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Desde las bibliotecas puedes descargar libros gratis,las obras completas de grandes autores en castellano. Exclude inactive (FIDE rated only): Exclude Rated players Search only unrated: Sort by: Name Country Rating (FIDE rated only). No wages, lots of gossip, long hours of complete boredom. A personal life is frowned upon. Skill with a needle required. Must be firm in saying no to kingly advances. Royal favor if successful. For a young woman of noble birth who wished to be at the center of court life, an appointment as Lady- in- Waiting to a princess, duchess or queen was highly coveted. To be selected as a Lady- in- Waiting was considered a mark of high social standing and an honor for the family. These ladies were not servants, but more like companions. They were mostly unpaid. After all, it was a privilege to be chosen. They did get free room and board, as did their horses. They were also given the proper clothes befitting the status of whom they were waiting upon. Free clothes.) And if they were of noble birth, these ladies had servants of their own, who were also given board. But it wasn. It was the close and intimate proximity to power and status. To be able to whisper a favor in a royal ear, or to gain the confidence and trust of your mistress, or to petition on behalf of one? So what exactly does a Lady- in- Waiting do besides keep the Queen company? The skills required to be a Lady- in Waiting are not difficult and certainly not far afield of the skills already taught to young noblewomen at the time. Embroidery appears to be at the top of the list. Ladies spent a great deal of time on their needlework and sewing. They made undergarments and shifts for themselves, others, their queen or princess. For instance, Katherine of Aragon made Henry VIII. She continued doing so even when Henry had taken up with Anne Boleyn. To appease Anne, Henry no longer sent cloth to Katherine. During the reign of her daughter, Elizabeth I, a story was circulated that Anne, while Queen, instructed her ladies to sew shirts as well. But they were not for the King, instead these handcrafted shirts were to be given to the poor. How pious and charitable of Anne . Anne embroidered entwined H and A. Many an hour was spent planning plays and other court entertainments. The latest Tweets from Lady in Waiting . She is free and complete in her identity as an image bearer of God. Want to watch this again later? Sign in to add this video to a playlist. Autoplay When autoplay is enabled, a suggested video will automatically play next. Definition from Wiktionary, the free dictionary. Jump to: navigation, search. Buy Lady in Waiting: Becoming God's Best While Waiting for Mr. Right, Expanded Edition on Amazon.com FREE SHIPPING on qualified orders. And of course, a knowledge of games, especially games of chance, came in enormously handy to keep your queen or princess amused. So not too terribly difficult . You did not get to see your own family or, if you were married raise your children. Your time was not your own. You put your mistress. She expected complete and utter devotion to her service. Which Lady- in- Waiting are You? The most noble women in England are on the top rung. They are the Ladies of the Privy Chamber. The Privy Chamber is the queen. In the case of Queen Elizabeth, she would receive honored guests and ambassadors here, conduct business with her counsellors and in the evening pursue entertainments such as music and games with her ladies. Only the most senior ladies had access to the Privy Chamber. Four of the Ladies of the Privy Chamber are given the exalted title of . These are the most senior ladies . They saw to the more intimate needs of the queen . In the case of Queen Elizabeth, Ladies- in- Waiting may have slept in the Privy Chamber with her. One may have even slept with her in her bed or in a trundle nearby. The lowest rank, if you will, is Ladies of the Presence Chamber. They are there more for show. The Presence Chamber is a large room, often known as the Throne Room. It was open to the public for those who had business before the court. An unmarried Lady- in- Waiting was called a Maid of Honor and often dressed in white. She carried the Queen. Women knew instantly where they ranked by where they were assigned. There. Henry VIII had stipulated in his Act of Succession that if Elizabeth had no children, then the crown was to go to the heirs of his younger sister, Mary Tudor Brandon. Needless to say, Katherine was very