying international marketing might join a cultural club, for example, while a student pursuing a digital marketing degree might join a technology club. This type of engagement can help students deepen their learning and
broaden the ways in which they apply their knowledge.
Lastly, students in on-campus marketing graduate programs may benefit from face-to-face, in-person learning with classmates and professors. For some people, being able to ask questions, take advantage of professor's office hours, and discuss materials with classmates in person allows for a richer experience. And, having a specific schedule and a requirement to be on campus at certain times might provide the structure and support that some students need.
Did You Know?
Because professionals in the field strive to appeal to people, marketing has a decidedly social science side. As such, many on-campus programs have labs in which students work together to discover human phenomena that impact the marketing process.
Marketing vs Advertising: What's the Difference and What Can I Expect from a Program?
Keep in mind that advertising is a component of marketing. Marketing includes a broad range of methodologies, venues, and formats designed to promote ideas, sell products, establish brand awareness, and otherwise extend messages to a broader audience. Advertising, meanwhile, is just one of many marketing methods that employs print, television, or radio to sell a product or idea.
Marketing and advertising degree programs are therefore slightly distinct. In many cases, schools combine marketing and advertising curricula. Students might take just one or two courses in advertising as a part of their marketing degree, or they might concentrate their studies in advertising and take numerous courses in the subject.
Types of Marketing Degrees at the Graduate-level of Education
At the graduate level of education, students can pursue a graduate certificate, which takes six months to one year to earn; a master's degree, which takes up to two years to earn; or a doctorate degree, which takes four to five years to earn. Each of these levels of education has on-campus options.
Certificate programs in marketing typically include four to seven courses. These are designed to help students specialize their knowledge in a specific area of marketing and/or advertising or to introduce them to the fields. For students who only have the resources to devote six months to one-year to on-campus education, this could be a great option.
Master's degree programs in marketing typically include ten to fifteen courses. These are designed to have students study marketing broadly or an area of marketing, such as advertising or analytics, in depth. A master's degree in marketing commonly includes curricula to help students develop their management and leadership knowledge and skills.
Doctorate degree programs in marketing typically include 80 to 100 credit hours divided between coursework and research (thereby making it difficult to assess precisely how many courses students have to take as many of the credits might derive from students conducting and presenting research). In these programs, students typically focus on learning how to conduct research and become educators in the field of marketing.
Options for On-Campus Marketing Graduate Programs
At the master's and doctorate degree levels, many on-campus marketing graduate programs offer customizable curricula to enable students to pursue their areas of interest. Students may also be able to concentrate their learning or follow particular tracks of learning. Students might pursue, for example:
- A marketing management degree
- A business and marketing degree
- A sales and marketing degree
- A marketing law degree
Similarly, students might pursue a degree in marketing with an emphasis in a subject such as marketing analytics, consumer behavior, or design. Or they might pursue a degree in a subject such as marketing analytics and focus their learning even more deeply by studying audit analytics, business analytics, or marketing analytics. Students who are business professionals might pursue a Master of Business Administration or Doctor of Business Administration with an emphasis in marketing.
Ultimately, there are numerous options and ways for students to direct their learning at the graduate level of education.
Curricula in On-campus Marketing Graduate Programs
The courses students take in their on-campus marketing graduate programs depends on their focus and type of degree. However, we can give you a sense of some core subjects graduate marketing courses and programs might cover:
- Marketing management
- Consumer analysis and insights
- Branding strategy
- Internet Marketing
- Advertising and promotions
- Marketing analytics
Students might take elective courses in subjects such as:
- Global marketing
- Digital entertainment
- Marketing for entrepreneurs
- Business law and ethics
- Retail strategy
No matter the subject matter, projects and assignments in the classroom are likely to provide real-world experiences, and students in the classroom may be encouraged to interact with students of various backgrounds, nationalities, and interests. Case studies are typically global in nature and cover the spectrum of industries. Individual and group assignments will likely be designed to help students conduct in-depth analyses, think critically, and communicate effectively.
Marketing Degree Requirements
On-campus marketing graduate programs have different requirements depending on the program level.
In certificate programs, students typically complete core and elective courses and do not have to write a cumulative research paper or take a cumulative exam.
At the master's degree level, students typically complete core and elective courses and submit and defend a final project, which might be a thesis, presentation, portfolio, or other type of research project. Depending on the program, they might also have to take a comprehensive exam.
At the doctorate degree level, students typically complete core and elective courses, submit and defend a dissertation, and may have to take a comprehensive exam. They might also have to participate in an assistantship as a teacher or researcher.