popular, especially with European princes and English nobles. The attention went straight to silly young Katherine. Elizabeth was not happy with Katherine. Shop for lady in waiting on Etsy, the place to express your creativity through the buying and selling of handmade and vintage goods. Lady in Waiting goes beyond pop psychology and clings to the steadfast foundation of God's Word. Creatively integrating biblical truth with an honest evaluation of the struggles facing today's single woman, this book will add. Directed by Christian Taylor. With Virginia McKenna, Rodney Hudson, Beth McGee-Russell, Emma Wilson. But when Elizabeth came to the throne, she demoted Katherine to Maid of Honor . Katherine (silly, silly girl) is said to have complained bitterly. She knew how out of favor she had fallen with her new status. Fortunately for Katherine, the political winds shifted again. Mary, Queen of Scots, became a larger threat than Katherine. To keep Katherine close, Elizabeth restored Katherine (temporarily) to her exalted position as Lady of the Bedchamber. Queen Elizabeth had 2. Ladies- in- Waiting. The Queen normally chose her ladies, although the King might interfere if a favor was needed of a noble family or a woman had caught his eye. The Unintended Consequence of Being a Lady- in Waiting. While most noble ladies who are appointed to Lady- in- Waiting serve with piety and discretion, a few ladies have been the source of a great deal of grief for their queen, princess or royal duchess. That. The Lady would find it very difficult to thwart the King in these matters. Closeness to the King gave one power at court and the closest one could be is in his bed. Lady in Waiting is a historical novel by Rosemary Sutcliff and first published in 1957. It is set in the Elizabethan era and beginning of the Stuart period of England, telling the life story of Sir Walter Raleigh, primarily.Many were discreet, a pleasant diversion, and made no demands. If they were unmarried and should happen to find themselves with child, they were often married off to a knight or lesser noble quickly with a quiet settlement. Mary Boleyn and Bessie Blount, Maids of Honor to Katherine of Aragon, come to mind, as does Mary Shelton, Lady- in- Waiting to Anne Boleyn. Some Ladies were not so discreet. Alice Perrers, Lady- in- Waiting to Queen Philippa, became the hated and despised mistress of Edward III. Anne Boleyn, Lady- in- Waiting to Katherine of Aragon. Jane Seymour, Lady- in- Waiting to Anne Boleyn. Katherine Howard, Maid of Honor to Anne of Cleves. Henry VIII certainly dipped into his wives. They mostly handle the queen. You often see her holding the many flower bouquets the queen receives or her umbrella. It appears sewing or card- playing are no longer necessary skills. Her Majesty, Queen Elizabeth has nine Ladies- in- Waiting. Diana, Princess of Wales had eight. The Princess Royal has eleven. Camilla, Duchess of Cornwall has three. Sophie, Countess of Wessex has one. The younger royals, The Princesses of York and the Duchess of Cambridge have no Ladies- in- Waiting. Have we reached the end of a centuries- old tradition, found somewhat antiquated in our more republican and plebeian world? While the modern royals are cutting back on their Ladies of Waiting, it still remains an unpaid, yet prestigious position. Comments Comments. Gorilla - Wikipedia. Gorillas are ground- dwelling, predominantly herbivorousapes that inhabit the forests of central Africa. The eponymous genus Gorilla is divided into two species: the eastern gorillas and the western gorillas (both critically endangered), and either four or five subspecies. Gorilla trekking - Individual & Group Tours in Uganda - Africa. Gorilla Safari / Gorilla Trekking in Uganda - Gorilla Tours.com. Will the Rothschild’s giraffe survive? When it comes to rare mammal species in Uganda, almost immediately the mountain gorilla is. Our friends, the Grammar Gorillas, need help identifying parts of speech. If you click on the right word in the sentence, our friends get a banana. And you know, a gorilla with a banana is a gorilla with appeal. Beginner (nouns and verbs only). Gorilla Safari Offers Encounter Africa Safaris offer the lowest prices for gorilla safaris to see mountain gorillas in Uganda and Rwanda. We combine gorilla treks with wildlife viewing, chimp trekking, cultural and community interaction to give you an authentic African. They are the largest livingprimates. The DNA of gorillas is highly similar to that of humans, from 9. Although their range covers a small percentage of Africa, gorillas cover a wide range of elevations. The mountain gorilla inhabits the Albertine Rift montane cloud forests of the Virunga Volcanoes, ranging in altitude from 2,2. Are you looking for a gorilla safari in Uganda, Rwanda and Congo, Africa Tours Adventures is a specialist and trusted travel agent based in Uganda with over 5 years hands on safari experience. We are devoted to providing excellent service and customer satisfaction. Female gorillas have been documented engaging in lesbian sex for the first time. The behaviour was observed by scientists during a research trip to the Rwandan section of the Virunga mountain range in central Africa. The wild mountain gorillas, observed by a team led by Dr Cyril Grueter of the. Contact Us Blog Home Gorilla Trekking 2 Days Rwanda Gorilla Tour 3 Days Gorilla Trekking in Congo 4 Days Gorilla trekking & Wildlife Safari 3 Days Uganda Gorilla Safari 5 Days Gorilla Trekking Uganda 7 Days Gorillas, Wildlife & Chimps Safari 8 Days Gorilla. Jahrhunderts brach an dieser Stelle epidemieartig die Schlafkrankheit aus. Zwischen 1925 – 1947 wurden das menschenverlassene Areal am Kazinga-Kanal zu Wildschutzgebieten. Lowland gorillas live in dense forests and lowland swamps and marshes as low as sea level, with western lowland gorillas living in Central West African countries and eastern lowland gorillas living in the Democratic Republic of the Congo near its border with Rwanda. BC) a Carthaginian explorer on an expedition on the west African coast to the area that later became Sierra Leone. More recently, a third subspecies has been claimed to exist in one of the species. The separate species and subspecies developed from a single type of gorilla during the Ice Age, when their forest habitats shrank and became isolated from each other. Mountain gorillas are the most severely endangered, with an estimated population of about 8. Adult males are 1. Female gorillas are shorter, with smaller arm spans. Occasionally, a silverback of over 1. Obese gorillas in captivity can weigh as much as 2. Adult males also have a prominent sagittal crest. The eastern gorilla is more darkly coloured than the western gorilla, with the mountain gorilla being the darkest of all. The mountain gorilla also has the thickest hair. The western lowland gorilla can be brown or grayish with a reddish forehead. In addition, gorillas that live in lowland forests are more slender and agile than the more bulky mountain gorillas. The eastern gorilla also has a longer face and broader chest than the western gorilla. Due to novel sequences, though, it is different enough to not conform with the human ABO blood group system, into which the other great apes fit. The range of the two species is separated by the Congo River and its tributaries. The western gorilla lives in west central Africa, while the eastern gorilla lives in east central Africa. Between the species, and even within the species, gorillas live in a variety of habitats and elevations. Gorilla habitat ranges from montane forests to swamps. Eastern gorillas inhabit montane and submontane forests between 6. In addition, eastern lowland gorillas live in montane bamboo forests, as well as lowland forests ranging from 6. Nests tend to be simple aggregations of branches and leaves about 2 to 5 ft (0. Gorillas, unlike chimpanzees or orangutans, tend to sleep in nests on the ground. The young nest with their mothers, but construct nests after three years of age, initially close to those of their mothers. Diets differ between and within species. Mountain gorillas mostly eat foliage, such as leaves, stems, pith, and shoots, while fruit makes up a very small part of their diets. Their home ranges average 3. Leaves and pith are commonly eaten, but fruits can make up as much as 2. Since fruit is less available, lowland gorillas must travel farther each day, and their home ranges vary from 2. Eastern lowland gorillas will also eat insects, preferably ants. Termites and ants are also eaten. Gorillas rarely drink water . Gorilla remains have been found in leopard scat, but this may be the result of scavenging. Troops tend to be made of one adult male or silverback, multiple adult females and their offspring. Silverbacks also have large canine teeth that also come with maturity. Both males and females tend to emigrate from their natal groups. For mountain gorillas, females disperse from their natal troops more than males. However, male mountain gorillas sometimes stay in their natal troops and become subordinate to