Career Options in the Fields of Marketing and Advertising
With a graduate degree in marketing, you can work in numerous capacities. For example, you might work as:
- An Advertising, Promotions, and Marketing Manager: In 2017, professionals in this position earned a median annual salary of $129,380.i
- A Market Research Analyst: In 2017, professionals in this position earned a median annual salary of $63,230.ii
- A Public Relations Specialists: In 2017, professionals in this position earned a median annual salary of $59,300.iii
The Bureau of Labor Statistics predicts that these positions will grow between nine and 23 percent between 2016 and 2026.iv
Guess What? While each of the positions listed above requires professionals to have only a bachelor's degree, the Bureau of Labor Statistics shows in their 2017 audit that 23 percent of market research analysts and marketing specialists held master's degrees. On average, they earned a mean annual wage of $25,000 more than professionals who held only a bachelor's degree.v
Find Schools With Marketing Graduate Programs
There are obvious benefits to pursuing a graduate-level marketing degree on campus. In-person interaction and access to campus resources name just a couple. Click on certificate, master's degree, and doctorate degree program icons to learn more about each program and to narrow your search by degree level or location. When you find a program of interest, use our onsite form to contact schools directly.
i bls.gov/ooh/management/advertising-promotions-and-marketing-managers.htm#tab-4 | ii v bls.gov/ooh/business-and-financial/market-research-analysts.htm | iii bls.gov/ooh/media-and-communication/public-relations-specialists.htm | iv bls.gov/ooh/management/advertising-promotions-and-marketing-managers.htm#tab-8 | v bls.gov/careeroutlook/2015/article/should-i-get-a-masters-degree.htm |
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Master of Science: Digital Marketing
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Master of Science in Communication: Integrated Marketing Communication
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Partner a deep understanding of the latest marketing tactics with a foundation of business and management knowledge in the Dual Degree MBA/MSIMC program. Students earn both MSIMC and MBA in Marketing degrees.
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Colorado Technical University
Master of Business Administration - Marketing
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The Marketing Department offers high-quality M.A. and Ph.D. programs to a small number of students each year. The faculty is actively engaged in resea...
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This program prepares professionals to foresee marketplace changes and to develop strategic approaches in businesses.
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LSB College
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Emerson College
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Ball State University
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Students can develop an individualized program to further career goals. Each of the three degrees contains a research component.
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Syracuse University
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Working at the frontier of knowledge and being engaged with its dissemination to others is one of the most rewarding professions you can choose. The PhD Program at the Whitman School of Management at Syracuse University is committed to building a great...
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University of Oregon
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Medill's IMC program centers on a customer-centric, data-driven method of communicating with consumers, investors and employees.
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Montclair State University
The post-MBA certificate in Marketing is composed of four (4) courses from the following list, for a total of 12 semester hours (SH): MKTG 530 Expo...
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The programme enables you to acquire a thorough understanding of marketing theories and how to apply them to real-world business challenges in an inte...
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Regent's University London
MA Marketing
Explore how dynamics of human behaviour affect marketing decisions, and how to create products, services and brands people want.
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What have we been up to. . .? Olivia is currently on a Josh kick. You explain that one to me, she'll holler with me, snuggle with him. They both then stare at me with with a smug face, I could be making that up but don't bother to tell me. He discovered that if he "hums" to her she will do it right back to him. It is the cutest thing, & I never get sick of it. I'm just delighted that her little brain is catching on to "copying". . . it is the only way she will learn to communicate with us. She is growing like a weed and a delight to our life. We now have to use a detangle spray for the mop that is wildly abundant on the top of her head. . . so I get to comb my "alive" doll. The poor child puts up with it.
What to expect when you're expecting. . . . now, I have never read that particular book front to back but I'm seriously thinking of writing my own version & in the pages I will put down what really goes on when you're an alive incubator. They never say that you will fart outloud in Dick's Sporting Goods and then have to run for cover, they don't clue you in about getting dyslexia, ( I now say slouser tracks instead of trouser slacks) . . .or that your dreams get wild and crazy. I dreampt the other night that my brother & I were at a giant city that was in complete ruin. In the middle was a roaring river where, my bro snatched up a sting-ray, bite off its left back leg and we proceeded to float the river on the sting-ray. Sting-rays most certainly do not have legs, but mine do. And breathing. . . I no longer breathe. . . I gasp & pant & huff my way around the house. It's a lot of fun. Addictions??? Never did that book say just how much you LOVE Wendy's homestyle chicken sandwiches. I keep the place in business. . . . nor, does it tell you that you won't recognize your husband.
Need a whole paragraph for this one. . . maybe its because I'm 30 & spouting out kids, but nowadays there just aren't cute men, sorry boys, or it could be the fact that I'm married & much to worried about laundry to bother even noticing. Last Saturday morning we drove to Inman & went to a little lumber store. After awhile I haul sis out & Josh stays inside to wait the line out. I'm sitting in the front seat with Olivia on what little lap I have left, when out of the corner of my eye I see this guy. . . . . . you know. . .black cap, gray sweatshirt, wind pants over long legs. . . & this fat whale is thinking. . .hmmmmmm. . . . . not to shabby. . . as he goes to the trunk of another car (to get some nails I now know). . . .nice view. . .hate to see you leave, love to watch you go type of deal. . . I of course quit looking and start jabbering to small daughter because I already have a cute hubby, inside waiting in line, only he wasn't waiting in line, the cute guy was Josh, same guy I ate b&g with at my breakfast table an hour ago, same guy's shorts I fold every other day. . . same guy I'm MARRIED to. You would think I could figure that out in about a second or a little less, wouldn't you? . . . and I have 13 weeks left to go.
|The Cherub
|2 peas in a pod
|. . . Little Man. I think these tend
to look a little "alienish" but perhaps
you can make out the face.