the silverback. If the silverback dies, these males may be able to become dominant or mate with the females. This behaviour has not been observed in eastern lowland gorillas. In a single male group, when the silverback dies, the females and their offspring disperse and find a new troop. Joining a new group is likely to be a tactic against this. This likely serves as protection from leopards. Younger males subordinate to the silverback, known as blackbacks, may serve as backup protection. Blackbacks are aged between 8 and 1. The bond that a silverback has with his females forms the core of gorilla social life. Bonds between them are maintained by grooming and staying close together. Relationships between females may vary. Maternally related females in a troop tend to be friendly towards each other and associate closely. Otherwise, females have few friendly encounters and commonly act aggressively towards each other. Males in all- male groups, though, tend to have friendly interactions and socialise through play, grooming, and staying together. The gestation period lasts 8. Female mountain gorillas first give birth at 1. Gorillas mate year round. This serves to urge the male to mount her. If the male does not respond, then she will try to attract his attention by reaching towards him or slapping the ground. By 1. 2 months old, infants move up to five meters (1. They enter their juvenile period at their third year, and this lasts until their sixth year. At this time, gorillas are weaned and they sleep in a separate nest from their mothers. Sounds classified as grunts and barks are heard most frequently while traveling, and indicate the whereabouts of individual group members. Screams and roars signal alarm or warning, and are produced most often by silverbacks. Deep, rumbling belches suggest contentment and are heard frequently during feeding and resting periods. They are the most common form of intragroup communication. The ritualized charge display is unique to gorillas. The entire sequence has nine steps: (1) progressively quickening hooting, (2) symbolic feeding, (3) rising bipedally, (4) throwing vegetation, (5) chest- beating with cupped hands, (6) one leg kick, (7) sideways running, two- legged to four- legged, (8) slapping and tearing vegetation, and (9) thumping the ground with palms to end display. Colo, a female western gorilla at the Columbus Zoo and Aquarium is the oldest known gorilla, at 5. December 2. 01. 5). A few individuals in captivity, such as Koko, have been taught a subset of sign language. Like the other great apes, gorillas can laugh, grieve, have . Gorillas are now known to use tools in the wild. A female gorilla in the Nouabal. A second female was seen using a tree stump as a bridge and also as a support whilst fishing in the swamp. This means all of the great apes are now known to use tools. Great apes are endowed with semiprecision grips, and have been able to use both simple tools and even weapons, by improvising a club from a convenient fallen branch, for example. Scientific study. American physician and missionary Thomas Staughton Savage obtained the first specimens (the skull and other bones) during his time in Liberia. Other species of gorilla were described in the next few years. He brought dead specimens to the UK in 1. On his first trip, he was accompanied by his friends Mary Bradley, a mystery writer, her husband, and their young daughter Alice, who would later write science fiction under the pseudonym James Tiptree Jr. After their trip, Mary Bradley wrote On the Gorilla Trail. She later became an advocate for the conservation of gorillas, and wrote several more books (mainly for children). In the late 1. 92. Robert Yerkes and his wife Ava helped further the study of gorillas when they sent Harold Bigham to Africa. Yerkes also wrote a book in 1. In 1. 95. 9, he conducted a systematic study of the mountain gorilla in the wild and published his work. Years later, at the behest of Louis Leakey and the National Geographic, Dian Fossey conducted a much longer and more comprehensive study of the mountain gorilla. When she published her work, many misconceptions and myths about gorillas were finally disproved, including the myth that gorillas are violent. Western lowland gorillas (G. The SIVgor Simian immunodeficiency virus that infects them is similar to a certain strain of HIV- 1. The first gorilla genome was generated with short read and Sanger sequencing using DNA from a female western lowland gorilla named Kamilah. This gave scientists further insight into the evolution and origin of humans. Despite the chimpanzees being the closest extant relatives of humans, 1. For example, gorillas have featured prominently in monstrous fantasy films such as King Kong. Pulp fiction, such as Tarzan and Conan the Barbarian, has featured gorillas as physical opponents of the titular protagonists. Conservation status. All species (and sub- species) of gorilla are listed as Critically Endangered on the IUCN Red List. In 2. 