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A long time ago I discovered that Peanut Butter was irresistible to every Rottweiler we ever had. Every morning for almost 20-years, without fail, all of our Rotts have gotten a spoonful along with whatever pills they were taking at the time. While not every one of our Rotts has been taking medicine all the time they have always been given 6-7 Brewer's Yeast pills to help ward off fleas. I've mentioned that to our Vet and they seem to have heard this from others as well. I can't say there's scientific proof to support this but on the other hand, none of our Rotts have ever had fleas and none have ever worn flea collars.
The real enjoyment I get in giving them their "peanut butter pills" is the look on their faces when they get to clean the spoons. As what happened this morning when I also happened to have the camera in the kitchen after taking the picture out the window. Please note the dreamy look in Ruff's eyes as he gets the first taste.
Immediately followed by his little girlfriend, Bess.
Ruff and Bess have become inseparable. The earlier lessons in future warrior queen training that Ruff was giving the little girl have turned into a genuine love between these two. They go outside together and hang out together as I often see looking out the kitchen window. So I put the telephoto lens on the camera and grabbed this shot this morning of the two of them sitting side by side at the far end of the pool.
Around the house, day and night, but what seems mostly while I'm watching the television, my companions are always engaged in playful contact. And as a few pictures show, they seem oblivious to old man Axl who may be laying nearby.
Love is a beautiful thing to see. And I am so glad that Ruff has found a new companion to take the place of his first love, Sassy. He doesn't get to play with Axl very much any more because the old man has trouble moving around.


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Whether it's a working breakfast, networking event, corporate lunch, celebratory dinner or special cocktail party, our events team will be with you every step of the way.
Elegant, discreet and well-equipped with AV, our room is ideal for parties, afternoon presentations, product launches, meetings and all manner of entertaining. We can accommodate from 8 to 40 seated and up to 70 for drinks.
The Private Dining Room is proving popular for breakfast meetings. Guests have three buffet options to choose from, which include our healthy smoothies, along with continental and cooked breakfasts.
For morning meetings the room can be used any time between 7am – 11am. It is equipped with a wall mounted flat screen for presentations, and Wifi, which are both complimentary.
The room can hold meetings of 8 to 40, including theatre style seating.
The whole of Boulestin can be hired for larger breakfast meetings or networking events.
Whether it's a wedding reception, networking event or Champagne and canapé evening, our whole restaurant and private courtyard can be hired exclusively.
We can seat up to 80 using the entire restaurant, or up to 120 standing.
Our outside courtyard is the star of the show, tucked away at the back of the restaurant, it's a real hidden gem with its original Victorian gas lamps, away from the hustle and bustle of busy St James's.
To make an enquiry contact us on 0207 930 2030, or email Yurena Bethencourt at [email protected].


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Thursday, December 17, 2009
Transformers: Animated Coming to Japan
Transformers: Revenge of the Fallen has finally been released in Japan. As part of the inserts is news that Transformers: Animated is coming to the country as their next toy line starting March 2010 and I assume translated cartoons will start airing at some point. Their name for the toy line has not been revealed. I expect they will get all versions of the toys that were released stateside with Tomy Takara adding their own additions as they see fit. For Transfans in North American this mostly means that you will get a second chance to fill out collection gaps and grow it with Japan exclusive releases. (via TFormers)
3 comments:
Are Japanese fans now coming to know him as Optimus Prime? He's been known as Convoy there for a long time. Or do they know him by both names?ReplyDelete
Just out of curiousity, I went to Google Translate and translated "Optimus Prime" to Japanese. This is what I got.ReplyDelete
Translated back to English, it's Convoy!
Maybe this will give TFA a chance at a revival in America.... I can dream...ReplyDelete
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Getting into the swing of this now! Here are some photos from the first couple of weeks of August. As before click the map and photos to see large versions.
Getting into the swing of this now! Here are some photos from the first couple of weeks of August. As before click the map and photos to see large versions.
|St-Quentin-de-la-Poterie was a fascinating little place. In the past it had been a commercial pottery town, making tiles and such like. Several years after the heavy industry closed down independent artisan pottery studies started opening up, now there is something like 23 studios. If you like ceramics, it's worth checking out! Anyway, to the photos, this was a mosaic on the street.
|Cevennes National Pak. This is in Bédouès where our campsite was. We liked this area and the nearby town of Florac which had an interesting market.
|We went for a walk from the campsite in Bédouès, Cevennes. We never like to take the same route back but we hadn't got a map so we took a bit of a risk. Turned into the longest walk ever, didn't get back to the van until sometime after 10pm! At the bottom of the campsite was this river and lots of wild greens for fresh juice.
|Gorges du Tarn, on the edge of the Cevennes National Park. The gorge is over 30 miles long and very beautiful, went for a good walk, although the trees generally obscured the view of the gorge, we got the best views from walking along the road.
|Flamingos in the Carmargue area, not far from Montpelier.
|The amphitheatre in Nímes, over 2000 years old and still standing
|In a little place called Ginac, campsite by the river.