00. 4, a population of several hundred gorillas in the Odzala National Park, Republic of Congo was essentially wiped out by the Ebola virus. The researchers indicated in conjunction with commercial hunting of these apes, the virus creates . The Gorilla Agreement is the first legally binding instrument exclusively targeting gorilla conservation; it came into effect on 1 June 2. See also. References^ ab. Groves, C. P. Wilson, D. E.; Reeder, D. M., eds. Mammal Species of the World: A Taxonomic and Geographic Reference (3rd ed.). Baltimore: Johns Hopkins University Press. Songs of the Gorilla Nation. Geographici Graeci Minores. Retrieved on 2. 01. Groves, Colin (2. Taylor & Michele L. Gorilla Biology: A Multidisciplinary Perspective(PDF). Cambridge University Press. Evolutionary Anthropology, 1. Fathom / Cambridge University Press. Retrieved 1. 1 September 2. R.; Ruvolo, M.; Steiper, M. Molecular Phylogenetics and Evolution. Retrieved 1. 5 September 2. Mol Biol Evol pii: msu. Prince- Hughes, Dawn (1. Songs of the Gorilla Nation. Archived from the original on 1. February 2. 00. 8. Uganda, Rwanda Gorilla Tours, Wildlife Tours and Safaris. Fast and accurate responses with. Uganda and Rwanda Tour. Safari. . Learn More. About us Here > > >. Our major purpose is that as many as. Uganda. Rwanda. with a professional and. We. emphasize giving plenty of quality. Uganda. Rwanda. both before and after the tour. WHY WE DO THE THINGS WE. DO? We delight in providing the most. We. emphasize giving plenty of quality. Uganda. and Rwanda both before and after the. Although we delight in earning. We will be most glad when at the end. Uganda and Rwanda. Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite : Nature Communications. Investigation of CMAH gene loss in mammalian evolution. We examined hominid and mammalian genomes with genome alignments and gene loss analysis. The mammalian gene loss events were analysed with the program CAFE (Computational Analysis of gene Family Evolution. Ensembl 7. 2. The gene loss event in hominins was further verified with independent genomic analysis; the sequence reads of the Neanderthal. Denisovan genomes. Tro. 3 at the CMAH gene locus with the University of California, Santa Cruz (UCSC) genome browser. The ferret. 54 and New World monkey. CMAH gene loss events are published. Dog CMAH does not appear to be functional in certain breeds of dogs. Ex vivo culture of RBCs. Bone- marrow- derived CD3. On day 6 or 7 of culture, cells were transduced with lentivirus harbouring either the chimpanzee (Pan troglodytes) CMAH complementary DNA (c. DNA) sequence in p. LVX- Puro (Clontech) or the empty vector, p. LVX- Puro. The CMAH c. DNA sequence with a C- terminal tobacco etch virus (TEV) cleavage site and FLAG- c- myc tags was codon- optimized for human expression and synthesized (Gene. Art). CMAH c. DNA was amplified using primers listed in Supplementary Table 3 and cloned into p. LVX- Puro. Transduction with lentiviral vectors and subsequent selection on 2. Figure 1: Expression of chimpanzee CMAH in human red blood cells (RBCs) introduces the non-human sialic acid variant Neu5Gc on the cell surface. Free parasite papers, essays, and research papers. These results are sorted by most relevant first (ranked search). You may also sort these by color rating or. Sacculina is a genus of barnacles that is a parasitic castrator of crabs. The adults bear no resemblance to the barnacles that cover ships and piers; they are. Friederich Karl Wiehe Published for the Institute for studies of the Jewish question, Berlin (1938) Contents. Twitch is the world's leading video platform and community for gamers. More than 45 million gamers gather every month on Twitch to broadcast, watch and chat about gaming. Notes on Labor 1875-1900. First read the following short pieces: Shoe worker and bobbin boy - List of the 75 wealthiest individuals in Human History. On day 1. 8, cells were replated on a fresh MS- 5 stromal cell layer and then collected on day 2. Cells were stored at 4. Cells were resuspended in either 5. The following antibodies were used at the indicated dilutions: anti- Neu. Gc (1: 5,0. 00, Siamab), phycoerythrin(PE)- conjugated anti- DARC (1: 1. Miltenyi Biotec), anti- CD7. PE (1: 1. 0, Miltenyi Biotec), fluorescein isothiocyanate- conjugated anti- glycophorin A (GPA); 1: 5. Clone 2. B7, STEMCELL Technologies) and anti- glycophorin C (GPC)- fluorescein isothiocyanate (1: 5. BRIC 1. 0, Santa Cruz). Cells were incubated for 1. Unstained cells and cells stained with DARC, CD7. GPA and GPC antibodies were resuspended in 1. Neu. 5Gc- stained cells were incubated in anti- chicken Ig. Y- Alexa Fluor 4. Life Technologies) at 1: 1,0. Control samples were similarly incubated in anti- mouse Ig. G2a- PE (1: 1. 0, Miltenyi Biotec), anti- chicken Ig. Y- Alexa Fluor 4. Ig. G- Alexa Fluor 4. Life Technologies). Cells were washed twice and subjected to flow cytometric analysis. The data were analysed in Flow. Jo 4 version 1. 0. Tree Star). Measurement of sialic acid by HPLCFor measurement of cell surface sialic acid, RBC and c. RBC ghosts were prepared for sialic acid release as described. Cells were stored at . RBC ghosts were pelleted at 2. Sialic acid release from RBC ghosts was achieved by mild acid hydrolysis as described. In brief, RBC ghosts were resuspended in 7. For formation of fluorescent derivatives, 7. The derivatization solution was made up of 7. Samples were run on an HPLC with a C1. Sialic acid elution was monitored by absorbance at 3. Data were acquired on Chemstation Rev A. Agilent Technologies). Eluted sialic acids were compared with a sialic acid reference panel, and Neu. Ac and Neu. 5Gc solutions of known amounts for quantitation of sialic acids. For measurement of sialic acid in invasion assays, neuraminidase treatment of RBCs was performed in 4. Supernatants were collected and sialic acid was quantified as described previously. In brief, cell supernatants were treated with 2. To terminate the reaction, 1. Mein Kampf, ( German: “My Struggle”) political manifesto written by Adolf Hitler. It was his only complete book and became the bible of National Socialism. Morgellons Parasite Disease Support Forum, Page 117. In this view Forum topics are ordered by the Date of the Last message posted within the thread, and messages. Una criatura extraterrestre ha llegado a un verde planeta en busca de esp Type de personnage de la com. Samples were analysed on the HPLC as described above, but elution was performed using a running buffer consisting of 1. Pk H SSUDatt. B/BSD- GFP (SSUD: small subunit of the D- type ribosomal RNA gene) was generated in our lab as follows. The targeting vector, p. DEF- DSSU- h. DHFR6. Pk H (provided by A. W. Thomas; Biomedical Primate Research Center, Rijswijk) to form Pk H SSUD. The resulting vector, p. SSUD- CG6- att. B, was integrated into Pk H SSUD to form Pk H SSUDatt. B. In the final step, p. BSD- GFP- INT- att. P6. 2 was integrated into Pk H SSUDatt. B. Pk YH1 was generated in our laboratory from the parental Pk H strain by growth for 3 months in human O+ RBCs supplemented with 1. The percentage of reticulocytes was decreased to 2. Parasites were grown at 2% haematocrit in complete RPMI (RPMI supplemented with 2. In brief, RBCs or c. RBCs were washed three times in incomplete RPMI and treated with 1. RBCs were then washed twice in incomplete RPMI and once in PBS p. H 7. 4. For neuraminidase treatment for invasion assays, human RBCs were washed in wash media (RPMI supplemented with 2. The neuraminidase- treated RBCs were then washed extensively in complete RPMI before use. The neuraminidase- treated RBCs were subjected to a second neuraminidase treatment to remove and quantify the remaining sialic acid. This second treatment was performed at 2. U neuraminidase at 5. Synchronised schizont- stage parasites were purified by magnetic separation on a MACS cell separator (Miltenyi Biotec) and added to RBCs at . Assay plates were kept in a modulator incubator chamber gassed with 1% O2, 5% CO2 and balance of N2, and reinvasion and growth were monitored over time by thin smear of mock wells. At trophozoite/schizont stage, final parasitaemia was estimated by light microscopy from counts of at least 1,0. May- Gr. The parasitized erythrocyte multiplication rate (final parasitaemia (after single round of invasion)/initial parasitaemia) was determined for each strain in each cell type. Synchronised schizont- stage parasites at . Invasion assays were seeded in triplicate 2. To quantify inoculum parasitaemia, a subset of samples was fixed immediately with formaldehyde and stored at 4. The remaining samples were incubated for 4. Inoculum and post- invasion samples were then stained with SYBR Green I and analysed by flow cytometry. Expansion rates were expressed as parasitized erythrocyte multiplication rate. For MGSA invasion inhibition assays, human RBCs were plated in a 9. MGSA (Pepro. Tech) of varying concentrations made up in incomplete RPMI were added to respective wells. Schizont- stage parasites purified by magnetic separation, as described above, were added to each well at . After allowing for a single round of invasion, parasitaemia was measured the following day by either SYBR green I or Vybrant Dye. Cycle Violet (Life Technologies) staining and flow cytometry. The parasitaemia of MGSA- treated samples was normalized to a mock- treated control for each strain. The data were analysed in Graph. Pad Prism 5. Protein expression plasmids. Region II of Pk. DBP. PCR products were cloned into the mammalian expression vector, p. SDP3. 2, a version of p. TT6. 6 with a modified multiple cloning site and with the rat COMP domain downstream of the multiple cloning site. For the GST- COMP- expressing plasmid, GST was amplified from p. ET- 4. 2a(+) (Supplementary Table 3) and cloned into p. SDP3. 2. For the COMP- negative control plasmid, Nhe. I- and Not. I- digested p. SDP3. 2 was modified with T4 DNA polymerase (New England Biolabs) to generate blunt ends and then ligated. Plasmid constructs were verified by sequencing. HEK2. 93- 6. E culture and protein expression and purification. HEK2. 93- 6. E cells, a kind gift from Yves Durocher (National Research Council, Canada), were maintained in serum- free Freestyle 2. Life Technologies) supplemented with 0. Pluronic F- 6. 8 (Life Technologies) and 2. Cells were grown in suspension in Erlenmeyer flasks at 1. Recombinant protein expression by transient transfection was achieved as described. In brief, cells were transfected at a cell density of 1 . DNA and the transfection reagent, linear PEI (Polysciences, Inc.), were diluted separately in complete Freestyle 2. The DNA was vortexed for 1. Cultures were processed on day 5 or 6 post transfection by pelleting and collecting the culture supernatant containing the expressed protein. Protein expression was confirmed by western blot using Strep- Tactin- HRP (IBA). Culture supernatant containing recombinant protein was used in protein overlay assays. For flow cytometry- based- binding assays, Pk. DBP. Bound protein was eluted by 2. Buffer exchange into PBS p. H 7. 4 was achieved using centrifugal filter units. Protein concentration was determined from absorbance measurements at 2. Membranes were blocked in PBS. Membranes were probed with the respective antibodies at the indicated dilutions. The DARC western blot was probed with anti- rabbit- HRP (GE Healthcare) at 1: 1. Neu. 5Gc western blot was probed with anti- chicken/turkey- HRP (Life Technologies) at 1: 5,0. All membranes were developed with Super. Signal West Pico chemiluminescent substrate (Thermo Scientific) and imaged by autoradiography. Protein overlay assay. Untreated and enzyme- treated rhesus macaque, and human RBC ghosts and c. RBC ghosts were prepared by cell lysis, and multiple washes in cold 5. Protein content of macaque and human RBC ghosts was quantified by Bradford assay. RBC/c. RBC ghosts were stored at . Before use, c. RBC ghosts were treated with DNase I (New England Biolabs). Samples of enzyme- treated or untreated macaque and human RBC ghosts or c. RBC ghosts, not treated with reducing agent or heat- denatured, were electrophoresed; 1. Electrophoresed protein was transferred to nitrocellulose membrane. Membranes were blocked in PBST- 1. HEK2. 93- 6. E culture supernatant containing recombinant protein with Tween 2. Membranes were washed, blocked in PBST- A for 1. Bound protein was detected by autoradiography. Flow cytometry- based binding assay. RBCs or c. RBCs were washed three times in PBS. Cells were pelleted in a 9. Samples were incubated for 1. Samples were washed three times in blocking buffer then incubated in anti- mouse Ig. G- Alexa Fluor 4. Samples were washed twice, then resuspended in 1. Data were analysed by Flow. Jo 4 version 1. 0. Whole- genome sequencing. Genomic DNA was extracted from schizont- stage Pk H and Pk YH1 parasites using the QIAamp DNA blood kit (Qiagen). Genomic DNA was used to construct Illumina sequencing libraries for both P. |
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December 2016
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